KWAME NKRUMAH UNIVERSITY OF SCIENCE & TECHNOLOGY, KUMASI

DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY: M. Phil. BTC 561

FERMENTATION AND ENZYME TECHNOLOGY 2020/2021 (Handout 5)

ENZYME SCREENING, PRODUCTION AND MODIFICATION

Commercial enzyme production has grown during the past century in volume and number of
products in response to expanding markets and increasing demand for novel biocatalysts.
Microorganisms constitute the major source of enzymes, but several enzymes are also obtained
from renewable animal and plant sources. Traditional enzyme production relied on the natural
hosts as raw materials, however genetic engineering has now given a choice for producing
sufficient quantities of enzymes in selected production hosts including microorganisms and
transgenic plants.

Production of a new microbial enzyme starts with screening of microorganisms for desirable
activity using appropriate selection procedures. The harsh environment to which several enzymes
are subjected during process applications has given impetus to screening of extremophiles for
enzymes having desirable features of activity and stability.

The level of enzyme activity produced by an organism from a natural environment is often low
and needs to be elevated for industrial production. Increase in enzyme levels is often achieved
by mutation of the organism. An alternative strategy that has gained favour is production of the
enzyme in a recombinant organism of choice whose growth conditions are well optimised and
whose GRAS status is established. Random or site-directed mutagenesis with the purpose of
engineering the activity and stability properties of an enzyme prior to its production is becoming
a common practice.

The microorganisms used for enzyme production are grown in fermenters using an optimized
growth medium. Both solid state- and submerged fermentation are applied commercially, however
the latter is preferred in many countries because of a better handle on aseptic conditions and
process control. The enzymes produced by the microorganism may be intracellular or secreted
into the extracellular medium.

Isolation and purification, i.e., downstream processing of enzyme from the raw material
constitutes the subsequent key stage in the production process. The desired level of purification
depends on the ultimate application of the enzyme product. The industrial bulk enzymes are
relatively crude formulations while speciality enzymes undergo a thorough purification to yield a
homogeneous product. A traditional downstream processing scheme involves stages of
clarification for separation of the enzyme from the solids comprising the raw material,
concentration to reduce the process volumes, and purification to separate it from other soluble
contaminants. In case of the intracellular enzymes, disruption of cells or Thissue for release of
the product is among the primary separation steps. There is a choice of different separation
techniques for each stage. Chromatography is the major technique for high-resolution purification
of enzymes.

Some separation techniques allow integration of the downstream processing stages required for
purification thus reducing the number of steps and hence the production costs. The enzyme is
finally formulated as a liquid or solid product. In either case, stabilizing additives are added for
rendering long shelf life to the product. Some enzymes are immobilised to solid supports or
enzyme crystals are cross-linked to render them insoluble and stable for repeated or long term
use in a process application. Large-scale production of enzymes has to comply with the standards

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set by International Organization of Standardization for ensuring quality and production efficiency,
and also environmental management control, whenever applicable.

ENZYME SOURCE

Figure: Origin, Purification and Uses of Enzymes

Enzymes Are Ubiquitous

Enzymes are essential components of animals, plants and microorganisms, due to the fact that
they catalyse and co-ordinate the complex reactions of cellular metabolism. Up until the 1970s,
most of the commercial application of enzymes involved animal and plant sources. At that time,
bulk enzymes were generally only used within the food-processing industry, and enzymes from
animals and plants were preferred, as they were considered to be free from the problems of
toxicity and contamination that were associated with enzymes of microbial origin. However, as
demand grew and as fermentation technology developed, the competitive cost of microbial
enzymes was recognized and they became more widely used. Compared with enzymes from
plant and animal sources, microbial enzymes have economic, technical and ethical advantages,
which will now be outlined.

Economic Advantages
The sheer quantity of enzyme that can be produced within a short time, and in a small production
facility, greatly favours the use of microorganisms. For example, during the production of rennin
(a milk-coagulating enzyme used in cheese manufacture) the traditional approach is to use the
enzyme extracted from the stomach of a calf (a young cow still feeding on its mother’s milk). The
average quantity of rennet extracted from a calf’s stomach is 10 kg, and it takes several months
of intensive farming to produce a calf. In comparison, a 1 000-litre fermenter of recombinant
Bacillus subtilis can produce 20 kg of enzyme within 12 h. Thus the microbial product is clearly
preferable economically, and is free from the ethical issues that surround the use of animals.
Indeed, most of the cheese now sold in supermarkets is made from milk coagulated with microbial
enzymes (so is suitable for vegetarians).

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A further advantage of using microbial enzymes is their ease of extraction. Many of the microbial
enzymes used in biotechnological processes are secreted extracellularly, which greatly simplifies
their extraction and purification. Microbial intracellular enzymes are also often easier to obtain
than the equivalent animal or plant enzymes, as they generally require fewer extraction and
purification steps.

Animal and plant sources usually need to be transported to the extraction facility, whereas when
microorganisms are used the same facility can generally be employed for production and
extraction. In addition, commercially important animal and plant enzymes are often located within
only one organ or tissue, so the remaining material is essentially a waste product, disposal of
which is required.

Finally, enzymes from plant and animal sources show wide variation in yield, and may only be
available at certain times of year, whereas none of these problems are associated with microbial
enzymes.

The primary consideration in the production of any enzyme relates to the choice of source. In
most cases, the desired activity can be obtained from several sources. Traditionally, however, the
choice of source has been more restricted for some enzymes. For example, the enzyme rennet
was until recently obtained from the stomach of suckling calves; the corresponding microbial
enzyme led to an off-flavor in the cheese produced. Today, recombinant DNA technology is used
to produce the calf enzyme in microorganisms.

Microorganisms represent an attractive source of enzymes as they can be cultured in large

quantities in a relatively short period by established methods of fermentation (see also Microbial
Cell Cultivation). However, the level of production of a particular enzyme varies in different
microorganisms, and moreover the enzymes often differ in composition and properties. One
usually finds that the closely related organisms have enzymes with nearly similar properties, while
unrelated organisms have enzyme systems that differ widely. The most critical feature of the
organisms for producing industrially significant enzymes is their GRAS (generally regarded as
safe) status, which implies that they must be non-toxic, non-pathogenic and generally should not
produce antibiotics.

The GRAS listed microorganisms include fewer than 50 bacteria and fungi. Examples are the
bacteria including Bacillus subtilis, B. licheniformis, and various other bacilli, lactobacilli,
Streptomyces species, the yeast Saccharomyces cerevisiae, and the filamentous fungi belonging
to the genera Aspergillus, Mucor, Rhizopus, etc. In case of Bacillus, mutants are selected that
can no longer form spores. Since Aspergillus cultures are frequently inoculated with conidia,
enzyme production using these fungi relies on good spore formation. Most of the bulk enzymes
(hydrolases) are secreted by the microorganisms directly into the culture medium, while some
enzymes e.g., penicillin acylase and glucose isomerase are intracellular. For some applications,
it may not be necessary to isolate the enzymes but the microbial cells themselves are used as
enzyme source. The organism is preferred which gives high yields of enzyme in shortest possible
fermentation time. The production strains used in industry are normally modified by genetic
manipulation to have high levels of production.

A common trend in the industry today is that the gene coding for the enzyme with desired
characteristics is transferred into one of the selected microbial production which have all the
required features of safety and high expression levels and for which the growth medium has been
optimised, hence avoiding the need for optimization of individual enzyme producing strains.

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A further short cut in the search for the right enzyme has been made in eliminating the step of
screening, isolation and cultivation of microorganisms which may either be present in low number
or produce low levels of the activity. Instead, DNA is directly isolated from an environmental
sample and the possession of the desired activity is located using an appropriate gene probe.
The gene is cloned and expressed in the desired production organism.

Despite the advantages of microorganisms as enzyme source, some enzymes are still
economically produced from plant and animal sources. This is possible because of sufficiently
high amounts of these enzymes in such sources and also as a means to convert inexpensive,
renewable material like agricultural and slaughter waste into value added products.

Examples of the enzymes isolated from plants include several proteases such as papain, ficin
and bromelain, and peroxidase. As mentioned above, rennet has been among the most
industrially significant enzymes obtained from animal tissue. The other enzymes obtained from
animal sources e.g., proteases like trypsin, chymotrypsin and urokinase, lactate dehydrogenase,
lysozyme, etc., have diverse applications in industry, analysis, and therapy.

In recent years, protein production in transgenic animals and plants has attracted attention.
Focus on transgenic animals (e.g, sheep, cattle) has been for the production of therapeutic
proteins. The expression of the foreign gene is targeted to the mammary gland so that the protein
is secreted directly into the milk.

Although both pharmaceutical and industrial proteins have been expressed in transgenic plants,
they are suggested to be ideal bioreactors for production of the latter category of proteins.
Production of bulk enzymes like α-amylase, xylanase, phytase, etc. combines the advantages of
low production costs of plant biomass with the minimal purification requirements for such
products.

The Dutch company, Gist Brocades has taken the lead in industrial scale plant-based production
system for phytase, an enzyme used as a feed additive in livestock farming for the purpose of
breaking down the antinutritional factor, phytin into myoinositol and phosphate. This production is
done with an idea of not having to extract the enzyme from the plant but rather to supplement the
feed directly and thereby avoid the need to add the enzyme exogenously.

Commercial sources of enzymes are obtained from three primary sources, i.e., animal tissue,
plants and microbes. These naturally occurring enzymes are quite often not readily available in
sufficient quantities for food applications or industrial use. However, by isolating microbial strains
that produce the desired enzyme and optimizing the conditions for growth, commercial quantities
can be obtained. This technique, well known for more than 3,000 years, is called fermentation.

Today, this fermentation process is carried out in a container vessel. Once fermentation is
completed, the microorganisms are destroyed; the enzymes are isolated, and further processed
for commercial use. Enzyme manufacturers produce enzymes in accordance with all applicable
governmental regulations,

Technical Advantages
Microbial enzymes often have properties that make them more suitable for commercial
exploitation. In comparison with enzymes from animal and plant sources, the stability of microbial
enzymes is usually high. For example, the high temperature stability of enzymes from thermophilic

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microorganisms is often useful when the process must operate at high temperatures (e.g. during
starch processing).

Microorganisms are also very amenable to genetic modification to produce novel or altered
enzymes, using relatively simple methods such as plasmid insertion. The genetic manipulation of
animals and plants is technically much more difficult, is more expensive and is still the subject of
significant ethical concern, especially in the U.K.

Enzymes May Be Intracellular or Extracellular

Although many enzymes are retained within the cell, and may be located in specific subcellular
compartments, others are released into the surrounding environment. The majority of enzymes in
industrial use are extracellular proteins from either fungal sources (e.g. Aspergillus species) or
bacterial sources (e.g. Bacillus species). Examples of these include α-amylase, cellulase,
dextranase, proteases and amyloglucosidase.
Many other enzymes for non-industrial use are intracellular and are produced in much
smaller amounts by the cell. Examples of these include:
• Asparaginase,
• Catalase,
• Cholesterol oxidase,
• Glucose oxidase and,
• Glucose-6-phosphate dehydrogenase.

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Production of Enzymes
Enzymes are now produced for a variety of applications, going from bulk high tonnage processes
in which the enzymes are considered as commodities to small-scale applications for refined uses
and research where enzymes are considered specialties.
Level of production and type of application define the kind of process for its production. Specialty
enzymes to be used in medicine and health-care products are usually required in high levels of
purity and in rather small quantities, while enzymes used in the bulk production of food, feed,
fabrics and fuel are usually produced as rather crude preparations in high tonnage. The increasing
use of immobilised enzymes for large-scale processes has increased the demand for purer
enzyme preparations. The production process will depend on the source and localization of the
enzyme.

Enzymes from plant and animal origin will simply be extracted from the corresponding tissues or
recovered from the corresponding fluids; microbial enzymes will be produced by fermentation and
recovered either from the spent fermentation medium (extracellular enzymes) or from the cell
paste after extraction by cell rupture or permeabilisation.

A generalised scheme for enzyme production is presented in Fig. 2.1.

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The production process can be divided into four stages:
• Enzyme Synthesis: it represents the propagation stage of the producing cells.
• Enzyme Recovery: it represents the extraction of the enzyme from the producing
cell system and involves solid–liquid separations, cell extraction and/or
concentration.
• Enzyme Purification: it represents a series of operations after enzyme recovery
aiming to remove unwanted contaminants (mainly accompanying proteins).
• Enzyme Product Formulation: it consists in different operations aimed to give the
enzyme product its final presentation; it includes final polishing operations,
stabilization and standardization.

DISCOVERING ENZYMES
Nature provides a vast amount of microbial enzyme resources. Our ability to tap into such
immense biodiversity depends on the tools available to expand the search for new enzymes by:
(i) Metagenome screening;
(ii) Genome mining in more than 2,000 sequenced microbial genomes; and
(iii) Exploring the diversity of extremophiles.

Metagenomic Screening
Although numerous microbes inhabit the biosphere, less than 1% can be cultivated through
standard laboratory techniques. Metagenomics has appeared as an alternative strategy
conventional microbe screening by preparing a genomic library from environmental DNA and
systematically screening such a library for the open reading frames potentially encoding putative
novel enzymes.

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Metagenomic screening is mostly based on either function or sequence approaches. Function
based screening is a straightforward way to isolate genes that show the desired function by direct
phenotypical detection, heterologous complementation, and induced gene expression. On the
other hand, sequence-based screening is performed using either the polymerase chain reaction
(PCR) or hybridization procedures. Usually, the common procedure is to use a set of degenerated
primers that have been designed based on consensus amino acid sequences.

Studies from different habitats such as volcanic vents, arctic tundra, cow rumen, marine
environments, and termite guts have yielded microbial enzymes with potential for biocatalytic
applications, such as lipase, oxidoreductase, amidase, amylase, nitrilase, beta-glucosidase,
decarboxylase, and epoxide hydrolase.

Function-based screening of metagenomic libraries is somehow problematic mainly due to

insufficient, biased expression of foreign genes in Escherichia coli as this bacterium is usually
used as surogate host. To overcome such hurdles, alternative bacterial host and expression
systems are currently being examined including Streptomyces lividans, Pseudomonas putida and
Rhizobium leguminosarum, among others.

Hit rate (probability of identifying a certain gene) also depends on other factors such as size of
the target gene and the assay method. Enzyme activities are usually assayed on agar plates
supplemented with different substrates Sensitivity of agar plate-based screening can be improved
by using cell lysates, screening for genes giving resistance to toxic compounds, or linking the
target activity to the expression of a reporter gene such as green fluorescent protein (GFP) or β-
galactosidase. Also, development of flow cytometry-based screens as SIGEX are leading the way
as they enable more rapid screening of large metagenomic libraries [39]. Genes obtained through
sequence-based screening are limited to those having homology to the sequences used as
probes.

Microbial Genomes
Recent success of genome sequencing programs has resulted in an explosion of information
available from sequence databases, thus creating an opportunity to explore the possibility of
finding new natural products (including enzymes) by database mining [40]. The next generation
sequencing platforms, (454 from Roche, Solexa from Illumina or SoLiD from ABI), hold promise
to reduce time and cost of genome sequencing. Using these platforms, as well as cutting edge
approaches such as resequencing, helps to complete multiple whole genomes in less than two
weeks. So far, more than 2,000 genome sequences and draft assemblies are available in the
database.

Two approaches are being followed to discover new enzymes. On one hand, genome hunting is
based on searching for open reading frames in the genome of a certain microorganism.
Sequences that are annotated as putative enzymes are subjected to subsequent cloning, over
expression and activity screening. Another approach, called data mining, is based on homology
alignment among all sequences deposited in databases. Using different bioinformatics tools (e.g.,
BLAST), a search for conserved regions between sequences yields homologous protein
sequences that are identified as possible candidates for further characterization.

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Gene Expression and Mutagenesis of Recombinant
Enzymes
Improved Product Safety
In the course of screening for new enzyme producing microorganisms, it sometimes turns out that
growing of technically interesting microorganisms under industrial conditions will not be possible.
Either the microorganism is pathogenic or toxic or not safe to handle. Enzyme purification could
be prohibitively expensive, e. g. because the enzyme was cell associated or contaminated with
undesirable compounds. Using production strains without long-term experience will anyhow be
accompanied by a higher risk of harmful by-products in the final enzyme isolate. The cloning of
enzymes following heterologous expression makes it possible to confine oneself to a small
number of production strains which already have been used for enzyme production for decades
and even might be considered as GRAS.

Chemical Modification of Enzymes

Chemical modification of enzymes was in fact the first method available to alter enzyme properties
in the sixties. Since the mid-eighties, interests on chemical modification have been rekindled. One
major goal for chemical modification is to increase enzyme stability and activity, particularly in
non-aqueous applications for biocatalysis in organic synthesis.

Examples: Various approaches were reported on covalent modification of enzymes: incorporation

of vanadium into phytase for example, which has the in vivo role of catalysing phosphate ester
hydrolysis is sufficient to convert phytase to a vanadium-dependent peroxidase that could
catalyse enantio-selective sulfoxidation.

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According to very recent reports the production of enzymes containing synthetic amino acids in
vivo (!) might also be technically feasible in the near future, thereby resulting in proteins with
“properties that natural proteins do not possess”. These approaches will even more extend the
variability of enzymes available for industrial exploitation.

Recent Developments in Enzyme Manufacturing

The application of genetic engineering techniques in enzyme manufacturing is dramatically
sparking the exploitation of new enzymes and the development of new enzyme properties.
Enzyme expression is dramatically increased by the use of strong expression or multi-copy
systems. New enzymes not accessible before can be cloned into and produced from an well-
known host organism. Thereby, enzymes from almost any source in nature become accessible,
including exotic sources such as extremozymes, exhibiting unusual properties such as extreme
thermostability.

Combinatorial approaches of rational protein design and directed evolution methods turns out to
efficiently alter the properties of enzymes: enzyme stability, catalytic mechanism, substrate
specificity and range, surface activity, folding mechanisms, cofactor dependency, pH and
temperature optima, kinetic parameters have been successfully modified. Recently, also the
enzyme activities were switched. Protein shuffling and related techniques dramatically increase
the variability of enzymes and might lead to enzymes not present in nature so far.

Apart from manufacturing enzymes from MO also plants are investigated for the production of
enzymes. Furthermore, enzymes could be chemically modified, e. g. by incorporation of cofactors,
chemical glycosylation.

Applying these methods, the variability in enzyme structure is dramatically increased and enzyme
properties are significantly enhanced. This in turn will keep up with the demands of the enzyme
applying industry and open up new areas of application. Thus, these methods are mainly
contributing to technical and economic goals. However, the safety of enzyme manufacturing might
also be improved by restricting to few well-known and safe-to-use production strains which are
used as hosts for genes from various sources.

Recent Developments in Enzyme Manufacturing

The exploitation of new types of enzymes, improvements of enzyme properties and of the
production process7 are overall goals of innovation in the enzyme manufacturing industry.
Although considerable improvements had been made in process engineering, large scale
fermentation and downstream purification of enzymes remained to be a significant bottleneck in
innovation. Until the mid-1980s, screening for interesting enzymes was mainly confined to groups

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of organisms which promised a realistic chance of developing a cost-efficient production process.
Microorganisms which are adapted to extreme environments and therefore, are difficult to be
accessed and cultured, but which are producing enzymes with promising properties, could not be
exploited.

Moreover, strain improvement in general as well as of enzymes properties in particular were

largely restricted to statistical methods such as induced mutagenesis. These methods are both
time-consuming and undirected. Only when genetic engineering techniques was introduced into
routine research and development of enzyme manufacturing companies a promising tool became
available to circumvent these problems. The introduction of genetic engineering is most probably
the most important breakthrough in enzyme manufacturing during the last 30 years. Much
progress has been made since the first enzyme from genetically modified microorganisms (GMM)
was introduced onto the market in 1987.

Table 1: Tools in Protein Engineering

Genetically Engineered Enzymes

The significant progress in genetics and in process technology enables the enzyme industry to
offer products with improved properties and often at reduced costs. Falch claimed that genetic
engineering enables us to select host organisms and cultivation conditions that are safe to the
manufacturing personnel, to the user of the product, and to the environment at large. Often an
enzyme with interesting properties is found in an organism which is insufficiently characterized or
is related to a toxin-producing or to an opportunistic pathogenic organism. Such uncertainty can
now be avoided by transfer of the enzyme gene to a safe host using protein engineering tools
(Table 1).

Genetic engineering opens new avenues to enzymes with improved stability, activity or specificity
and productivity. Due to this fact, enzymes naturally occurring in other organisms may now be
produced in largescale fermentation processes. Enzymes of animal or plant origin may be
produced independently of the supply of animal and plant tissue.

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Chymosin, the active protease in calf rennin, is already being produced by enzymes found in
minute concentrations in exotic microorganisms often difficult to grow may be produced by
selecting host microorganisms which are easy to cultivate on cheap raw materials and producing
a broth from which the enzyme is easy to purify. According to Leisola et al., several enzymes
have already been engineered to function better in industrial processes. These include
proteinases, lipases, cellulases, α-amylases and glucoamylases.

Otten and Quax in their paper outlined that, xylanases have received great attention in the
development of environment-friendly technologies in the paper and pulp industry, where large
scale production of xylanases is facilitated with the progress of genetic engineering. The same
authors emphasized that, recent breakthroughs in genomics have helped to overcome the
problems such as limited enzyme availability, substrate scope, and operational stability. Genes
encoding xylanases have been cloned in homologous and heterologous hosts with the objectives
of overproducing the enzyme and altering its properties to suit commercial.

Aravindan et al., highlighted that protein engineering of lipases was studied based on the
sequence information since the mid of 1980s. Many lipases have been engineered for
thermostability, protease stability and for oxidative stability. A. niger was developed as an
important transformation host to overexpress food enzymes since it has been considered GRAS
(Generally Recognized As Safe) by the US Food and Drug Administration.

Another example is the successful application of laccases in food processing. Several production
strategies can be adopted along with media and process optimisation to achieve production of
high amounts at reduced costs. Concomitantly, over-expression of laccase in suitable host
organisms would provide means to achieve high titers. Use of inducers could also enhance
production capabilities.

Table 2 shows some examples of enzymes produced by genetically modified microorganisms

and their application in food industry.

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Table 2: List of Commercial Enzymes from Genetically Modified
Microorganisms Used in Food Industry.

Genetic Engineering
From the perspective of an enzyme-producing company, genetic engineering serves as a
core technology which is offering the following advantages (MENRAD et al., 1999: 289):
• The exploitation of new types of enzymes and new source organisms, even
enzymes from organisms which are difficult to handle or non-culturable (e. g.
extremophiles).
• Drastic shortening of development times from screening to marketing.
• Significant cost reductions in the development and production process of
enzymes.
• Genetic engineering is a prerequisite for the optimisation of enzyme molecular
properties by protein engineering.
• Improved product safety and fewer production risks, due to the production of
enzymes from a wide variety of different source organisms in a small number of
well-characterised enzyme production organisms.

Table 7 gives an overview how these advantages are achieved by genetic engineering
techniques.
The rapid growth of biocatalysis is also sparked by efforts to engineer the (micro) environments
of enzymes (e. g., non-aqueous environments).

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Increasing Enzyme Expression
Before the era of genetic engineering, an increase in enzyme yield could only be attempted by
traditional strain selection from natural sources or by random mutagenesis induced by mutagenic
agents. Using genetic engineering techniques enzyme yield could be further increased
dramatically. This increase is made technically feasible either by multiplying the enzyme gene or
by constructing an artificial expression system.

Both approaches aim at increasing the transcription/translation of the enzyme gene(s) into
proteins on the cellular level. Multiplying enzymes genes could be done just by amplifying the
number of copies of the enzyme gene in the source organisms thereby not necessarily ending up
in a GMM as defined in by the regulator. Alternatively, the gene could be isolated from the source
organism, cloned onto a plasmid which is then introduced in a production strain. Whereas,
normally only one set of genomic genes (two sets in the case of diploid organisms) is present in
a microorganism, up to 1000 and more copies of plasmids and consequently of plasmid related
enzyme genes could be present in one cell.

The continuous improvement of artificial expression systems is sparked by the fast-growing

knowledge on regulatory DNA sequences. Strong promoters, enhancing sequences and efficient
terminators often derived from different organisms are used in order to enhance the transcription
rate of the enzyme gene. Frequently, ribosome binding sites are also optimised in order to
increase the translation rate. However, this approach could lead to changes in the amino acid
sequence of the enzyme protein.

Applying these techniques enzyme yield could be enormously increased. E. g. heterologous

expression of a xylanase in E. coli led to an accumulation of the enzymes up to 70% of total cell
protein.

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 “Self-Cloning consisting in the removal of nucleic acid sequences from a cell of an organism
which may or may not be followed by reinsertion of all or part of that nucleic acid (or a synthetic
equivalent) with or without prior enzymic or mechanical steps, into cells of the same species or
into cells of phylogenetically closely related species which can exchange genetic material by
natural physiological processes where the resulting microorganism is unlikely to cause disease
to humans, animals or plants.” (Council Directive 98/81/EC of 26 October 1998 amending
Directive 90/219/EEC on the contained use of genetically modified micro-organisms).

An increase in enzyme yield of 3 to 50 was reported in several studies. In case of a yeast β-

glucosidase a factor of 300 was achieved.

New Enzymes
Until the mid-1980 the access to new microbial enzymes was largely depended on to the
availability of new source organisms which could be used in a production process. Of course, the
variety of existing microorganisms is huge and it was estimated that probably much less than 1%
of all microorganisms are cultured until present. However, this approach is not only time
consuming but often turns out not to be feasible in practice because of economic reasons or
technical problems that cannot be hurdled. Consequently, the range of enzymes which are
accessible for large scale processing was considerably narrow.

Again, it was genetic engineering which enabled the manufacturer to tackle this problem. Using
these techniques, the gene for the enzyme of interest can be isolated and introduced into a well-
known production organism. Consequently, this has led to an enormous increase in the availability
of interesting enzymes: every organism which could be cultivated in the lab was accessible at this
point. In combination with polymerase chain reaction techniques (PCR), almost every enzyme in
nature, and recently also those from non-cultivatable organisms, are accessible and thereby
exploitable.

Screening and characterisation of new and potentially interesting enzymes is a big issue within
industry: According to Dalbøge and Lange (1998) between 1993 and 1997 more than 130 fungal
enzyme genes have been cloned at Novozymes alone.

Lipolase, a lipase from Novozymes is a well-known example. This enzyme was originally
produced by Humicola lanuginosa. Though, a large-scale process using this strain as production
organism was not economically feasible. Consequently, the lipase gene was isolated and
expressed in Aspergillus oryzae. Lipolase is presently being manufactured as a bulk enzyme
mainly for use in detergents.

The availability of this technique and the increasing knowledge on microorganisms living in
extreme habitats (extremophiles) also sparked the interest on enzymes from these organisms.
Adopted to such extreme conditions as Arctic sea, glaciers, hot springs or smokers in the deep
sea, these organisms must have enzymes that work under these extreme conditions
(extremozymes). As the properties of these enzymes are considerably different to those marketed
so far, extremozymes are of considerable importance for the industry.

Table 8 shows an overview on enzymes from extremophiles and their industrial importance.

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Extremophiles
Due to their capability to survive under environments of extreme conditions, both physical as
temperature (-2 to 12°C, 60–110°C), pressure or radiation, and geochemical such as salinity (2–
5 NaCl) and pH (<2, >9), extremophiles are a very interesting source of enzymes with extreme
stability under conditions regarded as incompatible with biological materials. Recent studies show
that the diversity of organisms in these extreme environments could be even greater than was
initially thought.
However, because the majority of these microorganisms have not yet been isolated in pure
culture, useful characterization of their enzymes still remains quite difficult.

Thermophilic proteases, lipases as well as cellulases and amylases are being used in many
different industrial applications. Extreme thermophiles (growing at 60–80 °C) are widely
distributed among several bacterial genera such as Clostridium, Thermus, Thermotoga, and
Bacillus, whereas most of hyperthermophiles belong to Archaea such as Pyrococcus,
Thermococcus or Methanopyrus, among others. The Taq DNA polymerase, isolated from the
thermophilic Thermus aquaticus, had 2009 sales of $500 million.

Thermophilic Enzymes
These enzymes are well adjusted to temperature above 50°C. Table 9 lists some thermophilic
enzymes. As perhaps an outstanding example amylase from Pyrococcus furiosus is one of the
most stable enzymes presently known with significant half-life at 130°C. Whether this already
represents the upper limits of thermal stability, remains however a question to be solved.
Physical limits seem to be reached around 250°C. At this temperature, peptide bond hydrolysis
reactions rapidly occur in (denatured) proteins. For comparison, the temperature optima of
“normal” mesophilic enzymes are in the range of 30° to 40°C with denaturation starting around
50° to 60°C. Interestingly, it could be shown that resistance to high temperature is often

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accompanied with resistance to proteases, chaotropic agents, low pH, oxidation and high salt
concentration.

The high stability of enzymes from extreme thermophiles generally resides in the amino acid
sequence and three-dimensional structure. This is shown by stability which is retained upon
purification and when genes for stable enzymes are expressed in mesophiles. A few proteins
might also require posttranslational modification to become fully thermostable. Up to present, this
means during the last 5 years, more than 100 genes from hyperthermophiles have been cloned
and expressed in mesophiles. As a consequence, these enzymes are highly attractive for
biotechnological applications, such as starch processing, leaching of low-grade ores, sugar
conversion, detergents, or as a tool for molecular biology (PCR): as these processes are carried
out at elevated temperatures.

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18
Psychrophilic Bacteria
These group of organisms optimally grow at or below 15°C, having an upper limit of growth of
about 20°C, and a lower limit of growth of 0°C or below. Psychrophilic enzymes have a high
specific activity at low and moderate temperatures, and are inactivated easily by a moderate
increase in temperature. Typically, the specific activity of these cold adopted enzymes is higher
than that of their mesophilic counterparts at temperatures of approximately 0 to 30°C. This
increased activity is accompanied by lower thermal stability.

These properties could be of interest for various applications. Cold-adapted cellulase could be
used in biopolishing and stone-washing of cotton garments. In fabric production, tissues often
have cotton fibre ends protruding from the main fibres which reduce smoothness and alter the
appearance of the garment. Treatment with cellulases could excise protruding ends. The current
treatment, however, is accompanied by a loss of mechanical resistance. A cold-adapted enzyme
would enable the decrease of processing temperature and rapid inactivation as a result of thermal
liability would be possible. The mechanical resistance of the final product would also be improved
as a result of rapid inactivation of the enzyme.

In the beverage industry, pectinases are added in the juice extraction process in order to reduce
viscosity and to clarify the final product. In the meat industry, proteases are applied to tenderise
meat, and in baking processes amylases, proteases and xylanases are used to reduce the dough
fermentation time. The use of psychrophilic enzymes in all these processes could be
advantageous not only because of their high specific activity, thereby reducing the amount of
enzyme needed, but also for their easy inactivation. Psychrophilic enzymes could therefore
become interesting alternatives to mesophilic enzymes in brewing and wine industries, cheese
manufacturing, animal feed and other applications.

Also, enzymes from psychrophiles have become quite interesting for many industrial applications,
partly because of the ongoing efforts to reduce energy consumption. Therefore, enzymes like
proteases, amylases or lipases have great commercial potential for development of detergents to
reduce wear and tear of textile fibers. Polymer-degrading activities (e.g., cellulases, xylanases,)
are quite interesting for the pulp and paper industry, as well as for saccharification of pre-treated
lignocellulosic biomass for the production of second-generation biofuels. These cold-active
enzymes also have potential for many other applications, such as extraction and clarification of
fruit juices, improvement of bakery products, polishing and stone washing of textiles, and
bioremediation of waters contaminated with oils or hydrocarbons.

Enzymes from halophiles have to cope with very high concentrations of salts (e.g., sodium or
potassium chloride). Such proteins have adapted to these environments by acquiring a large
number of negatively-charged amino acid residues of their surfaces to prevent precipitation. Such
a property has been taken advantage of by use of non-aqueous media.

Microorganisms that can survive under extreme pH values could be good sources of
thermoalkaliphilic enzymes, like proteases and lipases, particularly useful for applications as
additives in laundry and dishwashing detergents.

Enzyme Characterisation
Rapid developments in DNA sequencing technology make it likely that all industrially relevant
organisms will be completely sequenced in the near future, providing immediate access to useful
genes provided that the link between sequence and function can be established. Recent work by

19
Stewart and colleagues illustrates that this may not be easy; not all of the many ketone reductases
in Baker’s yeast could be identified even though the yeast genome sequence is available.

A similar important development is the structural genomics initiative, which has four key aims to:
• Organise known protein sequences into families;
• Select family representatives as targets;
• Solve the three-dimensional structure of targets by X-ray crystallography or NMR
spectroscopy;
• Build models for other proteins by homology to solved three-dimensional
structures (www.jcsg.org).

Here, the challenge will be to predict enzyme properties, such as substrate range or
thermostability, from (modeled) structure. Characterisation of enzymes with respect to substrate
range, kinetic parameters and optimal reaction conditions will still be necessary, and may benefit
from broadly applicable high-throughput methods, based on power-compensated isothermal
titration or sol-gel immobilised enzyme arrays.

Improving Enzyme Properties: Protein Engineering

Industry is interested in new enzymes exhibiting properties some of which are not easily
found/accessible in nature in order to meet the demands of the enzyme applying industry and to
open up new areas of application. Another approach towards these goals is to modify the
properties of enzymes which are already in use and for which a production process is already
established.

Before the introduction of genetic engineering, this approach was limited to induced random
mutagenesis, natural recombination, or fusion of protoplasts. These methods, however, are
randomly affecting the whole genome. Consequently, only a small number of genotype variants
created by this method will result in mutations in the gene of interest. Subsequent high throughput
screening is necessary to select mutants with interesting properties. Another disadvantage is that
this method do not provide a tool to specifically change the properties that are supposed to be
changed. Thus, protein engineering is, no doubt, is a more promising approach.

Protein engineering is based on three areas of intellectual endeavour: bioinformatics, including

functional genomics and proteomics, protein chemistry, and genetic engineering. Protein
engineering takes advantage of the fact that all functional properties of enzymes reside in the
structure of the protein. In turn the 3-D structure of proteins is largely determined by the sequence
of amino acid residues it comprises. It is the amino acid sequence that can be easily altered by
the means of genetic engineering techniques. Protein engineering comprises approaches and
methods to alter protein structure, usually by exchanging or deleting amino acids inside or
inserting amino acids into one given amino acid sequence. At present, this is done on a routine
basis by altering the corresponding nucleotide sequence in the enzyme gene.
Basically, three approaches exist for generating enzymes with improved properties:
• First, the enzyme properties can be modulated by an exchange of a single or a few
amino acid residues. This is mainly achieved by oligonucleotide-directed
mutagenesis.
• Second, by exchanging functional domains between related enzymes, which lead
to hybrid enzymes. This could technically be achieved by DNA shuffling.
• Third, the active site of an enzyme can be introduced into a small protein fragment
scaffold of the enzyme that is devoid of its original active site.

20
These modifications could be made on a “rational” basis directed to particular changes with
supposed impacts on the enzyme properties (rational protein design). Alternatively, modifications
are made randomly, however, restricted to the gene of interest and not affecting the whole
genome as described above (directed evolution; molecular evolution of enzymes).

As a prerequisite of rational protein design some hypothesis on the functional effects of well-
defined alterations is needed, usually affecting one or a few amino acids. The latter approach
takes into account the limited knowledge of structure, function relations in enzymes.

Virtually, no information on how enzyme structure relates to function is needed. This strategy is
even more useful as the problems and demand from enzyme application are usually complex and
multifactorial. According to industry, the directed evolution approach might therefore be the more
promising one.

Major goals for improving enzyme properties are:

1. Increase in catalytic activity (under conditions applied in practice),
2. Enzyme stability,
3. Improved substrate range.

Bioinformatics is a rapidly developing discipline that makes use of biological data stored in
computer databases, complemented by computational methods and data analysis, to retrieve
and/or derive new biological information. At the beginning bioinformatics was used in comparative
studies and has now developed further to predict structures and functions out of sequence
information at a high percentage of security.

Proteomics: The scientific study of an organism’s proteins and their role in an organism’s
structure, growth, health, disease (and/or the organism's resistance to disease, etc.). Those roles
are predominantly due to each protein molecule's tertiary structure/conformation.

Whereas academics might be more interested in explaining the structure-function relationship

and to find a causative explanation, industry is more focusing on the effects of alterations without
the need to fully understand the underlying mechanism.

Catalytic activity as described by distinct assay conditions, usually optimal for the enzyme, is
sometimes different compared to conditions applied to enzymes in practice. For example, the
“specific washing performance” of detergent enzymes is not only dependent on catalytic activity
but also on stability against temperature, detergents, bleaching agents, increased pH values,
availability of co-factors, presence of inhibitors.

Stability to pH and temperature are important factors e. g. for detergent enzymes. Starch
conversion into sugars needs low pH and high temperature, and so the demand for thermostable
α-amylase and amyloglucosidase from thermophile organisms is a logical consequence.
Enzymes which are able “to do their job” at lower temperatures are also of interest. For example,
in diary industry, ß-galactosidase is used at low temperature to reduce the amount of lactose.

Subtilisin, a bacterial serine protease often used in detergents, might illustrate the manifold goals
of protein engineering. Up to present, mutations in well over 50% of the 275 amino acids have
been reported in the scientific literature. Although enhanced stability has been the predominant
target, these alterations also resulted in changes in catalytic mechanism, substrate specificity,
surface activity, folding mechanisms and also in new activities.

21
Biocatalyst Engineering
Within a few years, the above developments will make templates available to model the structure
of most new enzymes. This will help protein engineering efforts to make specific changes that
cannot be selected for by molecular evolution, such as surface structures for immobilization. At
the same time, directed evolution can now provide us with enzymes tailored to the production
process.

Enzymes can also be modified by chemical means, introducing metals or modified cofactors,
which results in new catalytic activities]. Artificial enzymes and chemocatalysts designed on the
basis of knowledge gained from enzymatic catalysis are also rapidly ‘evolving’ and may well
complement or replace natural enzymes in the future. On the organism level, sequence and
structure knowledge may be combined with metabolic pathway analysis and engineering to
construct strains that direct their carbon flow towards specific for the commercial production of
chemicals.
A well-known example is the biotechnological production of indigo, a process that was brought
close to commercialisation by Genencor. Recently, the recombinant production strain was further
modified to increase indigo production and eliminate a red by-product.

Strategies to Improve Properties of Microbial Enzymes

The continuously expanding application of enzymes is creating a growing demand for biocatalysts
that exhibit improved or new properties. Although enzymes have favorable turnover numbers,
they do not necessary fulfill all process requirements and need further fine tuning to achieve
industrial scale production. Among those hurdles are substrate/product inhibition, stability, narrow
substrate specificity or enantioselectivity. Genetic modification is very important and recombinant
DNA techniques have increased production by 100-fold. Development of new/improved
biocatalysts is a challenging and complex task (Figure 1).
There are two major ways in which enzymes can be modified to adapt their functions to applied
ends:
(i) Rational redesign of existing biocatalysts and,
(ii) Combinatorial methods which search for the desired functionality in libraries
generated at random.

Figure: Discovery and Development of Biocatalysts.

22
Rational Design
This approach includes site-directed mutagenesis to target amino acid substitutions, thus
requiring knowledge of detailed information about the 3-dimensional structure and chemical
mechanism of the enzymatic reaction, some of which may not be available. However, the
increasing growth of databases containing protein structures and sequences is helping to
overcome this lack of information.

Comparison of the sequence of a new biocatalyst identified in a screening program with the
thousands deposited in the databases can identify related proteins whose functions or/and
structures are already known. Because new enzymes have evolved in nature by relatively minor
modification of active-site structures, the goals of homology-driven experiments include
engineering binding sites to fit different substrates as well as construction of new catalytic
residues to modify functions and mechanisms. A small number of variants are produced which
are then screened. Although in many cases, results are poor compared to natural enzymes, there
have been successes. Computational protein design starts with the coordinates of a protein main
chain and uses a force field to identify sequences and geometries of amino acids that are optimal
for stabilizing the backbone geometry. Because of the amazing number of possible sequences
generated, the combination of predictive force fields and search algorithms is now being applied
to functional protein design.

Directed Evolution
Combinatorial methods such as directed evolution create a large number of variants for screening
for enantioselectivity, catalytic efficiency, catalytic rate, solubility, specificity and enzyme stability,
but do not require extensive knowledge about the enzyme. Directed evolution is a fast and
inexpensive way of finding variants of existing enzymes that work better than naturally occurring
enzymes under specific conditions. Directed evolution includes an entire range of molecular
biological techniques that allow the achievement of genetic diversity mimicking mechanisms of
evolution occurring in nature. It involves random mutagenesis of the protein-encoding gene by
different techniques including the error-prone polymerase chain reaction (PCR), repeated
oligonucleotide mutagenesis, or chemical agents, among others. Error prone PCR accomplishes
introduction of random point mutations in a population of enzymes.

Such molecular breeding techniques (DNA shuffling, Molecular BreedingTM) allow in vitro random
homologous recombination, typically between parent genes with homology higher than 70%. After
cloning and expression, a large collection of enzyme variants (104–106) is typically generated
and is subjected to screening or selection.

23
 Figure: The Process of Directed Evolution.
The methods employed to generate diversity are highlighted in green, the host organisms
for directed evolution are shown in orange, and the screening and selection methods are
shown in blue:
• epPCR, error-prone PCR;
• SeSaM, Sequence Saturation Mutagenesis;
• IvAM, In vivo Assembly of Mutant libraries;
• CSM, Combinatorial Saturation Mutagenesis;
• StEP, Staggered Extension Process;
• RACHITT, RAndom CHimeragenesIs on Transient Templates;
• DOGS, Degenerate Oligonucleotide Gene Shuffling;
• ITCHY, Incremental Truncation for the Creation of HYbrid enzymes;
• SCRATCHY, ITCHY+ DNA Shuffling;
• SHIPREC, Sequence Homology-Independent Protein RECombination;
• USERec, USER friendly DNA Recombination;
• RM-PCR, Random Multi-recombinant PCR;
• SISDC, Sequence-Independent Site-Directed Chimeragenesis;
• CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast;

24
 • IVOE, In vivo Overlap Extension;
• MORPHING, Mutagenic Organized Recombination Process by Homologous IN
vivo Grouping;
• OSCARR, One-pot, Simple methodology for Cassette Randomisation and
Recombination;
• MAP, Mutagenesis Assistant Program;
• PROSAR, Protein Sequence Activity Relationship;
• 3DM, 3-Dimensional Matching;
• ConSurf, Conservation Surfacemapping;
• PELE, Protein Energy Landscape Exploration;
• FoldX, Force field;
• CASTER, Combinatorial Active-site Saturation Test aid software;
• CASTp, Computed Atlas of Surface Topography of proteins;
• HTS, High-Throughput Screening;
• MTP, MicroTiter Plate;
• GFP, Green Fluorescent Protein;
• IVC, In vitro Compartmentalization;
• FACS, Fluorescence Assisted Cell Sorting.

All the approaches mentioned are not mutually exclusive as the fields of rational, semi-rational
and random redesign of enzymes are moving closer. Thus, directed evolution techniques make
use, where possible, of smaller enzyme variant libraries designed by rational or semi-rational
methods to reduce the screening effort but without compromising the likelihood of finding better
variants.

One option is to target the active site residues (about 10–15 amino acids) and those closest to it
(another 20–30 amino acids) as mutations closer to these regions seem to be more beneficial.
Another strategy, called CASTing, is based on a combinatorial active site testing, in which
libraries are generated from groups of two or three residues made from the active site residues.
Hits obtained from these initial libraries are combined and new libraries are generated and
screened in an iterative way. By changing the nucleotide components during PCR, the amino acid
alphabet can be reduced yielding new proteins composed by only 12 amino acids. Screening of
such libraries can lead to remarkable improvements in activity by using saturation mutagenesis
at homologous enzyme positions.

As the fitness of a gene will increase more rapidly in a breeding population with high genetic
variability that is under the influence of selection, DNA Family Shuffling, an improvement in the
breeding technique based on recombination of several homologous sequences, yielded an
improvement in activity of about 30 to 80 times higher than when each gene was shuffled
independently.

Further techniques can create shuffle exons or domains, loop regions, random truncations or
insertions and deletions of codons. Random redesign techniques are being currently used to
generate enzymes with improved properties, such as: activity and stability at different pH values
and temperatures, increased or modified enantioselectivity, altered substrate specificity, stability
in organic solvents, novel substrate specificity and activity and increased biological activity of
protein pharmaceuticals and biological molecules.

25
Directed evolution increased the activity of glyphosate-N-acetyltransferase by 10,000-fold and at
the same time, its thermostability increased 5-fold. Proteins from directed evolution work were
already on the market in 2000. These were GFP of Clontech and Novo Nordisk’s LipoPrime®
lipase.

Enzyme Discovery and Screening

High-throughput screening methods for enzymes have been recently developed to perform a key
role in the search for new enzymes and biocatalysts (Wahler and Reymond 2001). For such
processes the modern trend being set is towards robotic handling and miniaturisation of
instruments as it improves reproducibility and authenticity of the resulting data. Basically there
can be two main approaches to enzyme screening: one can be the forward approach and the
other is the reverse approach.

The forward approach starts with the native enzyme of interest followed by its purification and
characterisation, whereas the reverse approach starts with the gene of interest followed by its
amplification, recombinant expression, and purification.

Forward Enzyme Screening Approach

In the forward enzyme screening approach, environmental samples are assayed for enzyme of
interest, based on their physicochemical properties such as temperature, pH, solvent stability,
and their substrate specificity or substrate enantioselectivity or stereospecificity. For this purpose,
recent advances in enzymological techniques have seen the development of high throughput
analytical instruments like parallel capillary electrophoresis and thermistor arrays to study the
physicochemical properties of particular enzyme of interest. Thermistors are small thermometers
that monitor temperature changes in wells of microtiter plates and provide an advantage over the
traditional infrared (IR) thermography, which monitors temperature from IR radiation.

On the simple principle that enzyme reactions can be assayed by recording changes in their
physicochemical properties, Copeland and Miller attached the fluorescent pH indicator to a solid
support to explore the screening of peptide- based catalysts prepared by combinatorial chemistry
techniques. The indicator was either directly attached to the bead or incorporated into a polymeric
gel.

Klein and Reymond in 2001 performed enantioselective assay for alcohol dehydrogenases using
fluorogenic substrates to measure the rate of oxidation of each enantiomer to ketone in the
presence of bovine serum albumin, which catalyses β-elimination to form the strongly fluorescent
product umbelliferone (Klein and Reymond 2001).

In similar lines Olsen et al. in 2000 reported an elegant imaging experiment that relies on the
fluorogenic phospholipid substrates for phospholipase A2 (PLA2) to visualise the activity and
localisation of this enzyme in zebrafish larvae, based on the principle of fluorescence resonance
energy transfer (FRET) (Olsen et al. 2000).

A similar FRET-based assay has been reported to detect enzyme activities in cells using
fluorescence-activated cell sorting (FACS). Another interesting example is in the use of
fluorogenic and chromogenic substrates for enzyme assays. For example, Wahler et al. employed
fluorogenic substrates to identify caspase activity in bacteria. Further progress in enzyme
research in this area was made by Aw et al. who described continuous assessment of enzymatic
activity using nanodroplet microarrays (Aw et al. 2010). By uniformly coating fluorogenic
substrates on slides, they successfully generated surfaces capable of detecting enzymatic activity

26
offering an unprecedented ability for performing solution- phase enzymatic assays in nanoliter
volumes on microarrays, in contrast to microliter volumes typically required in microplate-based
assays, thereby reducing the amounts of reagents required anywhere from a hundred-fold to a
thousandfold. This new approach thus provides a potentially more cost effective and label-free
enzyme screening technique.

A single slide is able to accommodate several thousand assays, facilitating the assessment of
both dose- and time-dependent inhibition parameters in a single run. High-throughput screening
for enzymes is often aimed not only at activity but also at substrate specificity for the application
of enzymes in fine chemical synthesis.

Progress in analysis of substrate specificity and enantioselectivity of enzymes was by Hwang and
Kim (2004), who developed a new high-throughput screening method using staining solution of
CuSO4/MeOH for screening ω-transaminase for multiple substrate specificities and their
enantioselectivities based on UV-visible spectrometry (Hwang and Kim 2004). Staining solution
of CuSO4/MeOH forms a blue complex with the α-amino acid produced during ω transaminase
reaction. This complex can be easily quantified using UV–vis spectrophotometer at 595 nm. In
similar lines, the idea of computer-aided substrate screening of enzymes is new and innovative
combination of computational skills and enzymology which was recently developed by Xu et al.
based on the enzyme structure, and they successfully screened substrates of Candida antarctica
lipase B (CALB).

In this method restricted molecular docking was employed to predict the energetically favorable
poses of substrate–enzyme complexes. Thus, in the future this approach combined with
homology modeling, wherein structure of an enzyme can be modeled from its primary amino acid
sequence based on a template structure, can be applied to screen substrates for novel and newly
characterized enzymes whose X-ray structure is still undetermined.

Some of the other latest innovations in detection and quantification of enzymes include the use
of microfluidics, which involves the manipulation of liquid samples in miniaturised systems, often
on a chip. Moreover in 2010, a research team at the Georgia Institute of Technology has
developed a technique called gelatin zymography to detect and quantify the nature of mature
cathepsin K in femtomoles (Li et al. 2010). Thus, we can use the aforementioned techniques with
other enzymes too with modifications done on the enzyme of interest.

Reverse Enzyme Screening Approach

The recent progress in sequencing of genomes of various organisms and the availability of this
multitude of data conveniently structured in freely accessible databases like the National Center
for Biotechnology Information (www.ncbi.nlm.nih.gov/) and the UniProt (www.uniprot.org/) in the
World Wide Web has made it possible to selectively screen for genes expressing enzyme of
interest followed by their primer base polymerase chain reaction (PCR) amplification and
expression in recombinant host. The PCR-based amplification of gene can follow three strategies.

First is amplification of genes whose sequence is already known by conventional primer design
and by traditional PCR and, second, amplification of homologous enzyme genes from
uncharacterised genomes though the design of degenerate primers followed by PCR amplification
by a process known as gene walking. For example, Hallin and Lindgren (1999) designed two sets
of PCR primers using consensus regions in gene sequences encoding the two forms of nitrite
reductase (Nir), a key enzyme in the denitrification pathway, and successfully amplified nir genes
in nine species within four genera of nitrifying organisms.

27
The third strategy is amplification of genes from distantly related organisms. In this regard, a
recent methodology has been developed to isolate genes coding for functionally equivalent
enzyme from distantly related species by Rose et al. (2003) through a new primer design strategy
for PCR amplification, based on consensus- degenerate hybrid oligonucleotide primers
(CODEHOPs). They have also developed an interactive computational program to design
CODEHOP PCR primers from conserved blocks of amino acids within multiple aligned protein
sequences.

Each CODEHOP consists of a pool of related primers containing all possible 3 to 4 nucleotide
sequences encoding highly conserved amino acids within a 3′ degenerate core, and a longer 5′
non-degenerate clamp region contains the most probable nucleotide predicted for each flanking
codon. The primer design software and the CODEHOP PCR strategy have been utilised for the
identification and characterisation of new gene orthologs and paralogs in different plant, animal,
and bacterial species.

As seen from the above examples, PCR is the most valuable tool to amplify genes of enzyme of
interest. Developments in conventional PCR techniques are self-sustained sequence replication
(3SR), nucleic acid sequence-based amplification (NSBA) (Compton 1991), Strand Displacement
Amplification (SDA) (Walker et al. 1992), and loop-mediated isothermal amplification PCR
(LAMP) (Notomi et al. 2000).

PCR uses heat denaturation of double- stranded DNA to promote next round of DNA synthesis;
NSBA and 3SR eliminate this step of heat denaturation by using a set of transcription and reverse
transcription reactions for gene amplification. SDA eliminates the heat denaturation by employing
a set of restriction enzyme digestions with modified nucleotides as substrate.

Notomi et al. (2000) have developed loop-mediated isothermal amplification PCR (LAMP) which
has emerged as the most cost- effective solution for the technique known as real-time PCR. This
method can amplify a few copies of DNA to 109 in less than an hour under isothermal conditions
and with greater specificity. This method relies on single temperature incubation of 60 to 65°C for
displacement DNA synthesis negating the requirement for expensive thermal cyclers. It is
performed by Bst DNA polymerase from Bacillus stearothermophilus, with high strand
displacement activity and a set of two specially designed inner and two outer primers. The inner
primers are called the forward inner primer (FIP) and the backward inner primer (BIP),
respectively, and each contains two distinct sequences corresponding to the sense and antisense
sequences of the target DNA.

Detection of amplification products of the targeted DNA can be easily done by photometry for
turbidity caused by increasing quantity of magnesium pyrophosphate in solution or by the use of
SYBR Green which specifically intercalates only to double-stranded DNA and emits green light
(λmax = 520 nm) (Mori et al. 2001).

Another approach of gene amplification, worth mentioning here, for enzymes, whose sequence
is not yet available, is that the sequencing of the N- and C-terminal of proteins, which have not
yet been sequenced, can lead to the design of primers and amplification of the gene of the enzyme
by degenerate primers coupled with 3′ or 5′ RACE (rapid amplification of cDNA ends) PCR.

RACE is a technique which can be used to obtain the full-length sequence of an RNA transcript
found within a cell. RACE can provide the sequence of an RNA transcript from a small known
sequence within the transcript all the way to the 5′ end (5′ RACE-PCR) or 3′ end (3′ RACE-PCR)

28
of the RNA (Sambrook et al. 2001). The PCR product can then be used for cloning and expression
of the enzyme gene.

Sakamoto et al. (2011) used this technique and amplified endo-β-1,3-glucanase (GLU1), from the
fruiting body of the fungi, Lentinula edodes. The glu1 gene was isolated by rapid amplification of
cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of
GLU1 protein.

The next step in the reverse enzyme approach, after amplification of the gene of interest for the
enzyme, is cloning the amplified gene in a suitable vector followed by its transformation into
competent hosts. Traditionally the steps in cloning of an enzyme gene involve DNA isolation
followed by restriction digestion of the DNA into desired fragments and then ligating them into the
selected vector system. Though these aforementioned processes turn out to be successful, the
entire process are fairly time consuming. Thus, sufficient groundwork and experimentation have
been carried out to modernize the entire protocol.

The advances in the cloning techniques have led to the development of more specific, highly
reproducible, and guaranteed cloning strategies known as the zero background TA cloning,
TOPO cloning, and the Gateway® cloning. TA cloning is the cloning technique that negates the
use of restriction enzymes. The technique is dependent on the ability of adenine and thymine to
hybridize in the presence of a DNA ligase.

With the property of Taq polymerase terminal transferase activity, deoxyadenosine is added to
the 3′ end of double-stranded PCR-amplified DNA duplex leaving a 3′ overhang. Such inserts can
be directly cloned to linearised vectors with 5′-deoxythymidine overhang with the aid of a DNA
ligase (Chen and Janes 2002).

A modified version of TA cloning is known as the zero background TA (ZeBaTA) cloning. The
improved system takes advantage of the restriction enzyme XcmI whose restriction site is
CCAATACT/TGTATGG to generate a T overhang after digestion and the negative selection
marker gene ccdB to eliminate the self-ligation background after transformation which is quite
common in the case of simple TA cloning (Chen and Janes 2002).

TOPO cloning is also a modified version of the TA cloning strategy and has been invented by
Invitrogen. In this method direct insertion DNA takes place in a matter of few minutes with the aid
of Taq or Pfu polymerase and vaccinia virus DNA topoisomerase I which specifically recognises
DNA sequence 5′-(C/T)CCTT-3′ into linearised plasmid vectors with 3′ T overhangs, without the
requirement for any ligation reaction. Taq polymerase has a nontemplate-dependent terminal
transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products.

The topoisomerase I remains covalently bound to the vector and binds to duplex DNA at specific
sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand of a covalent bond
between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase
I. The phosphotyrosyl bond between the DNA and enzyme can subsequently be attacked by the
5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase.

Figure 7.1 illustrates the TOPO cloning procedure.

29
 Figure: Schematic illustration of the TOPO cloning procedure

Gateway Cloning Technology is a powerful new methodology that can greatly facilitate enzyme
expression, cloning of PCR products, and analysis of gene function by replacing restriction
endonucleases and ligase with site-specific recombination. This technology had been invented
and commercialised by Invitrogen since the late 1990s. The overall reaction of Gateway ® cloning
has been illustrated in Fig. 7.2.

It mainly involves two site-specific recombination reactions known as the LR reaction and the BP
reaction. In the LR reaction a recombination reaction between an Entry Clone and a Destination
Vector occurs with the action of LR Clonase. An Entry Clone, containing a gene flanked by
recombination sites attL1 and attL2, recombines with a Destination Vector (pDESTTM) to yield
an Expression Clone and a by-product plasmid with attB1, attB2 and attP1, attP2 sites,
respectively. The by-product plasmid contains the ccdB gene and hence gives rise to no colonies
when using standard strains of E. coli. In BP reaction the gene of interest in the Expression Clone
(between attB sites) is transferred into a Donor Vector (containing attP sites) to produce a new
Entry Clone (attL sites) catalyzed by the BP Clonase enzyme.

30
Figure: The overall process of Gateway ® cloning. (a) The LR reaction. (b) The BP reaction. The
sites labeled L, R, B, and P are, respectively, the attL, attR, attB, and attP recombination sites for
bacteriophage lambda in E. coli. attL1 reacts only with attR1, and attL2 reacts only with attR2.
attB1 reacts only with attP1 and attB2 with attP2 (www.lifetech.com/gateway)

Another series of dual expression vectors were constructed by Madzak et al. containing an hp4d
promoter which allows for expression of more than one expression cassettes in a single vector
and expresses different proteins efficiently and constantly without multiple influences by nutritional
and environmental factors in medium, such as carbon/nitrogen sources and pH values. This
cassette is integrated into the genome of a genetically modified Yarrowia lipolytica strain by
homologous recombination. This efficient hp4d promoter expression cassette system has been
applied to produce various enzymes, such as β-galactosidase, prorennin, cytokinin oxidase,
lipase, and laccases successfully (Chuang et al. 2010).

Transformation into competent host cell or expression host is the major vital step for a successful
cloning experiment, and enzyme expression level control is still a big challenge in enzyme
evolution (Cambon et al. 2010). If this fails we will not be able to recover or express the enzyme
of interest. The choice of host affects not only the expression of the enzyme but also the way in
which the product can be subsequently purified. Traditional strategies for recombinant enzyme
expression involve transfecting cells with a DNA vector that contains the template and then
culturing the cells so that they transcribe and translate the desired protein.

Typically, the cells are then lysed to extract the expressed protein for subsequent purification.
Both prokaryotic and eukaryotic in vivo protein expression systems which consist of a suitable
vector with an appropriate promoter and other necessary regulatory sequences along with the
gene of interest are widely used. The selection of the system depends on the type of protein, the
requirements for functional activity, and the desired yield. Escherichia coli have been used
extensively till date as expression hosts due to its rapid growth rate, capacity for continuous
fermentation, and relatively low cost. Problems associated with such host system are codon bias,

31
correct protein folding, and disulfide bridge formation. Moreover, formation of inclusion bodies in
such prokaryotic host system is another great obstacle. There are surplus of information available
over the Internet on the modern expression host systems developed in the last decade; some of
the most relevant developments in this area are discussed here. In this regard the most promising
modern expression hosts that were developed are the Rosetta-gami™ B host strains developed
by Novagen, as they successfully express the eukaryotic proteins that contain codons rarely used
in E. coli (Ren 2007). These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, and GGA
codons on a compatible chloramphenicol-resistant plasmid named pRARE.

Elorza et al. expressed human alpha galactosidase A, which contains many non- optimized
codons disulfide bonds in its native structure in Rosetta-gami™ B host strains (Xiao et al. 2007).
Mai et al. have developed the shuttle vector pIKM1 consisting of selection marker which is the
thermostable kanamycin cassette from Streptococcus faecalis plasmid pKD102for for
transformation of Thermoanaerobacterium strain JW/SL-YS485.

Kushnir et al. developed a new inducible protein expression system which was able to
overexpress functional proteins, based on the protozoan host Leishmania tarentolae. This strain
can co-express T7 RNA polymerase and tetracycline repressor and thus can be stably
transformed with the heterologous target gene under control of the T7 promoter/TET operator
assembly, which can initiate transcription upon addition of tetracycline to the culture medium
(Kushnir et al. 2005).

Enzymes from extremophiles are hard to express in normal preexisting host systems. Recently
Cambon et al. developed a new Yarrowia lipolytica JMY1212 expression system which is an
efficient tool for rapid and reliable kinetic analysis of improved enzymes (Cambon et al. 2010).
This strain allows for targeted integration of the expression cassette into the genome and
homogenous transformants.

Most recently a method designated Plasmid Artificial Modification (PAM) was proposed by
Suzuki and Yasui (2011). This PAM method promises higher transformation efficiency as the
presence of PAM plasmid encoding the modification enzymes ensures that recombinant plasmids
harboring the gene of interest are modified such that it is protected from restriction endonuclease
digestion in the target bacterium. Another example of an excellent expression system developed
by the company TAKARA (http://www.takara-bio.com) is the Brevibacillus expression system.
Brevibacillus is a Gram-positive microbe, characterised by its ability to secrete/produce large
amount of proteins. This system is recommended for the production of functional proteins that are
naturally produced intracellularly but become insolubilised and cannot undergo in vitro refolding
when produced by Escherichia coli. The target protein can be easily purified using a histidine-
tagged protein purification column.

The His-Tag can be removed by exposing the purified protein to enterokinase treatment. In similar
lines, Biomedal CASCADETM was developed as a bacterial protein expression system that
provides tightly regulated, high-level expression. The system makes use of linked regulatory
circuits to amplify gene expression levels when induced, maintaining low basal expression levels
under noninducing conditions. Its features are tight control of gene expression, activity at low
temperature, low sensitivity to media formulation, and expression of heteromultimeric proteins.

The trend has moved towards eukaryotic expression system, and in this regard Drosophila
expression system (DES®) by Invitrogen and Leishmania tarentolae expression system known
as LEXSY by Jena Bioscience were developed. Other than the aforesaid techniques, a new
system known as cell-free enzyme expression or in vitro expression has recently emerged as a

32
strong tool in enzyme research as it is simple, inexpensive, and can translate proteins or enzymes
in hours from PCR fragments without the need for Escherichia coli cloning, and thus it is easily
adapted for high-throughput, automation, and/or miniaturisation procedures. Cell-free protein
expression makes use of cellular extracts or purified molecular components to direct protein
synthesis from added DNA template in test tubes (He 2008). Finally, cell-free systems are capable
of synthesising large protein populations in a single reaction, offering an exploitable system for
developing proteomic tools. The cell-free environment is also helpful in the study of protein
translation and synthetic biology applications (He 2008).

PURExpress™ In Vitro Protein Synthesis Kit from New England Biolab and TNT® Quick Coupled
Transcription/Translation Systems are examples of such technology.

Other most promising recently developed and high-fidelity reverse enzyme screening methods
are the processes known as metagenomics, diversity generation by error-prone polymerase
reaction (PCR), and gene shuffling of an existing enzyme’s gene or gene family. Metagenomics
is the new rising science used to explore the unculturable microbes directly from the
environmental samples. It is the outcome of the strength of genomics, bioinformatics, and system
biology and is a multistage process that implies direct cloning of environmental DNA into large
clone libraries to facilitate the analysis of the genes and the sequences within the libraries
(Handelsman 2004).

Employing metagenomics, Tirawongsaroj and co-workers isolated novel thermostable lipolytic

enzymes from a Thailand hot spring metagenomic library (Tirawongsaroj et al. 2008). Chu et al.
also identified two novel esterase genes from a marine metagenomic library through functional
screening of lipolytic clones (Chu et al. 2008), and Wang et al. with the aid of bioinformatics and
the huge freely accessible databases available on the web designed metagenomic gene-specific
primers and isolated 23 truncated lipase gene fragments from 60 metagenomic samples (Wang
et al. 2010). They designed primers based on well-known conserved motifs of lipase genes.

From the aforesaid we can say that bioinformatics or in silico biology related to genes of enzymes
can play a prominent role in screening and functional analysis of novel enzymes. In 2005 Ishikawa
and co-workers discovered a novel restriction endonuclease by genome comparison and
application of a wheat germ-based cell-free translation assay from the hyperthermophilic
archaeon Pyrococcus abyssi. Their methodology involved identification by comparison of
candidate genes from the already sequence and related genomes of the hyperthermophilic
archaea Pyrococcus abyssi and Pyrococcus horikoshii by using certain conserved genes as
genomic landmarks (Ishikawa et al. 2005). This method is much faster and more economical than
the traditional method used to screen for restriction endonucleases wherein individual strains are
cultured and their extracts assayed for the ability to produce specific fragments from small DNA
molecules. The method is also beneficial in the case when we are dealing with extremophiles who
are fastidious and economically hefty to laboratory culture and maintenance.

33
Production of Recombinant Proteins in Microbial Hosts
In 2005, there were 4,200 biotechnology companies worldwide. Total revenue amounted to $63
billion. Recombinant DNA technology has been remarkably advanced by their development of
efficient and scale-up expression systems to produce enzymes from industrially-unknown
microorganisms and other living organisms using industrial organisms such as E. coli, Bacillus
subtilis and other species of Bacillus, Ralstonia eutropha, Pseudomonas fluorescens,
Saccharomyce cerevisiae, Pichia pastoris, Hansenula polymorpha, and species of Aspergillus
and Trichoderma.

Approximately 90% of industrial enzymes are recombinant versions. E. coli has been
extensively used as a recombinant host for many reasons including:
(i) Ease of quickly and precisely modifying the genome;
(ii) Rapid growth to high cell densities;
(iii) Ease of culture in cheap media; and,
(iv) Ease of reduction of protease activity.

E. coli can accumulate heterologous proteins up to 50% of its dry cell weight. However, it has
several drawbacks, such as: inability for post-translational modifications, presence of toxic cell
wall pyrogens or, sometimes, formation of inclusion bodies containing inactive, aggregated and
insoluble heterologous protein. Despite this, high level production and excretion has been
obtained with the following heterologous proteins: alkaline phosphatase (PhoA) at 5.2 g/L into the
periplasm, and levan fructotransferase LFT at 4 g/L into the medium.

The P. fluorescens culture of the Pfenex company is capable of producing 20 g/L of protein. S.
cerevisiae offers certain advantages over bacteria as a cloning host. This yeast can be grown
rapidly in simple media and to a high cell density, can secrete heterologous proteins into the
extracellular broth, and its genetics are more advanced than any other eukaryote.

Glucose oxidase from Aspergillus niger has been produced at 9 g/L by this yeast. Despite these
advantages, S. cerevisiae is sometimes regarded as a less than optimal host for large-scale
production of mammalian proteins because of drawbacks such as hyperglycosylation, presence
of α-1, 3-linked mannose residues that could cause an antigenic response in patients, and
absence of strong and tightly-regulated promoters.

P. pastoris has become one of the most extensively used expression systems. Over 700 proteins
have been produced by this yeast. Production of recombinant proteins reached 22 g/L for
intracellular proteins and 14.8 g/L for secreted proteins. Claims have been made that P. pastoris
can produce up to 30 g/L of recombinant proteins.

Among the advantages of this methylotrophic yeast over S. cerevisiae are:

(i) An efficient and tightly-regulated methanol promoter (AOX1) which yields
alcohol oxidase at 30% of soluble protein;
(ii) Less extensive glycosylation, due to shorter chain lengths of N-linked high-
mannose oligosaccharides, usually up to 20 residues lacking the terminal α-1,3-
mannose linkages;
(iii) Integration of multicopies of foreign DNA into chromosomal DNA yielding
stable transformants;
(iv) Ability to secrete high levels of foreign proteins; and,
(v) High-density growth and straight-forward scale-up.

34
Heterologous gene expression in the methylotrophic yeast H. polymorpha is similar to that of P.
pastoris. The promoter of the methanol oxidase gene is used to express foreign genes. As with
AOX1 in P. pastoris, the MOX gene in H. polymorpha is highly expressed and tightly-regulated,
giving enzyme levels up to 37% of total cell protein.

One major difference is that expression of the MOX gene is significantly derepressed in the
absence of glucose or during glucose limitation and therefore tight regulation of the MOX promoter
is lost in the high glucose conditions usually used for high-biomass fermentations. H. polymorpha
can produce 1.4 g/L of secreted glucoamylase and 13.5 g/L of phytase.

The development of molecular techniques for production of recombinant heterologous proteins in

filamentous fungi has been laborious and contrasts markedly with the success achieved in yeasts.
The ability to introduce or delete genes remains difficult, although some advances in
transformation have been reported, e.g., restriction enzyme-mediated integration and
Agrobacterium tumefaciens-Ti plasmid-mediated transformation. Levels of production of non-
fungal proteins have been low compared to those of homologous proteins.

Different strategies have been developed to overcome these problems, including the construction
of protease-deficient strains, introduction of a large number of gene copies, use of strong fungal
promoters, efficient secretion signals, and gene fusions with a gene which encodes part of or an
entire well secreted protein. Little research has been carried out on glycosylation in molds
although hyper-glycosylation does not seem to occur and low-mannose side chains are formed.

Aspergillus awamori produces 4.6 g/L of glucoamylase from A. niger. Aspergillus oryzae yields
3.3 g/L of Mucor rennin. Acremonium chrysogenum produces 4 g/L of Fusarium alkaline protease.
Trichoderma reesei produces 35 g/L of recombinant proteins. A recent entry into the field is
Chrysosporium lucknowense, which produces high levels of proteins (50 to 80 g/L), and from
which low-viscosity and low-protease mutants have been obtained.

Production of Enzymes: Introduction

The production of enzymes is central to the modern biotechnology industry. The traditional
industrial enzymes continue to have expanding markets, and the recognition of potential to use
biocatalysis in various industrial sectors for new applications generates demand for enzymes with
novel activities and/or improved stability. Man has utilized enzymes throughout the ages either in
the form of vegetables rich in enzymes, or as microorganisms used for a variety of purposes, for
instance in brewing, baking, and in cheese production. However, it was only in the 19th century
that the various biological conversions were ascribed to the action of enzymes. This was initiated
by the isolation of an enzyme complex from malt by Payen and Persoz in 1833, termed ‘diastase’
that converts gelatinized starch into sugars, primarily maltose.

The history of modern enzyme production really began in 1874 when the Danish chemist Christian
Hansen first produced rennet by extracting it from dried calves´ stomachs with saline solution.
Apparently, this was the first enzyme preparation of relatively high purity used for industrial
purposes. During the early part of the last century, in the Far East, an age-old tradition involving
the use of mould fungi called koji in the production of certain foodstuffs and flavour additives
based on soya protein and fermented beverages, formed the basis on which the Japanese
scienThist Takamine developed a fermentation process for the industrial production of fungal
amylase. The process included the culture of Aspergillus oryzae on moist rice or wheat bran, and
the product was called 'Takadiastase' which is still used as a digestive aid. The value of the
industrial enzymes market was estimated to $ 2 billion, and has increased at an average annual

35
rate of 3-5 percent during the past decade. A number of companies are competing in the industrial
enzymes business, Novozymes dominating with 45 percent of sales, followed by Danisco that
holds a 20 percent share of the market.

The industrial or bulk enzymes include proteases, amylases, lipases, etc. which are required in
large volumes, but have an inherently low unit value so that they demand significantly lower
manufacturing costs. On the other end of the scale is the therapeutics sector with products such
as urokinase, which are produced in lower volumes and at inherently greater manufacturing cost.
In between these two lie the diagnostic enzymes.

The technology for producing and using commercially important enzyme products combines the
disciplines of microbiology, genetics, biochemistry and engineering, have developed and matured
through time both singly and in an interactive manner. Demands for new enzymes arise from the
development of new processes or from the unsatisfactory performance of known enzymes in
established processes. The revolution in gene technology over the last two decades has had a
big impact on enzyme industry. Genetic engineering techniques have enabled enzyme
manufacturers to produce sufficient quantities of almost any enzyme no matter what the source,
while protein engineering allows the properties of the enzymes to be adjusted prior to production.

Table 1 lists some of the companies, which are producers of enzymes belonging to the different
categories.

Table Some of the Enzyme Manufacturing Companies

36
 Figure: The Biocatalysis Cycle
• The first step in the development of a biocatalyst process is the identification of a target
reaction.
• On the basis of the reaction constraints, the idel biocatalyst is defined, screened for,
characterized and modified.
• Subsequently, the biocatalyst is produced, applied in the bioconversion process.

37
Microbial enzymes are produced mainly by submerged fermentation under tightly controlled
environmental conditions. However, solid-state fermentation (SSF) has also a good potential
for the production of enzymes, especially those from filamentous organisms that are particularly
suited for surface growth. Some enzymes, particularly those related to lignocellulose degradation,
are currently being produced by SSF, but other hydrolases, namely amylases proteases and
phytase are being produced by SSF as well. SSF compares favorably with submerged
fermentation in terms of energy requirements, volumetric productivity and product recovery; it
represents a good option when production costs should be reduced as is the case of the microbial
enrichment of agricultural residues or the production of bulk inexpensive enzymes.

In most cases, submerged fermentation is the technology of choice for microbial enzyme
production. Submerged fermentation was vigorously developed after World War II for the
industrial production of antibiotics and since then it has represented the most relevant area of
bioprocess engineering. Comprehensive reviews on the subject can be found in several
textbooks. The technology is highly developed and automated and nowadays utilized for the
production of most industrial enzymes.

Submerged Fermentation can be conducted in different modes of operation. The most

traditional is batch fermentation, in which the bioreactor is filled with medium, inoculated and
incubated under controlled conditions to the point in which the product (enzyme) has been
synthesized to (or nearly to) its maximum level; then the cells are harvested for enzyme recovery,
if intracellular, or else discarded to recover the medium containing the enzyme, if extracellular.

Fed-batch fermentation is a variant of the former in which, after certain time of batch cultivation,
the bioreactor is fed with nutrients according to a controlled rate profile and up to a final volume
and the product is then recovered as above. This mode of cultivation is particularly appealing for
the production of enzymes because it allows the control of the metabolic responses of the
producing cells and operation is rather simple. The third mode is continuous culture, in which the
medium is fed continuously to the bioreactor and the fermented broth continuously removed at
the same rate. Steady state will eventually be obtained, so that the theory of the chemostat is
applicable to describe this operation. Continuous culture has been extensively used as a tool to
study enzyme regulation; however, despite its obvious advantages of higher productivity and
better control of operating conditions, the industry has been reluctant to adopt it, mainly because
of the hazards of contamination and mutation that can washout the producing strain.

Some relevant aspects to be considered for developing a fermentation process for

enzyme production are now analyzed.

Enzyme localization with respect to the producing microorganism is a key aspect in enzyme
production. The enzyme can be properly intracellular, periplasmic or excreted into the medium
during fermentation and this will define the downstream operations for its production. In principle,
enzyme excretion is an asset as will be analyzed in the forthcoming sections. Most enzymes are
intracellular but among extracellular enzymes there are many of technological significance;
actually, a significant part of the commodity enzymes are extracellular. There are enzymes that
are intracellular in one organism and extracellular in another; for instance, invertase is mainly
intracellular in Saccharomyces, while a significant portion of it is excreted in Candida and
Streptomyces; β-galactosidase is extracellular in molds while intracellular in yeasts. Intracellular
enzymes can be made extracellular by genetic engineering and protein engineering techniques.

Specific activity (units of enzyme activity per unit mass of microorganism) is a very relevant
parameter for enzyme production by fermentation and much effort has been devoted to increase

38
it by both genetic and environmental manipulations. Conventional mutation and selection, genetic
engineering, site-directed mutagenesis and directed evolution are powerful genetic tools to obtain
high producing microbial strains; in some cases, a substantial portion of the total protein
synthesized by the organism corresponds to the enzyme. High specific activity not only reduces
the cost of fermentation but also the cost of downstream operations. Significant increase in
enzyme specific activity can be obtained by adequate environmental manipulations, mainly
through medium design and optimization of relevant operation parameters like temperature, pH,
agitation and aeration rates.

Enzyme synthesis is subjected to different types of control by the producing strain (see ahead),
so by proper medium design the biological signals that trigger such mechanisms can be put under
our control. Most of the present applications of enzymes relate to hydrolases, which are enzymes
mainly associated to cell catabolism; therefore, their synthesis is controlled by induction and
catabolite repression.

Both controls are exerted at the level of transcription; the former requires the presence of the
inducer to block the repressor protein and allow the transcription of the structural gene coding the
enzyme; the later allows the cell to establish a hierarchy of substrate utilization by repressing the
expression of the genes coding the catabolic enzymes of one substrate while the other
(supposedly the better) is being consumed.

Catabolite repression is related to the substrate consumption rate and not to the substrate
structure. In Gram-negative bacteria, control is associated to the level of cyclic-AMP that acts as
a positive modulator by preventing the blockage by a repressor protein of the structural genes
coding the enzymes.

This is not a universal mechanism: in some enterobacteria cyclic-AMP has proven to have no
significant effect and in Gram-positive bacteria, yeasts and moulds other signal molecules, like
cyclic-GMP and polyphosphorylated nucleotides, are involved. These control mechanisms have
profound influence on enzyme synthesis.

Culture medium should have adequate levels of inducer, which can be the substrate, a
substrate analogue or the product of the enzyme reaction. Substrate analogues are more potent
inducers than the substrate itself because they are not acted upon by the enzymes they induce;
in the case of depolymerases, inducers are usually intermediate or end products of hydrolysis,
since the substrate as such cannot enter the cell to trigger the mechanism. Level and time of
addition of the inducer affects the level of enzyme synthesized and are operation parameters that
should be optimized.

Specific growth rate of the producing strain is also a relevant parameter for enzyme
production by fermentation. Many enzymes are synthesized as growth-associated metabolites
so that cell specific growth rate has a direct impact on enzyme specific rate of synthesis as shown
by the non-structured model of Luedeking and Piret (1959):

39
Where α prevails over β for growth associated metabolites.

Conditions that maximize the specific cell growth rate are often in compromise with those that
maximize the specific rate of enzyme production. In fact, it is usual that pH, temperature and the
level of dissolved oxygen optimal for growth differ from the corresponding optima for enzyme
production.
Compromise values are often used, but impressive increases in enzyme productivity have been
reported by profiling these variables during cell culture. In the case of enzymes whose synthesis
is non-growth associated, a two-stage culture can be envisaged and the variables optimized for
each stage.

Genetic stability and safety of the producing microbial strain are also relevant aspects to be
considered in enzyme production. This is particularly so in the case of recombinant enzyme
proteins because of structural and segregational instability of the cloning vector. Depending on
use of the enzyme, the producing strain must be considered safe for its application. For instance,
enzymes used in the food industry in USA should have the GRAS (Generally Recognized As
Safe) status conferred by the Food and Drug Administration (FDA).

A list of GRAS enzymes and the corresponding producing strains can be found
in://www.cfsan.fda.gov/∼dms/opa-enzy.html. To obtain a GRAS status for an organism is
costly and time-consuming so that sometimes it is a better option to clone the enzyme structural
gene into a GRAS host.

Morphological and rheological properties of the producing strain are also relevant for enzyme
production, especially for the case of mycelial microorganisms. Viscosity increase and non-
Newtonian rheology may reduce oxygen transfer rates and enzyme synthesis is usually related
to one particular growth morphology.

Industrial Enzymes: Production and Application

Industrial enzymes are produced by fermentation using microorganisms such as bacteria or fungi
under carefully controlled conditions. Currently, researchers are locating extremophile organisms
from around the world that produce enzymes of a promising industrial nature. These searches
range from the rain forests to remote arid regions to the bottom of the ocean. In some cases,
these bacteria or fungi can be modified to produce additional interesting enzyme varieties.

In practice, the great majority of microbial enzymes come from a very limited number of genera,
of which Aspergillus species, Trichoderma species, Bacillus species and Kluyveromyces species
predominate. Most of the strains used, have either been employed by the food industry for many
years or have been derived from such strains by mutation and selection. In choosing the
production strain several aspects have to be considered. Ideally the enzyme is secreted from the
cell. This makes the recovery and purification process much simpler compared to production of
intracellular enzymes, which must be purified from thousands of different cell proteins and other

40
components. Secondly, the production host should have a GRAS-status (Generally Regarded As
Safe). This is especially important when the enzyme produced by the organism is used in food
processes. Thirdly, the organism should be able to produce high amount of the desired enzyme
in a reasonable time frame. Most of the industrially used microorganisms have been genetically
modified to overproduce the desired activity and not to produce undesired side activities.

Leisola et al., divided the enzyme production process into following phases:
1. Selection of an enzyme.
2. Selection of a production strain.
3. Construction of an overproducing strain by genetic engineering.
4. Optimization of culture medium and production conditions.
5. Optimization of recovery process.
6. Formulation of a stable enzyme product.

According to the same authors, criteria used in the selection of an industrial enzyme include
specificity, reaction rate, pH and temperature and stability, effect of inhibitors and affinity to
substrates.

Enzyme production techniques can be used to tailor the chemical reactions of the enzymes for
specific types of industrial activity and operating conditions such as temperature, pH, and reaction
kinetics. Industrial production of enzymes requires a clear understanding of the associated
scientific and technological issues. These issues range from identification of the biological
sources for enzyme production to their genetic manipulation for overproduction, strategies for cell
cultivation, isolation, purification and stabilization.

A good example of a layout for large scale enzyme production of frutosyltransferase (FTase) from
Aureobasidium pullulans was presented in the work of Vaňková et al., as shown in Figure 3.

41
42
Figure 3: Process flowsheet of industrial production of frutosyltransferase (FTase).
Streams are denoted by alphanumeric codes where F denotes the cultivation part,
E the part of the cell FTase separation, and I the part of the medium FTase separation.

Since FTase of A. pullulans occurs in the periplasmic space of cells and so the part of the enzyme
is easily released to the cultivation medium. Therefore, the recovery of the enzyme was
considered from both the harvested cells and cultivation medium.

On the basis of application, industrial enzymes could be divided into four major categories, i.e.
detergent enzymes, technical enzymes, food enzymes and feed enzymes. The technical enzymes
segment could further be divided into textile enzymes, leather enzymes, pulp and paper enzymes,
fine chemicals enzymes, fuel ethanol enzymes and others. Enzymes can often replace chemicals
or processes that present safety or environmental issues.
For example, enzymes can:
• Replace acids in the starch processing industry and alkalis or oxidizing agents in
fabric desizing;
Reduce the use of sulfide in tanneries;
Replace pumice stones for “stonewashing” jeans;
Allow for more complete digestion of animal feed leading to less animal waste;
Remove stains from fabrics.
Clothes can be washed at lower temperatures, thus saving energy.

A report presented by the Enzyme Technical Association, outlined that enzymes can be used
instead of chlorine bleach for removing stains on cloth. The use of enzymes also allows the level
of surfactants to be reduced and permits the cleaning of clothes in the absence of phosphates.
Enzymes also contribute to safer working conditions through elimination of chemical treatments
during production processes. For example, in starch, paper and textile processing, less hazardous
chemicals are required when enzymes are used.

The major enzymes used in industrial enzymes market are amylase, lipase, protease, ligase,
phytase, cellulase, xylanase, between others. Food enzymes constitute the major market share
of all four categories of industrial enzymes.

The development of commercial enzymes is a specialized business which is usually undertaken

by a handful of companies which have high skills in:
• Screening for new and improved enzymes.
• Fermentation for enzyme production.
• Large scale enzyme purifications.
• Formulation of enzymes for sale.

The enzyme industry is in an excellent position to contribute to a cleaner environment. First of all,
enzyme technology offers industries and consumers an opportunity to replace processes using
aggressive chemicals with mild, non-toxic enzyme processes. Secondly, the biotechnological
processes used to produce enzymes have a minimal impact on the environment.

43
Large-Scale Fermentation of Enzymes
Use of an aerobic submerged culture in a stirred tank reactor is the typical industrial process
for enzyme production involving a microorganism that produces an industrial enzyme.

Figure 1 shows a flowchart of a typical production process. In the following, relevant aspects of
the fermentation process as displayed in Figure 1 are briefly described: the role of the organism
in enzyme fermentation, the media as the “raw material”, the requirement of sterile environments
for enzyme production and the fermentation process itself.

Organism and Enzyme Production

The basic mechanisms of enzyme synthesis, including transcription, translation, and post-
translational processing, seem to be highly conservative. However, several differences exist
between various classes of organisms, as well as some fundamental differences between
prokaryotic and eukaryotic organisms. The enzymes themselves differ enormously in molecular
mass, number of polypeptide chains, isoelectric point, and degree of glycosylation. In addition, a
variety of enzyme-producing species exist. Although all the differences may influence the
characteristics of synthetic patterns, the basic mechanisms underlying enzyme synthesis are
similar enough to allow a general treatment of the microbiological production process. However,
the differences in production kinetics among various species are large enough to make individual
optimisation programs necessary.

Different organisms may also differ in their suitability for fermentation; such process
characteristics as viscosity or recoverability, legal clearance of the organism, and available
knowledge about the selected organism, must be considered. As cellular enzyme expression is
heavily influenced by regulatory mechanism, enzyme synthesis rates range from no synthesis to
maximum synthesis allowed by the synthetic apparatus, as in a normal control loop. The
complexity of mechanisms ranges from relatively simple and well-understood induction and
repression systems to very complex global regulatory networks. Process development must deal
with the complexity of the enzyme synthesis system either by changing the structural
characteristics, including structural elements of the regulatory system, or by selecting optimal
environmental conditions.

Media
Substrates for industrial applications are usually included in complex media, which must be
supplemented with special compounds, such as a nitrogen source, various nutrient salts, or
certain trace elements. The main sources for microbial processes consist of sugars such as
sucrose or hexoses. They represent the source for both carbon and energy. Typical feedstock
materials are molasses, unrefined sugar, sulfite-liquor from cellulose production plants,
hydrolysates of wood and starch, or fruit juices, such as the grape juice used in wine making. In
many cases starches from various cereals (barley, corn, rice, rye, and wheat) or tubers (potatoes)
are used as inexpensive raw materials. Starch-containing substrates from wheat are mostly used
in raw form, i.e. in the form of grist, and therefore contain a number of additional substances.
Nitrogen, phosphorus and potassium are important nutrients to maintain growth. They are
added in the form of inorganic compounds such as phosphates, ammonium compounds, or
potassium chloride. Soy meal, fish meal, cotton seed, low-quality protein materials such as
casein or its hydrolysates, millet, stillage, and especially corn steep liquor are also used as
low-price nutrients. In addition, these chemically complicated mixtures contain trace elements and
growth promoters.

44
A number of trace elements are provided in sufficient quantity by the tap water that is used for the
preparation of the medium. In general, the raw materials are dissolved or suspended in water and
the resulting medium is heated, filtered and sterilized. The complex composition of the media
used in industry causes considerable problems. For downstream processing (harvest,
concentration, and purification of product) or for analytical assays during the process, additional
pre-treatment of the raw material is needed to avoid unfavourable side effects.

Figure 1: Flow-Chart of a Fermentation Process

Sterilisation
Most bioprocesses for the production of enzymes are based on pure cultures. Because
contamination would prevent proper functioning of the process, bioprocessing must be carried
out under aseptic conditions. Solid substrates, such as grist used for amylase production or
treated soil used in mushroom cultivation, are kept in rooms at elevated temperatures for an
appropriate amount of time. Liquid media are sterilized in situ, e. g. in the reaction vessel or in
separate containers, usually by external heating. In some cases, media are prepared in a
concentrated form and steam is injected directly into the solution, with the resulting condensate
making up the final volume.

45
The temperature for sterilisation is normally above 100°C for an appropriate period. Flow
sterilization through heat exchangers also applies temperatures far above 100°C, but residence
times are shorter than in batch sterilisation. In general, the duration of the heat treatment does
not only depend on the material to be processed but also on the pH of the medium and the initial
number of viable germs or spores. In aerobic processes the culture must be supplied with sterile
air. Air is usually filtered through glass wool filters, sintered materials, or membranes of
appropriate design.

Inoculation
After inoculation with the microorganisms, the process should start immediately and the reaction
should proceed fast. The amount of active cell culture added is therefore critical and depends on
the size of the batch. Scaling-up from the original starter culture to the inoculation broth is done
in several steps in large-scale industrial processes. The starter culture is kept deep frozen (-70 to
-90 °C) for preservation. In fungal inoculates proper wetting of the spores is achieved by adding
small amounts of surfactants to the broth. If inoculation by spare suspensions is not optimal,
mycelial pellets be used for start-up. Bacterial spores must be activated by thermal treatment
before they can be used for inoculation. During the exponential growth phase of the bioprocess
cells can be harvested for following inoculations.

Fermentation
For enzyme production, economy of scale leads to the use of fermenters with a volume of 20 to
200 m3. The higher energy yield from aerobic combustion results in the use of aerobic processes
which require continuous transfer of poorly soluble oxygen into the culture broth.

Process design is the complicated task of choosing the optimal conditions for maximal process
outcome. The number of interdependent factors is high and the available physiological knowledge
is seldom complete. The relationship between synthesis rate and growth rate could be very
complex and depends on the presence of inductors or the absence of repressors. The use of
genetically developed strains may considerably ease process design. The designed process is
usually first tested on a pilot-plant scale and optimised in a number of fermentation runs; it is then
scaled up to production size. The total synthesis rate depends not only on growth rate but also on
biomass concentration.

Two types of cultivation can be distinguished:

In batch culture, the growth rate cannot be controlled by dosed feeding, because all substrates
are added at the beginning. Batch processes are rarely used today.

In fed-batch processes, a low initial biomass concentration should be chosen to maintain the
desired growth rate for a certain period, without exceeding the transport capacity of the
equipment. In addition, fed-batch processes can be designed in a way that the enzyme
concentration at harvesting is higher than in batch or continuous cultures, and the productivity of
fed-batch processes can be increased several-fold compared to batch processes. Fed-batch
processes probably constitute the most frequently used process type.

Continuous cultures are ideally suited for high productivity because the excess of biomass is
continuously withdrawn, and both synthesis rate and biomass concentration will be optimal.
Therefore, in principle, continuous culture is preferred for biotechnological production. However,
enzyme concentrations are lower than those reached in fed-batch cultures. In addition, the use of
continuous cultures is limited by technical reasons such as the higher contamination risk and the
problem of strain degeneration.

46
 ENZYME REACTORS
Types of Reactors, Modes of Operation
More than 80% of the commercial value of enzymes is linked to their applications as process
catalysts. Hydrolytic reactions conducted mainly with the enzyme dissolved in the aqueous
medium has been the traditional way of using enzymes, this technology still representing a major
share of enzyme processes. However, in recent decades the use of enzymes in organic synthesis
has widened its scope of application to unprecedented levels.

Enzyme reactors can operate batch-wise or continuously; fed-batch operation has been
proposed. Batch processes with the enzymes (usually hydrolases) dissolved in an aqueous
reaction medium, despite its wide application have several drawbacks, since enzymes are poorly
stable and hard to recover in such systems, leading to low productivity; besides, such processes
are characterized by a rather low added value so that process optimization is critical for being and
keeping competitive.

Poor stability is usually the limiting factor in any enzyme process so that enzyme stabilization
during reactor operation is a major concern and among the many strategies for enzyme
stabilization enzyme immobilisation is the most relevant.

Immobilised enzymes can be used in batch processes but in this case the enzyme is
recovered to be used in subsequent batches until the accumulated inactivation makes
necessary to replace the spent biocatalyst. As a consequence, specific productivity (mass of
product/mass of biocatalyst · time of operation) is increased and bioreactor design becomes
flexible to suit the particular needs of a given process.

Immobilised enzymes, despite the complexities introduced by the heterogeneous nature of the
catalytic process, are usually much more stable than their soluble counterparts, are easily
recovered from the reaction medium and reused, product contamination with biocatalyst is
avoided and continuous operation becomes feasible.

Continuous operation of enzyme reactors is to a large extent linked to immobilised

enzymes since stability should be high to justify such an operation. The notable exception
is the case of starch liquefaction with bacterial α-amylase where the soluble enzyme is used in
the continuous liquefaction of cornstarch in the manufacture of high-fructose syrup; the enzyme
is continuously dosed to a tubular reactor where hydrolysis and starch gelatinization occur
simultaneously. In this case, the low cost of the enzyme and its little significance in operation cost
allows a certainly quite inefficient use of the biocatalyst. But in most cases continuous processes
are conducted with immobilised enzymes.

Despite the advantages of continuous processes with immobilised enzymes, industry has been
reluctant to adopt it. Reluctance to modify existing conventional processes and the costs
associated to process control, yield losses during immobilisation and mass transfer limitations
during operation have precluded a more extended use of immobilised enzymes as process
biocatalysts.

This situation is changing as new processes emerge based on biocatalyst stability and new
strategies of enzyme immobilisation are developed. Several reactor configurations have been
proposed and used for conducting enzyme-catalyzed processes, as shown in Fig. 5.1. Batch

47
operations with soluble enzymes are conducted mainly in stirred tank reactors (BSTR) provided
with mixing elements and systems for temperature and pH control.

Batch operations with immobilised enzymes are also conducted in stirred tank reactors,
but in this case a device is included to allow biocatalyst retention after product recovery at the
end of each batch. A bottom stainless steel screen is the most used system but other options
exist like basket type reactors and also in-situ and ex-situ biocatalyst retention in ultrafiltration
modules.

Recirculation Batch Reactors (RBR) have been used in occasions; the enzyme is packed in
the form of a narrow bed through which the reaction medium is circulated up to the point when
the desired conversion is attained. This configuration allows a smooth operation when the reaction
involves proton production or consumption and the enzyme is sensitive to pH variation. Since the
conversion per pass is low (and controllable at will), so it is the pH change that is controlled in the
recirculation chamber; in this way the enzyme is never in direct contact with the acid or base used
for pH control. The options for continuous operation with immobilised enzymes are many, as
shown in Fig. 5.1.

48
The most used are the packed-bed column reactors where the immobilised enzyme is fixed within
the reactor while the substrate stream passes through, and the stirred tank reactor where the
enzyme is retained in the reactor by an appropriate screen or recovered by ex-situ filtration or
centrifugation and recycled back into the reactor. An alternative is the expanded or fluidized bed
reactor where the enzyme particles are retained by a hydrodynamic balance between gravity and
drag forces promoted by upflow substrate stream.

BASIC DESIGN OF ENZYME REACTORS

Design Fundamentals
The decision on what type of reactor to use is the first step in design and sometimes can be taken
by a merely qualitative analysis, attending to the different characteristics of each type and its
suitability to the particular process. Continuous processes are usually selected for rather large-
scale production. Continuous packed bed reactors (CPBR), if considered in a plug-flow regime,
will be a better choice than continuous stirred tank reactors (CSTR) in most cases by strictly
kinetic considerations (see next section); however, they have the drawback that reaction
conditions (i.e. pH, temperature) are difficult to control especially when scaling-up to high-level
production.

The opposite holds for CSTR; continuous fluidized bed reactors (CFBR) can be considered
somewhat in between. Properties of the biocatalyst will also be determinants: if sensitive to
compression CPBR can be inadequate because bed compaction and channeling may occur; if
sensitive to shear forces, CSTR can be inadequate because of biocatalyst attrition. The properties
of the feed stream are also determinants: if a limpid substrate solution is used, CPBR is adequate,
but if it has a considerable amount of suspended material bed clogging will hamper operation and
make necessary frequent washing cycles that will significantly reduce productivity.

Selection of the most adequate reactor configuration for the hydrolysis of penicillin G with penicillin
acylase can be used as an illustrative example. The enzyme catalyzes the hydrolysis of penicillin
G to 6 aminopenicillanic acid (non-competitive inhibitor) and phenylacetic acid (competitive
inhibitor). The former is a weak acid poorly dissociated but the latter is rather strong so that
protons are produced as reaction proceeds. The enzyme is quite sensitive to pH and optimal pH
is around 8 (it varies between 7.5 and 8.5 according to the source).

With this information one can make an educated guess of what reactor configuration is the more
adequate. For kinetic considerations CSTR is inadequate since both products of reaction are
inhibitors but it offers a good option for pH control during reaction; CPBR, though sound from a
kinetic perspective, is also inadequate because of the difficulty in controlling pH throughout the
biocatalyst bed. BSTR, despite its reduced productivity as a consequence of a higher proportion
of unproductive time, is a better choice since it is similar to CPBR from a kinetic perspective and
similar to CSTR with respect to pH control.

RBR is even a better choice than CPBR since in this case pH control is exerted in a different
chamber and so there is no direct contact of the enzyme with the base added to control the pH.
In fact this kind of reactor has been proposed as the best option to perform the hydrolysis of
penicillin G. A similar analysis can be done for the production of 7-amino-3-desacetoxy
cephalosporanic acid from cephalosporin G with immobilised penicillin acylase.

49
Enzyme reactor design refers to the determination of the reactor size (operational volume)
required to perform a given production task. For an already available reactor, design refers to the
evaluation of its performance in the fulfilment of the production task. A scheme for the construction
of a model for enzyme reactor design and performance evaluation was already presented in Fig.
3.1.

There are basically four components on that scheme that refers to material balance, enzyme
kinetics, enzyme inactivation and mass transfer limitations. The first two are fundamental
components of this scheme: the material balance, that reflects the magnitude of the process and
the mode of operation, and the kinetic model, that describes the rate of the reaction catalyzed by
the enzyme. A basic design of enzyme reactor operation can be made considering these two
components. However, other components can be taken into consideration, like non ideal flow
patterns within the bioreactor, enzyme inactivation and mass transfer limitations in the case of
heterogeneous catalysis (i.e., immobilised enzymes). Non-ideal flow has been thoroughly
analyzed for chemical reactors and biochemical reactors.

Enzyme inactivation during bioreactor operation is a very important component of enzyme reactor
design since, irrespective of their robustness, enzyme biocatalyst will always be forced to operate
down to the point where a significant fraction of its initial activity has been lost. Mass transfer
limitations in heterogeneous enzyme catalysis has been extensively studied as well and applied
to enzyme reactor design.

Basic Design of Enzyme Reactors under Ideal Conditions.

• Batch Reactor;
• Continuous Stirred Tank Reactor under Complete Mixing;
• Continuous Packed-Bed Reactor under Plug Flow Regime

Basic design and performance evaluation of an enzyme reactor refers to ideal conditions of
operation, this is:
• Ideal flow regime (completely mixed if CSTR or BSTR; plug-flow if CPBR),
• Controlled and constant operational variables (pH and temperature),
• Full retention of enzyme activity throughout its operation and absence of mass
transfer limitation in the case of heterogeneous catalysis.

Departure from ideal behavior can be produced by non-ideal flow patterns;

• (Incomplete mixing in the case of stirred reactors, backmixing in the case of packed bed
reactors),
• Non-isothermal operation because of heat transfer limitations,
• Variable pH because of insufficient mixing and control,
• Enzyme losses during operation because of thermal inactivation or elution from the
support if immobilised,
• Presence of partition effects or diffusional restrictions in the case of heterogeneous
catalysis.

Basic design of enzyme reactor operation, both batch and continuous, will be presented first as a
background to include afterwards the effects of enzyme inactivation and mass transfer limitation.
A generalised kinetic expression for one-substrate reactions was presented that considers all
possible mechanisms for one-substrate reactions (or reactions in which one of the substrates is
clearly limiting as it occurs in most hydrolytic reactions in aqueous medium):

50
Batch Enzyme Reactor
Operation is discontinuous: the reactor is filled with the reaction medium containing the
substrate(s) and operating conditions adjusted; then the enzyme is added and the reaction is left
to proceed until the desired conversion has been obtained. Afterwards the enzyme, if dissolved
in the reaction medium, is inactivated, the reactor is emptied and the reacted medium containing
the product subjected to downstream operations. If the enzyme is immobilised, the biocatalyst is
retained within the reactor by a proper screen, and then washed to be ready for the following
batch.

Continuous Packed-Bed Reactor (CPBR)

Operation is continuous with a constant flow-rate of reaction medium fed to the reactor where the
biocatalyst is packed forming a submerged bed. The reactor can be fed from the bottom or the
top. At laboratory scale it is often preferred to use bottom feeding because it is easier to maintain
the level of liquid above the biocatalyst bed; it also precludes from bed compaction. At large scale
top feeding is frequently used to reduce the energy requirements for pumping. Bed compaction
can be controlled in this case by periodic reversion of flow which also serves to the purpose of
cleaning the biocatalyst. Under no enzyme inactivation, the reactor will reach steady state after
three to five residence times so for the most part of the time the operation can be considered in
steady-state.

ENZYME RECOVERY
Once the enzyme has been synthesized by the producing organism, it must be recovered, which
implies its separation from the cell system (see Fig. 2.1).

Solid–Liquid Separation
First downstream operation is the solid–liquid separation of the cells from the spent fermentation
medium. Separation can be done conventionally by centrifugation or dead-end filtration. Filtration
is more adequate for multicellular organisms of filamentous morphology, like molds and
actynomycetes, but filter-aid should be used because of the compressible nature of the cell cake
formed. Plate and frame and rotary filters are frequently used. Centrifugation is more adequate
for unicellular organisms, like bacteria and yeasts, but usually prior flocculation is required
because of the small size of the individual cells.

Tubular or disk-type centrifuges are the most used. These operations, though widely employed in
bioprocesses, suffer from some drawbacks because microbial cells are small, compressible, and
their density is similar to that of the spent medium. Therefore, alternatives to these operations
have been developed. Among them, cross-flow microfiltration is outstanding: flow is tangential to
the membrane which severely reduces cake formation and membrane clogging, operating costs
are lower and the operation is modular and scalable. Variables in cross-flow microfiltration for
enzyme recovery have been thoroughly analyzed. Improvements of the original design of
microfilters have been developed and proven useful for enzyme recovery.

If the enzyme is excreted during cell growth, recovery will proceed from the liquid phase; if the
enzymes remain associated or within the cell, recovery will proceed from the solid phase (see
Fig. 2.1). Excretion is desirable from a process perspective, so efforts have been made for
genetically engineered cells to export otherwise intracellular enzymes.

51
Concentration of Extracellular Enzyme Crude Broths
Excretion of the enzyme is favorable because of cell membrane selectivity that acts as a powerful
purification step, since only few proteins are excreted. Therefore, the clarified fermentation broth
has a rather high degree of purity that in many cases suffices the requirements for a particular
enzyme use so that further purification steps are not required.

The main concern here is the low protein concentration. This represents the main drawback of
extracellular enzymes, since concentration of the starting material has a profound impact on final
production cost. Even under dense culture conditions, protein concentration rarely exceeds a few
grams per liter so that the enzyme broth has to be concentrated at least by one order of magnitude
prior to further purification steps or formulation.

Many of the operations suitable for enzyme concentration have some potential for protein
fractionation (purification). Only those mainly devoted to concentration will be analyzed here; the
others will be considered.

The most obvious operation for carrying this out is vacuum evaporation: technology is
conventional and has been traditionally applied for the concentration of foodstuffs and
pharmaceuticals. Enzymes are labile molecules so that evaporation must be conducted under
rather high vacuum to prevent denaturation. Other gentler methods of concentration are becoming
more relevant.

Concentration by water freezing has also been considered and high throughput crystallisers have
been designed. The method is not competitive with evaporation in economic terms and protein
occlusion within the ice-water crystals reduces yield so that it is not a viable option except for the
case of very labile enzymes. Water removal by freeze drying is acceptable as a final polishing
step for highly purified enzymes, but it is not economically viable at that step; besides, the
presence of dissolved salts produce eutectic mixtures and foaming is a problem because of
enzyme denaturation.

Concentration by foaming has also been performed taking advantage of the tensoactive
properties of proteins. Foaming can be induced by air or inert gas bubbling through the enzyme
solution and proteins will accumulate at the gas–liquid interface as predicted by Gibbs law:

This technique has also the potential for protein fractionation, but this has not been fully
appreciated, maybe because yields of recovery are low due to enzyme inactivation at the gas
liquid interface.

The most relevant operation for enzyme concentration is ultrafiltration. Ultrafiltration is a

membrane filtration operation, where pressure difference across the membrane is the driving
force. Molecules are separated on the basis of their size in the range from 1,000 to 100,000 Da,
so covering most of the enzymes. Despite its potential of fractionation, ultrafiltration has been
mainly used to concentrate enzymes by removing solvent (water) and smaller size solutes.

52
Ultrafiltration membranes are made of different materials, among which polyacrilonitrile,
polysulphone, polyamide and polyvinylidene fluoride are outstanding in terms of stability,
mechanical strength and chemical compatibility. Ultrafiltration membranes are characterized in
terms of its molecular weight cutoff and can be isotropic or anisotropic, the latter being best in
terms of flowrate and control of operation. Rejection coefficient, this is, the fraction of the solute
(protein) concentration that is retained by the membrane is also used to characterize it.

Membranes are composed by a selective skin of about 0.2µm width supported by a sponge-
like structure of about 50 to 150µm width that confers mechanical strength. Ultrafiltration
equipment come in different formats and sizes. Large size units are usually composed by stacked
flat sheets or hollow fiber bundles of about 1 mm in diameter. Units are quite compact, exhibiting
large area to volume ratios, and are modular so that they can flexibly adapt to process
requirements. Operating pressures range from 100 to 500 KPa and capacities range from 10 to
200L/m2 · h.

Ultrafiltration is a mild operation; therefore, enzyme inactivation is kept to a minimum. Its main
problem is the phenomenon of concentration polarization, which refers to the buildup of protein
near the membrane as a consequence of the flow through it. Increase in protein concentration
near the membrane establishes a gradient that promotes back diffusion of the protein away from
the membrane surface.

At high protein concentration or flux, the membrane can be saturated and transmembrane flow
determined by the protein back diffusion rate, which is slow enough to severely affect the
operation. A secondary membrane composed by retained solutes (proteins) builds up increasing
the hydraulic resistance and reducing membrane selectivity. This latter aspect is of major concern
when using ultrafiltration for protein fractionation.

To minimize concentration polarization, the judicious selection of the membrane and the flow
regime near the membrane are important to reduce the film thickness and to scour deposits; low
flux and low protein concentrations are advisable if practical. The effect of concentration
polarization can be managed by offering a large area for filtration as it occurs in the very compact
modules now available. Selective removal or replacement of low molecular weight solutes in the
enzyme preparation can be done by diafiltration in which ultrafiltration proceeds against a solution
of defined composition.

Ultrafiltration can be considered now as the best option for enzyme concentration in most cases:
it is a mild operation that causes no significant losses and the advances in the field of material
science and process engineering provide now very flexible options to select the most adequate
membrane and equipment for each particular case.

Extraction of Intracellular Enzymes

Enzyme extraction will be determined to a great extent by the type of container cells and the
location of the enzyme within the cell structure. Enzymes from animal cells or tissues are easy to
extract since such cells are devoid of cell wall; therefore, osmotic shock and other rather mild
extraction methods are quite effective.

Enzymes from plant cells or tissues require more rigorous conditions because they are endowed
with a thick cellulose wall; however they have a rigid structure that can be efficiently ruptured by

53
applying shear forces. Microbial cells, especially bacteria and yeast, are particularly difficult to
disrupt because of the resilient nature of their cell envelopes.

However, some are periplasmic and can be extracted rather easily by gentle procedures like
osmotic shock; actually, a differential release can be obtained in such cases aiding substantially
to enzyme purification. On the other extreme, some enzymes are bound or contained within
membranes or other internal cell structures; in those cases, extraction not only requires cell
disruption but also special extractive procedures involving detergents or solvents that can
certainly be harmful for the enzyme.

Notwithstanding, most intracellular enzymes are cytoplasmic and their recovery implies cell
disruption or permeabilisation, which is not an easy task. Methods for intracellular enzyme
recovery can be divided into those that produce cell rupture by mechanical forces and those that
produce cell permeabilisation by membrane damage.

Some new methodologies have arisen, like the extraction of thermostable enzymes by
thermolysis. The most relevant methods for intracellular enzyme extraction are listed in Table.
2.1. Not all of them are amenable for large-scale operation, whether for economical or
technological reasons.

Mechanical disruption methods are the most studied and frequently represent the best option in
terms of process economics. They are well endowed for large-scale operation; validation is rather
simple and conditions of operation can be optimized (see ahead).

Their main drawback is that drastic conditions are required for efficient cell breakage so that
enzyme inactivation during operation is a major concern.

54
Depending on the method, temperature may rise significantly and very efficient heat transfer is
required, which is not easy to attain especially at large scale of operation (Foster 1995). Among
the many options available, the use of homogenizers and bead mills outstand. High-pressure
homogenizers are intensively used in the food industry. Actually, equipment similar to those used
in milk homogenization can be used for cell disruption and are available in all kind of sizes and
design (see www.directindustry.com). They have been extensively used for enzyme and
recombinant protein extraction and perform well especially with bacterial cells being also efficient
with yeast cells, but not applicable with highly filamentous organisms.

Homogenizers work by forcing the cell suspensions under high pressure through a very narrow
passage in the valve, which then impinges on a hard-impact ring.

Disruption of the cell wall occurs by a combination of compression, highly turbulent eddies and
strong shear forces. Most of the studies on mathematical modelling of cell disruption (see ahead)
have been performed in homogenizers, presumably because the number of relevant operational
variables is small: pressure is the key variable and also temperature and the number of passages
through the homogenization valve are relevant.

Bead milling is another important method for cell disruption which is considered one of the most
efficient techniques for cell disruption. It works very well with yeast and is also effective with
bacteria and filamentous organisms. Cells in the form of a paste or slurry are mixed with small
size beads (made of glass, ceramic or metal) that act as abrasives, and subjected to intense
stirring, rupture being produced by a combination of compression and shear forces.

Process can be continuous or discontinuous and temperature control can be exerted by an outer
jacket or by recycling through a heat exchanger. There are a number of equipment that have been
specifically designed for cell disintegration and are available in different sizes and designs (see:
www.glenmills.com).

Mathematical modelling of this type of operation has been hampered by the large number of
variables involved (see ahead). Digestion of cell envelopes is a more selective technique for the
release of intracellular compounds. Contrary to mechanical methods, cell rupture is not required
and in most cases, cells are merely permeabilised. The system of choice should be dictated by
the chemical composition of the cell envelopes. Among cell permeabilisation methods, those
involving alkali or organic solvent treatment are usually too harmful, costly and non-specific, so
that their use is limited.

Cell permeabilisation by selective enzyme digestion is a more promising technology that can be
of interest for selectively recovering labile and expensive intracellular enzymes. Lysozyme has
been routinely used for disrupting bacterial cell wall peptidoglycans, though it has been postulated
that can also act as a an activator of pre-existing autolytic wall enzymes; it has also been used in
combination with β-glucanase for the degradation of yeast cell wall. Helicase from Helix pomatia
has been routinely used for cell wall degradation of yeasts and molds. The chemical composition
of the cell envelopes, shown in Table 2.2, can be used to judiciously select the appropriate
enzyme or enzyme cocktail.

55
Former lytic enzyme preparations were expensive, non-specific and not readily available in large
quantities, but more specific microbial lytic enzymes have been developed that can be produced
more economically on a large scale. β-Glucanases from Cytophaga and Oerskovia have proven
to be quite effective in yeast cell wall degradation.

Selective extraction of recombinant proteins and enzymes have been performed with such
enzymes with considerable success; in many cases, no more than 20% of the intracellular protein
has been released which is a major saving in terms of further purification requirements.

Actually, the potential of this method relies very much on that selectivity, but enzyme cost is still
a major restriction that can be overcome by screening better lytic enzymes by directed evolution
and protein engineering.

Kinetics of extraction with lytic enzymes has been modelled and the process optimized. The
subject of enzymatic lysis of microbial cells has been reviewed.

Autolysis is a very appealing method for enzyme extraction in those organisms prone to autolysis,
like yeasts and bacilli. Under certain stress conditions (i.e., drying, high organic solvent or
electrolyte concentration) lytic enzymes (proteases, nucleases and glucanases) are induced that
partially digest its own cell wall being then the intracellular content easily extracted by osmotic
shock. Autolysis has been used for long in the extraction of cellular proteins; it is the basis for
commercial production of some types of yeast extracts and it has been used efficiently to extract
yeast enzymes.

Invertase, both periplasmic and cytoplasmic, was efficiently extracted from bakers’ yeast cells
subjected to autolysis by drying. At a critical moisture content autolysis was triggered, the
efficiency of protein extraction and enzyme recovery depending on the time that the cells
remained below such critical moisture.

The process was scaled up to pilot level and the crude extract was used as raw material for the
production of a biocatalyst employed in the continuous inversion of sucrose syrup. The process
of autolysis has been optimized, the addition of exogenous papain notably increasing yield and

56
productivity by reinforcing the autolytic effect. It is an interesting alternative for extraction of yeast
enzymes: it is cheap, simple and readily scalable, the main drawback being its low productivity
as a consequence of the prolonged time required for autolysis.

There are many efficient methods for disrupting cells for the release of its intracellular content.
The problem with enzymes is that the method must be sufficiently rough to disrupt or distort the
cell envelopes, but gentle enough to preserve activity. This poses a compromise so that the
process can be optimized. A suitable objective function for optimization is the amount of active
enzyme recovered:

In principle, any extraction method can be optimized accordingly as long as suitable and validated
expressions for protein release and enzyme inactivation rates are available. In practice Eqs. 2.5
and 2.6 can be complex and depend on many operational variables. If both protein release and
enzyme inactivation are assumed to proceed according to first order kinetics:

57
Protein release by bakers’ yeast and Bacillus brevis cell disruption in an industrial homogenizer
has been carried out and modelled. The most relevant variable was pressure drop across the
homogenizer valve; kR depended on the pressure at exponents of 2.9 and 1.8 for the yeast and
the bacteria respectively. In the latter case, the recovery of a labile intracellular enzyme was
optimised by combining protein release and enzyme inactivation kinetics.

Protein release was also modelled in an industrial bead mill, but in this case kR depended on too
many variables:
• Bead size,
• Cell concentration,
• Beads to cell paste volumetric ratio,
• Temperature,
• Agitation speed,
• Agitator design,
• Recycling ratio.

Removal of Cell Debris

After extraction, the enzyme preparation is contaminated with undisrupted cells and cell debris
that have to be removed before purification. Common operations of solid–liquid separation, like
centrifugation and dead-end filtration, can be used but the drawbacks already mentioned with
respect to cell separation are augmented by the very small size of the cell fragments.
Microfiltration is a better option that has been used successfully.

Aqueous two-phase extraction is the most promising operation for cell debris removal that, despite
its potential for protein fractionation, will be revised in this section. It is basically a liquid-liquid
extraction system that considers two aqueous phases comprising either two polymers or a
polymer and a salt.

Phase separation is produced due to the phenomenon of polymer incompatibility that produces
two-phases by mutual exclusion. However, each phase is aqueous in nature, so in principle not
detrimental for enzyme activity as in conventional water-solvent two-phase systems.

Other advantages are the variety of polymers that can be used, its environmental benignity and
the availability and suitability of equipment for liquid–liquid extraction, which is a conventional
operation in the chemical and pharmaceutical industry.

The most common systems are polyethyleneglycol-dextran and polyethyleneglycol-salt. The

former is expensive, the medium is highly viscous and the process is hard to validate when crude
dextran fractions are used so the latter is preferred especially for large-scale operation, as long
as the enzyme withstands the high ionic strength required in the salt phase.

Several other biphasic systems have been tried with the purpose of using less expensive an more
environmentally benign polymers, but the polyethyleneglycol-salt system is still the most used.
Two-phase partitioning can be smoothly integrated to the extraction step and can also be
improved by combining with affinity ligation (affinity partition).

The two-phase system can be represented by a phase diagram, as the one shown in Fig. 2.2 for
the polyethylene glycol (MW 4000)-dextran (T500) system where the equilibrium is represented
by the curved line.

58
Figure 2.2: Phase Diagram Of The Polyethylene Glycol 4000-Dextran T500 System

All mixtures corresponding to points over the equilibrium curve (M) separate into two phases
whose composition is defined by points T and B, corresponding to the top and bottom phases
respectively. The volume ratio of top phase to bottom phase will be given by the relative values
of the traces BM and MT over the corresponding tie-line:

When used for cell debris removal, proteins invariably concentrate in the top phase (PEG in the
case of PEG/dextran and PEG/salt), while fragments do it in the bottom phase.

DOWNSTREAM PROCESSING
Downstream processing is a very important step in biotechnology because costs for collection,
concentration and purification of the final product are substantial. High product concentrations in
the supernatant or inside the cells and efficient purification are therefore important aspects in the
overall economy of enzyme manufacture.

The degree of purity of commercial enzymes ranges from raw enzymes to highly purified forms
and depends on the application. Raw materials for the isolation of enzymes are animal organs,
plant material and microorganisms. Often enzymes may be purified several hundred-fold but the
yield of the enzyme may be very poor, frequently below 10% of the activity of the original material.

In contrast, industrial enzymes will be purified as little as possible, only other enzymes and
material likely to interfere with the process which the enzyme is to catalyse, will be removed.

59
Unnecessary purification will be avoided as each additional stage is costly in terms of equipment,
manpower and loss of enzyme activity. As a result, some commercial enzyme preparations
consist essentially of concentrated fermentation broth, plus additives to stabilise the enzyme's
activity.

However, the content of the required enzyme should be as high as possible (e. g. 10% w/w of the
protein) in order to ease the downstream processing task. This may be achieved by developing
the fermentation conditions or, often more dramatically, by genetic engineering. It may well be
economically viable, e. g. to spend some time cloning extra copies of the required gene together
with a powerful promoter back into the producing organism in order to get “over-producers”.

Downstream processing involves isolation and purification steps and ends up in the formulation
of the enzyme preparation. Enzymes are universally present in living organisms; each cell
synthesizes a large number of different enzymes to maintain its metabolic reactions. The choice
of procedures for enzyme purification depends on their location.

On the one hand, isolation of intracellular enzymes often involves the separation of complex
biological mixtures. On the other hand, extracellular enzymes are generally released into the
medium with only a few other components. Enzymes are very complex proteins and their high
degree of specificity as catalysts is manifest only in their native state. The native conformation is
attained under specific conditions of pH, temperature, and ionic strength. Hence, only mild and
specific methods can be used for enzyme isolation.

Figure 2 shows the sequence of steps involved in the recovery of enzymes.

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Figure 2: Flowchart of Downstream Processing of Enzymes

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Preparation of Biological Starting Materials

Animal Organs
Animal organs must be transported and stored at low temperature to retain enzymatic activity.
Frozen organs can be minced with machines generally used in the meat industry, and the
enzymes can be extracted with a buffer solution. Besides mechanical grinding, enzymatic
digestion can also be employed.

Plant Material
Plant material can be ground with various crushers or grinders, and the desired enzymes can be
extracted with buffer solutions. The cells can also be disrupted by previous treatment with lytic
enzymes.

Microorganisms
Enzymes might accumulate inside the cells or be released into the medium. Most enzymes used
commercially are extracellular enzymes, and the first step in their isolation is the separation of the
cells from the solution. For intracellular enzymes, which are being isolated today in increasing
amounts, the first step involves grinding to rupture the cells. A number of methods for the
disruption of cells are known, corresponding to the different types of cells and the problems
involved in isolating intracellular enzymes. However, only a few of these methods are used on an
industrial scale.

Cell Disruption by Mechanical Methods

High-pressure homogenisation is the most common method of cell disruption. The cell suspension
is pressed through a valve and hits an impact ring. The cells are ruptured by shearing forces and
simultaneous decompression.

The wet grinding of cells in a high-speed bead mill is another effective method of cell disruption.
Glass balls with a diameter of 0.2-1 mm are used to break the cells. The efficiency of this method
depends on the geometry of the stirrer system

Cell Disruption by Non-Mechanical Methods

Cells may frequently be disrupted by chemical, thermal, or enzymatic lysis. The drying of
microorganisms and the preparation of acetone powders are standard procedures in which
the structure of the cell wall is altered to permit subsequent extraction of the cell contents.

Separation of Solid Matter

After cell disruption, the next step is separation of extracellular or intracellular enzymes from
cells or cellular fragments, respectively. This operation is rather difficult because of the small size
of bacterial cells and the slight difference between the density of the cells and that of the
fermentation medium. Continuous filtration is used in industry. Large cells, e. g. yeast cells, can
be removed by decantation. Today, efficient centrifuges have been developed to separate cells
and cellular fragments in a continuous process. Residual plant and organ matter can be separated
with simpler centrifuges or filters.

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Filtration
Pressure Filters: A filter press (plate filter, chamber filter) is used to filtrate small volumes or to
remove precipitates formed during purification. The capacity to retain solid matter is limited, and
the method is rather work-intensive. However, these filters are highly suitable for the fine filtration
of enzyme solutions.

Vacuum Filters: Vacuum filtration is generally the method of choice because biological materials
are easily compressible. A rotary vacuum filter is used in the continuous filtration of large volumes.
The suspension is usually mixed with a filter aid (e. g. kieselguhr) before being applied to the filter.

Cross-flow Filtration: In recent years, a new method of filtration, cross-flow filtration, has been
devised. In conventional methods, the suspension flows perpendicular to the filtering material. In
cross-flow filtration, the input stream flows parallel to the filter area, thus preventing the
accumulation of filter cake and an increased resistance to filtration. Cross-flow filtration can be
conveniently used in recombinant DNA techniques to separate organisms in a closed system.

Centrifugation
The sedimentation rate of a bacterial cell with a diameter of 0.5 mm is less than 1 mm/h. An
economical separation can be achieved only by sedimentation in a centrifugal field. The range of
applications of centrifuges depends on the particle size and the content of the solids.

Decanters (scroll-type centrifuges) work with low centrifugal forces and are used in the
separation of large cells or protein precipitates. Solid matter is discharged continuously by a screw
conveyer moving at a differential rotational speed.

Tubular bowl centrifuges are built for very high centrifugal forces and can be used to sediment
very small particles. However, these centrifuges cannot be operated in a continuous process.
Moreover, solid matter must be removed by hand after the centrifuge has come to a stop. A further
disadvantage is the appearance of aerosols.

Separators (disk stack centrifuges) can be used in the continuous removal of solid matter from
suspensions. Solids are discharged by a hydraulically operated discharge port (intermittent
discharge) or by an arrangement of nozzles (continuous discharge). Bacteria and cellular
fragments can be separated by a combination of high centrifugal forces, up to 15,000 × gravity,
presently attainable, and short sedimentation distances. Disk stack centrifuges that can be
sterilized with steam are used for recombinant DNA techniques in a closed system.

Extraction
An elegant method used to isolate intracellular enzymes is liquid-liquid extraction in an aqueous
two-phase system. This method is based on the incomplete mixing of different polymers, e. g.
dextran and poly(ethylene glycol), or a polymer and a salt in an aqueous solution. The first
extraction step separates cellular fragments. Subsequent purification can be accomplished by
extraction or, if high purity is required, by other methods. The extractability can be improved by
using affinity ligands or modified chromatography gels, e. g., phenyl-Sepharose.

Flocculation and Flotation

The flocculation of bacterial cells to form larger particles can be achieved by the addition of
mineral colloids, salts, or organic polymers. The neutralization of charges on the cell surface and
the formation of bridges between individual cells lead to agglomeration. Agglomerates can then
be removed by filtration or centrifugation.

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Concentration
The enzyme concentration in starting material is often very low. The volume of material to be
processed is generally very large, and substantial amounts of waste material must be removed.
Thus, if economic purification is to be achieved, the volume of starting material must be decreased
by concentration. Only mild concentration procedures which do not inactivate enzymes can be
employed. These procedures include thermal methods, precipitation, and to an increasing extent,
membrane filtration.

Thermal methods
Only brief heat treatment can be used for concentration because enzymes are thermolabile.
Evaporators with rotating components which achieve a thin liquid film (thin-layer evaporator,
centrifugal thin-layer evaporator) or circulation evaporators (long-tube evaporator) can be
employed.

Precipitation
Enzymes are very complex protein molecules possessing both ionisable and hydrophobic groups
which interact with the solvent. Indeed, proteins can be made to agglomerate and, finally,
precipitate by changing their environment. Precipitation is actually a simple procedure for
concentrating enzymes.

Precipitation with Salts: High salt concentrations act on the water molecules surrounding the
protein and change the electrostatic forces responsible for solubility. Ammonium sulfate is
commonly used for precipitation; hence, it is an effective agent for concentrating enzymes.
Enzymes can also be fractionated, to a limited extent, by using different concentrations of
ammonium sulfate. Sodium sulfate is another precipitating agent used.

Precipitation with Organic Solvents: Organic solvents influence the solubility of enzymes by
reducing the dielectric constant of the medium. The solvation effect of water molecules
surrounding the enzyme is changed; the interaction of protein molecules is increased; and
therefore, agglomeration and precipitation occur. Commonly used solvents are ethanol and
acetone.

Precipitation with Polymers: The polymers generally used are polyethylenimines and
polyethylene glycols of different molecular masses. The mechanism of this precipitation is similar
to that of organic solvents and results from a change in the solvation effect of the water molecules
surrounding the enzyme. Most enzymes precipitate at polymer concentrations ranging from 15 to
20 %.

Precipitation at the Isoelectric Point: Proteins are ampholytes and carry both acidic and basic
groups. The solubility of proteins is markedly influenced by pH and is minimal at the isoelectric
point at which the net charge is zero. Because most proteins have isoelectric points in the acidic
range, this process is also called acid precipitation.

Ultrafiltration
A semipermeable membrane permits the separation of solvent molecules from larger enzyme
molecules, because only the smaller molecules can penetrate the membrane when the
osmotic pressure is exceeded. This is the principle of all membrane separation processes
including ultrafiltration. In reverse osmosis, used to separate materials with low molecular
mass, solubility and diffusion phenomena influence the process, whereas ultrafiltration and

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cross-flow filtration are based solely on the sieve effect. In processing enzymes, cross-flow
filtration is used to harvest cells, whereas ultrafiltration is employed for concentrating and
desalting.

Desalting: The desalting of enzyme solutions can be carried out conveniently by diafiltration.
The small salt molecules are driven through a membrane with the water molecules. The permeate
is continuously replaced by fresh water.

Purification
For many industrial applications, partially purified enzyme preparations will suffice; however,
enzymes for analytical purposes and for medical use must be highly purified. Special procedures
employed for enzyme purification are crystallization, electrophoresis, and chromatography.
However, crystallization and electrophoresis are not relevant for large scale purifications.
Chromatography, in contrast, is of fundamental importance to enzyme purification. Molecules are
separated according to their physical properties (size, shape, charge, hydrophobic interactions),
chemical properties (covalent binding), or biological properties (biospecific affinity).

In gel chromatography (also called gel filtration), hydrophilic, cross-linked gels with pores of
finite size are used in columns to separate biomolecules. In gel filtration, molecules are separated
according to size and shape. Molecules larger than the largest pores in the gel beads, i.e. above
the exclusion limit, cannot enter the gel and are eluted first. Smaller molecules, which enter the
gel beads to varying extent depending on their size and shape, are retarded in their passage
through the column and eluted in order of decreasing molecular mass. Gel filtration is used
commercially for both separation and desalting of enzyme solutions.

Ion-exchange chromatography is a separation technique based on the charge of protein

molecules. Enzyme molecules possess positive and negative charges. The net charge is
influenced by pH, and this property is used to separate proteins by chromatography on anion
exchangers (positively charged) or cation exchangers (negatively charged). The ability to process
large volumes and the elution of dilute sample components in concentrated form make ion
exchange very useful.

For hydrophobic chromatography, media derived from the reaction of CNBr-activated

Sepharose with aminoalkanes of varying chain length are suitable. This method is based on the
interaction of hydrophobic areas of protein molecules with hydrophobic groups on the matrix.
Adsorption occurs at high salt concentrations, and fractionation of bound substances is achieved
by eluting with a negative salt gradient. This method is ideally suited for further purification of
enzymes after concentration by precipitation with such salts as ammonium sulfate.

In affinity chromatography the enzyme to be purified is specifically and reversibly adsorbed on

an effector attached to an insoluble support matrix. Suitable effectors are substrate analogues,
enzyme inhibitors, dyes, metal chelates, or antibodies. The insoluble matrix is contained in a
column. The bispecific effector, e. g. an enzyme inhibitor, is attached to the matrix. A mixture of
different enzymes is applied to the column. The immobilised effector specifically binds the
complementary enzyme. Unbound substances are washed out and the enzyme of interest is
recovered by changing the experimental conditions, for example by altering pH or ionic strength.

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Immunoaffinity chromatography occupies an unique place in purification technology. In this
procedure, monoclonal antibodies are used as effectors. Hence, the isolation of a specific
substance from a complex biological mixture in one step is possible. In this procedure, enzymes
can be purified by immobilising antibodies specific for the desired enzyme. A more general
method offers the synthesis of a fusion protein with protein A by “protein engineering.” Protein A
is a Staphylococcus protein with a high affinity for many immunoglobulins, especially of the IgG
class of antibodies. In this way, enzymes that usually do not bind to an antibody can be purified
by immunoaffinity chromatography.

Covalent chromatography differs from other types of chromatography by forming a covalent

bond is between the required protein and the stationary phases.

Recent Developments in Enzyme Purification

An apparent quote from Efraim Racker, an eminent Austrian biochemist, is “Don’t waste clean
thinking on a dirty enzyme.” This quote stresses on enzyme purification as the first criteria
before analysing its properties. The science, art, and practice of protein and enzyme purification
are known for more than a century, yet, in many respects, the field is only now evolving (Ward
and Swiatek 2009). Each enzyme possesses unique properties, which can be exploited for its
purification, and modern techniques aim for enzyme purification with a minimum of labor and cost
to remove all contaminants while retaining as much as possible of the enzyme of interest.

Chromatography is called the protein or enzyme purification workhorse for both native and
recombinant proteins (Smith 2005).

The outline of the general scheme of enzyme purification is illustrated in the Figure below. Today
numerous companies like Qiagen, Applied Biosystems, BioChrom, and Amersham Biosciences
offer improvements to these techniques by introducing new tweaks to improve efficacy of the
procedure. Here we discuss the strategies for purifying native and recombinant enzymes. The
generalised steps for enzyme purification are given in Fig. 7.3. Among column chromatographic
matrices, BioChrom has developed a polymeric material, Hydrocell. The high level of cross-linking
in the polymer beads is intended to give better and faster protein separation compared to
conventional silica-based HPLC columns. GE Healthcare has also recently launched a new
chromatography matrix for the purification of glutathione S-transferase (GST) and His- tagged
proteins which combines small-lbead technology with affinity purification. The relatively small
bead size results in distinctly separated protein elution peaks.

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 Fig. 7.3 Generalized Steps for Enzyme Purification

Another method of enzyme purification is affinity ultrafiltration. In affinity ultrafiltration, affinity

separation is combined with membrane filtration technique. The enzyme to be purified is
complexed with a macroligand composed of a soluble polymer along with target protein-specific
affinity ligands. The complex is trapped by an ultrafiltration membrane, whereas unwanted
proteins pass through the membrane (Galaev 1999). Several enzymes like urokinase have been
purified through this method.

Two-Step Affinity Purification System by Qiagen combines sequential affinity purification steps
and yields ultrapure (>98 %) protein. This system can be useful with enzymes in the presence of
high concentrations of chelating compounds or from eukaryotic cell lysates. In similar lines,
magnetic beads have become a popular choice for protein and enzyme purification due to their
speed, ease of use, and affordability. They possess an iron core surrounded by an inert polymer
material (Fig. 7.4). When subjected to magnetic field, the beads behave like magnets simplifying
purification procedures negating the use of centrifugation. Such beads also come along with
specific molecular tags suitable for affinity purification.

Some companies such as Dynal Biotech, Polysciences, and BioScience Beads have capitalised
on magnetic beads as one of their leading products (Smith 2005). For purification of small quantity
of proteins and enzymes, there is an increasing trend in the use of mass spectroscopy in protein
and enzyme analysis. For this purpose, Vivascience has developed Vivapure MALDI Prep Micro

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spin columns, which contain the C-18 (the number of carbons in the alkyl chains attached to silica
particles in conventional column chromatography membrane), for desalting and concentrating
protein or enzyme digests for mass spectroscopy.

Companies like Millipore are providing solutions for purification of bulk amount of enzymes. They
have developed the Amicon concentrators which can be used to concentrate and desalt from 4
to 15 ml of solution (Smith 2005).

A breakthrough in mass spectrometry was made by scientists at ETH Zurich, along with AB
SCIEX, a global leader in life science analytical technologies; they have developed MS/MSALL
with SWATHTM Acquisition, a groundbreaking that successfully quantifies nearly peptides and
proteins in a sample from a single analysis. These techniques employ data-independent methods
to systematically generate complete, high- specificity fragment ion maps that can be queried for
the presence and quantity of any protein of interest using a targeted data analysis strategy
(Framingham, Mass, April 17, 2012). This technique will thus aid in enzyme sequencing and
identification in a much faster rate in the near future.

Fig. 7.4 Magnetic Dynabeads from Dynal Biotech (Dynal Biotech)

• Recombinant protein expression and purification calls for production of large
amounts of an affinity-tagged protein so that a single purification step using affinity
chromatography is sufficient to achieve the desired level of purity.
(Smith 2005; Striving for purity: advances in protein purification. Nature Methods 2, 71–77 (2005))

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There is a variety of new commercially available fusion systems where enzyme genes are tagged.
This is designed to ease enzyme purification followed by cleavage of enzyme of interest. A variety
of tags are available for such purpose and are listed in Table 7.1. The tagged enzyme is purified
through washing in an affinity chromatography medium and the tagged enzyme is eluted. Modern
columns developed in this regard are His GraviTrap for histidine-tagged proteins. HiTrap columns
are suitable for use with a syringe or peristaltic pump for histidine-, GST-, MBP-, and Strep-
tagged proteins (HisTrap, GSTrap™, MBPTrap™, and StrepTrap™ columns, respectively).

After affinity purification of the recombinant enzyme, cleavage of the protein from the tag system
calls for proteases like factor Xa, thrombin, or enterokinase. The limitation is that they may cleave
the protein of interest also. Thus, new proteases were discovered that have made their way into
commercial expression vector systems.

PreScission protease by GE Healthcare Life Sciences (Amersham Pharmacia) is one such

product based on the rhinovirus protease. New England Biolabs markets a system named
IMPACT where the cleavage is affected by the activity of a self-splicing protein called an intein
(Chong et al. 1998).

For tag-free expression, Wacker Biotech ESETEC® technology provides an innovative and
highly efficient Escherichia coli expression system, which enables secretion of native or
recombinant enzyme products into the fermentation broth. This simplifies primary recovery and
purification processes. The system is based on a two-step export mechanism: first, the target
product is transported across the cytoplasmatic membrane into the periplasmatic space. During
this step the signal peptides are cleaved off, releasing the native product.

The second step is mediated by a unique feature of the proprietary WACKER Secretion Strain.
The correctly folded protein is secreted from the periplasmatic space across the outer membrane
into the culture broth for easy recovery. Although the tagged system allows for single-step enzyme
purification through affinity chromatography, it suffers from the disadvantage that affinity tags may
sometimes interfere with the postpurification use of the protein. The solution to such a problem is
known as Capture, Intermediate Purification, and Polishing (CIPP) technique.

In the capture phase, enzymes are isolated, concentrated, and stabilised. During the intermediate
purification phase, bulk impurities such as other proteins and nucleic acids, endotoxins, and
viruses are removed. In the polishing phase, trace amounts or closely related substances are
removed (GE Healthcare 2009). Another worth- mentioning innovation is in the use of adenosine
triphosphate (ATP)-affinity chromatography. This has been widely used to purify various ATP-
binding enzymes such as kinases and β- and γ-glutamate decarboxylase using affinity purification
by ATP-matrix attachment (Jeansonne et al. 2006; Bendz et al. 2007; Dhillon et al. 2009).

Recent trend in enzyme detection after purification has seen the development of Caliper Life
Sciences’ LabChip90, an automated electrophoresis system and an alternative to the
conventional slab gel electrophoresis system such as SDS-PAGE. It is less expensive than
traditional gel electrophoresis and increases sample throughput by two- to threefold. This system
integrates and automates all of the processing steps right from sampling to separation to detection
and to quantification via the software that comes with the instrument (Smith 2005).

Table 7.1: Fusion Vector Systems with Tags for Recombinant Protein Purification

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FORMULATION OF THE FINAL ENZYME PRODUCT

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Once the enzyme has been purified to the desired extent and concentrated, the manufacturer's
main objective is to retain the activity. Enzymes for industrial use are sold on the basis of overall
activity. Often a freshly supplied enzyme sample will have a higher activity than that stated by the
manufacturer. This is done to ensure that the enzyme preparation has the guaranteed storage
life. The manufacturer will usually recommend storage conditions and quote the expected rate of
loss of activity under those conditions. It is of primary importance to the enzyme producer and
customer that the enzymes retain their activity during storage and use. Some enzymes retain their
activity under operational conditions for weeks or even months. However, most enzymes do not.
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Most industrial enzyme preparations contain a relatively little amount of active enzyme, the rest
being due to inactive protein, stabilisers, preservatives, salts and the diluent which allows
standardisation between production batches of different specific activities.

The key to maintaining enzyme activity is maintenance of conformation, so preventing unfolding

aggregation of the enzyme molecules and changes in the covalent structure.

Three approaches are possible:

• Use of additives
• Controlled use of covalent modification
• Enzyme immobilisation.

In general, proteins are stabilised by increasing their concentration and the ionic strength of their
environment. Neutral salts compete with proteins for water and bind to charged groups dipoles.
This may result in the interactions between an enzyme's hydrophobic areas being strengthened
causing the enzyme molecules to compress and making them more resistant to thermal unfolding
reactions.

Not all salts are equally effective in stabilising hydrophobic interactions, some are much more
effective at their destabilisation by binding to them and disrupting the localised structure of water
(the chaotropic effect).

From this it can be seen why ammonium sulphate and potassium hydrogen phosphate are
powerful enzyme stabilisers whereas sodium thiosulphate and calcium chloride destabilise
enzymes. Many enzymes are specifically stabilised by low concentrations of cations which may
or may not form part of the active site, for example Ca2+ stabilises -amylases and Co2+ stabilises
glucose isomerases. At high concentrations (e. g. 20% NaCl) salt discourages microbial growth
due to its osmotic effect. In addition, ions can offer some protection against oxidation to groups
such as thiols by salting-out the dissolved oxygen from solution.

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Low molecular weight polyols (e. g. glycerol, sorbitol and mannitol) are also useful for stabilising
enzymes, by repressing microbial growth due to the reduction in the water activity, and by the
formation of protective shells which prevent unfolding processes. Glycerol may be used to protect
enzymes against denaturation due to ice-crystal formation at sub-zero temperatures. Some
hydrophilic polymers (e. g. polyvinyl alcohol, polyvinylpyrrolidone and hydroxypropylcelluloses)
stabilise enzymes by a process of compartmentalisation, whereby the enzyme-enzyme and the
enzyme-water interactions are somewhat replaced by less potentially denaturing enzyme polymer
interactions. They may also act by stabilising the hydrophobic effect within the enzymes.

Many specific chemical modifications of amino acid side chains are possible which may (or, more
commonly, may not) result in stabilisation.
A useful example of this is the derivatization of lysine side chains in proteases with N-
carboxyamino acid anhydrides. These derivatiszation result in polyaminoacylated enzymes with
various degrees of substitution and length of amide-linked side chains. This derivatization is
sufficient to disguise the proteinaceous nature of the protease and prevent autolysis.

Enzymes are much more stable in the dry state than in solution. Solid enzyme preparations
sometimes consist of freeze-dried protein. More usually they are bulked out with inert materials
such as starch, lactose, carboxymethylcellulose and other poly-electrolytes which protect the
enzyme during a cheaper spray-drying stage.

Other materials which are added to enzymes before sale may consist of substrates, thiols to
create a reducing environment, antibiotics, benzoic acid esters as preservatives for liquid enzyme
preparations, inhibitors of contaminating enzyme activities and chelating agents. Additives of
these types must, of course, be compatible with the final use of the enzyme’s product.

In order to ensure safe handling, stability, suitable mixing, functionality etc. in the various
applications, most enzyme preparations are formulated in a variety of liquid and granular forms.
Some enzyme preparations are immobilised. Often the precise details of the methods used to
stabilise enzyme preparations are kept secret or revealed to customers as a confidential
information only.

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The nature of enzyme products
The nature of enzyme products is influenced by the:
I. Enzyme itself and its properties “as active compound”,
II. Enzyme source, the fermentation media and conditions and the purification steps
(resulting in the “enzyme concentrate”),
III. Additives used to formulate the final “enzyme preparation”. Thus, one has to
differentiate between “enzyme”, “enzyme concentrate”, and “enzyme preparation”.
Table 1 describes the terminology that AMFEP (AMFEP´s Position on Roster of
Questions, 2002-02-27/PE) is using for enzymes.

Enzyme preparations vary considerably in terms of purity and formulation (additives) among
companies and depending on the particular application. Most enzyme applications do not require
high enzyme concentration of the isolate or the preparation.

A look on the range of main components of industrial enzyme preparations (Table 2) reveals, that
the contents of active enzyme protein in the final enzyme preparation is usually very low, typically
in the order of 1 - 5%. Sugars and inorganic salts are used for stabilising the finished product for
storage and distribution. Salts and sometimes carbohydrates such as starch, maltodextrins and
sugar alcohols are used to dilute extracted enzymes (the enzyme concentrate) to a standard
activity.

Preservatives are generally restricted to use in liquid preparations. Thus, by-products from the
fermentation process (other proteins, carbohydrates, sugars, salts), additives and preservatives
comprise up to 99% of the enzyme preparation. Manufacturing costs are not the only issue, in

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some cases limited purity and concentration are desirable in terms of formulation of the
preparation, improving stability/performance, facilitating homogeneous blending etc. The
increasing use of GMM and new recovery techniques have generally increased purity and amount
of enzyme present in the enzyme concentrate.

Table 3 gives an overview on the typical ranges of enzyme concentrations in enzyme concentrate
and enzyme preparation for different applications. The ranges for technical, food and feed
enzymes largely correspond with the typical ratios published in a study on detergent enzymes (2
- 10%) and the ratio given in Table 2 (1 - 5%)

Only in case that the active enzyme present in a concentrate reaches a purity of 80% or higher
(w/w on basis of dry matter), it becomes possible to determine the purity fairly accurate even if
the specific activity is unknown. This can be done by measuring the protein content in the
concentrate in combination with the relative amount of the enzyme protein in a SDS-PAGE profile.
For less pure products, this method does not result in accurate figures. Based on the limited data
presently available, it can be stated that the percentage of active enzyme in enzyme concentrates
may range between 2% and 70% (w/w on basis of dry matter) (AMFEP´s Position on Roster of
Questions, 2002-02-27/PE).

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The Enzyme Concentrate
Impurities in the enzyme isolate/concentrate result from the fermentation broth and subsequent
purification and consist of proteins, peptides and amino acids, carbohydrates, minerals
and other minor components. The relative amounts of these components vary considerably within
and between categories of enzyme concentrates. Enzyme content and purity is similar for
technical, food and feed enzymes. Enzymes used in personal care products, for therapeutic and
analytical or diagnostic application may generally be of higher purity and concentration. Ash
constituents comprise small amounts of minerals and diluents are additives for granulation, liquid
formulation, stabilisation, preservation etc. Table 4 and Table 5 show the approximate ranges of
impurities as stated by AMFEP.

Enzymes used for food and feed have to comply with purity specifications comprising limits for
heavy metals and contaminating microorganisms and absence of mycotoxins, antibiotics, and the
production strain. Some of the impurities may also fulfil a technical function during enzyme
application. This is particularly true for side activities.

An enzyme concentrate will typically contain other enzymes usually referred to as “side
activities” (not to be confused with “side activities” of a particular enzyme protein). The type
and range of these side activities are largely depending on the enzyme manufacturing conditions
and the production strain. These side activities may be more or less important in any given
application. An example of this is the use of amylases in baking. Fungal alpha-amylase is added
to dough as part of a “flour improver”. The objective is to hydrolyse starch to provide more
fermentable sugars for the yeast. Different enzymes, however, result in different effects on the
rheology of the dough and the final product quality.

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The reason for this is that the enzyme preparation contains a side activity of endo-beta-xylanase
which cleaves the hemicellulose in the flour. This enzyme is now manufactured solely for this
application.

Side activities can partly be inactivated during the enzyme manufacturing process. Nevertheless,
it is quite normal to find a variety of different activities in an industrial enzyme preparation. If a
preparation is marketed as a protease, e. g. glycosidases and lipases might be present as well.

Factors Affecting the Purity of Enzyme Concentrates

The purity of the enzyme concentrate is largely influenced by the:
(i) Fermentation and purification processing applied,
(ii) Production organism used,
(iii) Media used for fermentation.

Production Organisms
The particular production strain is obviously affecting the nature as well as the range of byproducts
present in the enzyme concentrate. As microorganisms are known to produce toxins, the
production organisms may themselves be sources of hazardous materials and have
therefore been a chief focus of attention by the regulatory authorities. Some of these toxins
are quite well known. However, there is always the possibility of introducing new toxins.
Therefore, production strains which are investigated not to produce toxins and which do have
a long history of safe use are preferred.

Media for Enzyme Production

Media used have a bearing on the cost of the enzyme and media components often find their
way into commercial enzyme preparations. Details of components used in industrial scale
fermentation broths for enzyme production are not readily obtained. Not surprisingly, as
manufacturers do not wish to reveal information that may be of technical or commercial value
to their competitors. Also some components of media may be changed from batch to batch
as availability and cost of e. g. carbohydrate feedstock change.

Such changes reveal themselves in often quite profound differences in appearance from batch to
batch of a single enzyme from a single producer. The effects of changing feedstock must be
considered in relation to downstream processing. If such variability is likely to reduce the efficiency
of the standard methodology significantly, it might be economical to use a more expensive defined
medium of easily reproducible composition.

Clearly defined media are usually out of question for large scale use on cost grounds but
may be perfectly acceptable when enzymes are to be produced for high value uses, such as
analysis or medical therapy where very pure preparations are essential. Less-defined complex
media are composed of ingredients selected on the basis of cost and availability as well
as composition. Waste materials and by-products from the food and agricultural industries
are often major ingredients. Thus, molasses, corn steep liquor, distillers solubles, and wheat
bran are important components of fermentation media providing carbohydrate, minerals, nitrogen
and some vitamins.

Extra carbohydrate is usually supplied as starch, sometimes refined but often simply as ground
cereal grains. Soybean meal and ammonium salts are frequently used sources of additional
nitrogen. Most of these materials will vary in quality and composition from batch to batch causing
changes in enzyme productivity.

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Enzyme Preparations
Enzyme preparations contain a varying amount of additives for the purpose of stabilising the
enzyme activity, preservation, granulation, coating or as (de)colouring aids. The choice of
formulation ingredients (additives) is not specific for a given application category, but certain
substances used for e. g. technical enzymes may not comply with food, feed and cosmetics
regulations, specifications etc. Some applications may require special substances and
technologies, e. g. in the case of immobilised enzymes or enteric coating of digestive aid
preparations.

AMFEP (AMFEP’s Position on Roster of Questions, 2002-02-27/PE) only outlined typical

kind of additives and their functionalities (Table 6) as product formulation often comprises
company specific and proprietary information.

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Enzymes going to be introduced to the market should conform to a number of quality procedures
including regulatory requirements. This is provided by the quality assurance within the
company. Enzyme products must be consistent as appropriate to their intended use. This may be
ensured by good manufacturing practice (GMP) and further checked by quality control. Additives
used in the manufacturing of food enzymes must e. g. be of food grade. In the context of EU
chemical legislation, additives have to be listed on EINECS or already notified.

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 IMMOBILISED ENZYMES
Enzyme immobilisation increases the number of enzyme molecules per unit area increasing the
efficiency of the reaction. The advantages of immobilising enzymes include the repetitive use of
a given batch of enzyme, better process control, enhanced stability, enzyme-free products,
increased stability of polar substrates, shifting of thermodynamic equilibria to favour ester
synthesis over hydrolysis, reduction of water dependent side reactions such as hydrolysis,
elimination of microbial contamination and the potential for use directly.

Enzyme Immobilisation
The use of enzymes in industrial applications has been limited by several factors, mainly the high
cost of the enzymes, their instability, and availability in small amounts. Also the enzymes are
soluble in aqueous media and it is difficult and expensive to recover them from reactor effluents
at the end of the catalytic process. This restricts the use of soluble enzymes to batch operations,
followed by disposal of the spent enzyme-containing solvent.

Over the last few decades, intense research in the area of enzyme technology has provided many
approaches that facilitate their practical applications. Among them, the newer technological
developments in the field of immobilised biocatalysts can offer the possibility of a wider and more
economical exploitation of biocatalysts in industry, waste treatment, medicine, and in the
development of bioprocess monitoring devices like the biosensor.

D´Souza stated that, immobilisation means associating the biocatalysts with an insoluble matrix
so that it can be retained in adequate reactor geometry for its economic reuse under stabilised
conditions. Immobilisation helps in the development of continuous processes allowing more
economic organization of the operations, automation, decrease of labour, and
investment/capacity ratio. Immobilised biocatalysts offer several other advantages; notable
among them is the availability of the product in greater purity. The same author highlighted that
the purity of the product is very crucial in food processing and pharmaceutical industry since
contamination could cause serious toxicological, sensory, or immunological problems.

The other major advantages include greater control over enzymatic reaction as well as high
volumetric productivity with lower residence time, which are of great significance in the food
industry, especially in the treatment of perishable commodities as well as in other applications
involving labile substrates, intermediates or products.

A large number of techniques and supports are now available for the immobilisation of enzymes
or cells on a variety of natural and synthetic supports. The choice of the support as well as the
technique depends on the nature of the enzyme, nature of the substrate and its ultimate
application. Commercial success has been achieved when support materials have been chosen
for their flow properties, low cost, nontoxicity, maximum biocatalysts loading while retaining
desirable flow characteristics, operational durability, ease of availability, and ease of
immobilisation.

Biocatalysts can be immobilised either using the isolated enzymes or the whole cells.
Immobilisation of whole cells, due to operational facility, has been shown to be an easier
alternative to immobilisation of isolated enzyme. On the other hand, immobilised cells showed
lower catalytic activity compared with immobilised enzymes. Enzymes are good catalysts in terms
of high catalytic and specific activity with ability to function under mild conditions. However, they
are not always ideal catalysts for practical applications because they are generally unstable and
they inactivate rapidly through several mechanisms.

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Techniques for immobilisation have been broadly classified into four categories, namely:
• Entrapment,
• Covalent binding,
• Cross-linking,
• Adsorption and,
• Combination of one or more of these physical techniques together with chemical
conjugation techniques.

It must be emphasized that in terms of economy of a process, both the activity and the operational
stability of the biocatalysts are important. They determine its productivity, which is activity
integrated over the operational time.

Immobilisation often stabilises the structure of enzymes, thereby, allowing their applications even
under harsh environmental conditions of pH, temperature and organic solvents, and thus enable
their uses at high temperatures in nonaqueous enzymology, and in the fabrication of biosensor
probes. In the future, development of techniques for the immobilisation of multi enzymes along
with cofactor regeneration and retention system can be gainfully exploited in developing
biochemical processes involving complex chemical conversions.

According to Ribeiro et al., the use of free lipase for biodiesel production results in technical
limitations, and it is practically unreliable, due to impossibility of their recovery and reuse, which
in turn increases the production costs of the process, besides promoting the product
contamination with residual enzyme. These difficulties can be overcome by the use of these
enzymes in its immobilised form, allowing the reuse of biocatalyst several times, reducing costs,
and further improving the quality of the product.

Alzohairy and Khan claimed that, recent advances in the design of immobilisation supporting
materials with tailorable pore size and surface functionality has enabled more precise control of
immobilisation of enzymes. New simulations of the surface characteristics of the target enzymes
can be used to aid in the design of appropriate support materials. As the structure and mechanism
of action of enzymes becomes available more controlled immobilisation methods will be
generated. The same authors concluded that, the development of cheaper and disposable array
biosensors, bioreactors and biochips for the simultaneous detection of clinically important
metabolites and rapid screening of diseases has attracted much attention during the recent past.
The use of more and more immobilised enzymes in clinical, biotechnological, pharmacological
and other industrial fields has great promise among future technologies.

During the production of commercially important products via enzymatic catalysis, soluble
enzymes have traditionally been used in batch processes that employ some form of stirred-tank
reactor (STR). In these processes, at the end of the batch run the product must be separated
from any unused substrate, and also from the enzyme catalyst. Removal of the enzyme at this
stage can be achieved by thermal denaturation (only if the product is thermostable) or by
ammonium sulfate precipitation or ultrafiltration. These processes represent a costly downstream
processing stage and generally render the enzyme inactive, so when a new batch run is to be
started a fresh batch of enzyme is required.

Immobilised enzyme systems, in contrast, ‘fix’ the enzyme so that it can be reused many times,
which has a significant impact on production costs. As a very simple example, if an enzyme is
mixed with a solution of warm (but not too hot) agar and this is allowed to set, the enzyme will be
entrapped (for the purposes of this example let us ignore the fact that the enzyme will gradually

88
leak out of this gel). The agar can then be cut up into cubes and these can be placed in a STR,
together with substrate, as shown in Figure 12. Again the reaction would be allowed to proceed
(and it might actually be slower due to diffusional constraints and other effects described later).
At the end of the batch run the catalyst can now be easily separated from the product by passing
the reactor contents through a coarse mesh. Immediately an important downstream processing
step has been carried out and, just as importantly, the active enzyme has been recovered so that
it can be reused for the next batch run. This ease of separation of enzyme from product is a major
advantage of all immobilised systems over their counterparts that use free (i.e. soluble) enzyme.

This physical advantage of ease of reuse of immobilised biocatalysts is one of the main reasons
why such systems are favoured commercially. However, immobilisation may also produce
biochemical changes that lead to enhanced biocatalyst stability, which may be manifested as:
• An increased rate of catalysis
• Prolonged duration of catalysis
• Greater operational stability to extremes of pH, temperature, etc.

The particular advantage(s) conferred by immobilisation will therefore differ from one system to
another. It should be noted that open there may be no biochemical advantage at all, and the
simple physical advantage of ease of separation of the biocatalyst from the product may be
sufficient to favour the commercial development of an immobilised process.

At this point one problem that will immediately spring to mind for most students is that they have
always been taught to fully mix all of the reagents of a reaction, yet the basic principle of
immobilisation is to partition the biocatalyst into a distinct phase, rather than mix it homogeneously
with the substrate. Will this not cause reaction rates to be low? The answer to this question is yes,
and the relationship between the activity of an immobilised system and a non-immobilised system
can be expressed as the effectiveness factor (η), where:

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Thus, an immobilised system with an effectiveness factor of 0.1 would show only 10% of the
activity of a non-immobilised system with the same amount of enzyme and operating under the
same conditions. At first sight this might appear to be a major problem. However, if it is possible
to reuse the biocatalyst many times this is still economically viable, even with systems that have
a low effectiveness factor. In principle, therefore, for economic viability:

Thus, if an immobilised system has an effectiveness factor of 0.1 (i.e. 10%) and we can reuse the
biocatalyst 10 times, we essentially achieve the same overall catalytic activity with both the
immobilised system and the immobilised one. However, if we are able to reuse the biocatalyst
100 times, we in fact obtain 10 times more total activity from the immobilised system than from
the equivalent non-immobilised system, so the immobilised system may be economically
preferable.

Once a biocatalyst has been immobilised it can also be put in a range of continuous-flow reactors,
enabling a continuous supply of substrate to be turned into product as it passes through the
reactor. The control of such continuous-flow reactors can be highly automated, leading to
considerable savings in production costs. For example, a STR can be easily modified to produce
a continuous-flow stirred-tank reactor (CSTR) (Figure 13a), in which the enzyme is held within the
reactor by a coarse mesh, and the product continuously flows out of the reactor as substrate is
pumped in. It is also possible to produce a packed-bed reactor (PBR) (Figure 13b), in which the

90
agar cubes are packed into a column and the substrate is pumped through the bed without any
need for stirring.

CSTRs and PBRs enable the enzyme to be reused many times before it needs to be replaced.
For example, in the production of high-fructose syrups, the immobilised glucose isomerase
enzyme would typically be used continuously for between 2 and 4 months, and only after this time
(when its activity would have dropped to 25% of the original level) would it need to be replaced.

The overall operating costs of continuous-flow reactors are often significantly lower than those of
equivalent batch processes. Batch reactors need to be emptied and refilled frequently at regular
intervals. Not only is this procedure expensive, but it also means that there are considerable
periods of time when such reactors are not productive (so-called ‘downtime’).

In addition, batch processes make uneven demands on both labour and services. They may also
result in pronounced batch-to-batch variations, as the reaction conditions change with time, and
they may be difficult to scale up, due to the changing power requirements for efficient mixing. Due
to their higher overall process efficiency, continuous processes using immobilised enzymes may
be undertaken in production facilities that are around 10 to 100 times smaller than those required
for equivalent batch processes using soluble enzymes. Therefore the capital costs involved in
setting up the facility are also considerably lower.

Immobilisation Techniques
It should be noted that although the agar entrapment method described here has provided a
useful example, it is not a particularly effective form of immobilisation. The high temperature
required to prevent the agar from setting may lead to thermal inactivation of the enzyme, and
the agar gel itself is very porous and will allow the enzyme to leak out into the surrounding
solution.

There are in fact thousands of different techniques of immobilisation, all of which are much more
effective than our example. In general, these techniques can be classified as belonging to one
of three categories (Figure 14):
• Adsorption
• Covalent Bonding
• Entrapment.

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Adsorption
The physical adsorption of an enzyme to a supporting matrix is the oldest method of
immobilisation. As early as 1916, J.M. Nelson and Edward G. Grifn described the adsorption of
yeast invertase on to activated charcoal, and the subsequent use of this preparation for sucrose
hydrolysis. Over the years a variety of adsorbents have been used, including:
• Cellulose,
• Sephadex,
• Polystyrene,
• Kaolinite,
• Collagen,
• Alumina,
• Silica gel,
• Glass.

Such immobilisation procedures are extremely easy to perform, as the adsorbent and enzyme
are simply stirred together for a time (typically minutes to hours). The binding forces that
immobilise the catalyst on the support may involve hydrogen bonds, van der Waals forces, ionic
interactions or hydrophobic interactions. Such forces are generally weak in comparison with
covalent bonds for example, a hydrogen bond has an energy content of about 20 kJ mol-1,
compared with 200 500 kJ mol-1 for a covalent bond. Tus, when using such methods, yields (i.e.
the amount of enzyme bound per unit of adsorbent) are generally low. In addition, adsorption is
generally easily reversed, and can lead to desorption of the enzyme at a critical time.

However, despite these limitations, such a method was used in the first commercial immobilised
enzyme application, namely DEAE–Sephadex-immobilised l-amino acid acylase, in 1969. DEAE–
Sephadex is an ion-exchange resin that consists of an inert dextran particle activated by the
addition of numerous diethylaminoethyl groups. Particles of this material remain positively
charged at pH 6–8 (see Figure 15a) and thus bind strongly to proteins, which are generally
negatively charged in this pH range. If the pH is kept constant, the enzyme and support will remain
ionically linked. However, when over time the enzyme loses its activity through denaturation, the
pH can be adjusted to a more acidic value, the old enzyme will be desorbed, and the pH can then
be readjusted back to pH 6–8 and a fresh batch of enzyme bound. Thus, the support matrix may
be used many times, giving the process significant economic benefits.

Clearly DEAE–Sephadex immobilisation is only of value for enzymes that have a neutral to
alkaline pH optimum. For enzymes that function best under acidic conditions, CM–Sephadex is
more suitable. This contains carboximethyl groups that remain negatively charged at pH 3.5 4.5
(Figure 15b). Proteins at this pH are generally positively charged and will thus ionically bind to the
support. Desorption of the enzyme will occur when the pH is adjusted to a more alkaline value.

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Due to the simplicity and controllability of this immobilisation procedure, combined with the
economic benefits of reuse of the support, ion-exchange materials are now widely used as the
method of choice in many industrial settings.

Covalent Bonding
Immobilisation of enzymes by covalent bonding to activated polymers is a widely used approach
since, although it is often a tedious procedure, it is capable of producing an immobilised enzyme
that is firmly bound to its support. The range of polymers and chemical coupling procedures that
are used is enormous.

The history of covalent bonding for enzyme immobilisation dates back to 1949, when F. Michael
and J. Ewers used the azide derivative of carboxymethylcellulose to immobilise a variety of
proteins. Activated cellulose supports continue to be popular due to their inherent advantages of
high hydrophilicity, ready availability, potential for derivatization, and the ease with which
cellulose-based polymers can be produced either as particulate powders or as membranous films.

It is often more effective not to build the reactive group into the cellulose itself, but instead to use
a chemical ‘bridge’ between the cellulose and the enzyme molecule. The requirements for such
a bridging or linking molecule are that it must be small, and that once it has reacted with the
support it must have a further reactive group capable of reacting with the enzyme. An example of
such a bridging molecule is glutaraldehyde, which contains two aldehyde groups, one at either
end of its (CH2)3 moiety. At neutral pH values the aldehyde groups will react with free amino
groups. Thus one end of the glutaraldehyde molecule may be attached to the support, and the
other to the enzyme.

Covalently immobilised enzymes are strongly bound to their support, so when the proteins
denature, they are default to remove (in contrast to adsorption, as described earlier). Therefore,
it is usual for both the enzyme and the support to be replaced. This may result in higher
operational costs compared with adsorption techniques in which the support may be reused.

Entrapment
The entrapment of an enzyme can be achieved in a number of ways:
• Inclusion within the matrix of a highly cross-linked polymer
• Separation from the bulk phase by a semi-permeable ‘microcapsule’
• Dissolution in a distinct non-aqueous phase.

An important feature of entrapment techniques is that the enzyme is not in fact attached to
anything. Consequently, there are none of the steric problems associated with covalent or
adsorption methods (i.e. the possibility of the enzyme binding in such a way that its active site is
obstructed by part of the supporting polymer matrix).

The example of an enzyme retained in agar, described earlier, is a useful illustration of

entrapment. A preferable alternative involves mixing the catalyst with sodium alginate gel and
extruding this into a solution of calcium chloride to produce solid calcium alginate particles. This
technique has the advantage of not requiring the use of high temperatures. However, although it
is a popular activity in teaching laboratories, outside that setting it is generally unsuitable for the
immobilisation of purified enzymes, as these are often able to leak out of the gel.

93
Entrapment techniques for purified enzymes are more likely to involve retaining the enzyme
behind some form of ultrafiltration membrane. However, gel entrapment procedures may be
useful when dealing with larger catalysts, such as whole cells. For example, gel-immobilised living
yeast cells have been used successfully in the manufacture of champagne by Moët & Chandon.

Immobilisation: Changes in Enzyme Properties

Earlier in this essay it was suggested that immobilisation might change the properties of an
enzyme to enhance its stability. Initially it was believed that such enhanced stability resulted from
the formation of bonds between the enzyme and the supporting matrix that physically stabilise
the structure of the protein. Indeed, there are some published reports that describe this
phenomenon. With regard to the stabilization of proteolytic enzymes, which often exhibit more
prolonged activity in the immobilised state, this is most probably explained by the fact that such
proteases in free solution are prone to autodigestion (i.e., enzyme molecules cleave the peptide
bonds of adjacent enzyme molecules), a process that is largely prevented when they are fixed to
a supporting matrix.

However, the effects of immobilisation are more often due to the supporting matrix changing the
microenvironment around the enzyme and/or introducing diffusional constraints that modify the
activity of the catalyst. Consider, for example, immobilisation of the enzyme by adsorption on to
a polyanionic (negatively charged) support such as cellulose. If the substrate is a cation (i.e.,
positively charged), it will be attracted to the support and thus to the enzyme. In this case the
enzyme might well display higher activity, as the substrate concentration in its microenvironment
would be higher than that in the surrounding bulk phase. Other cations would also be attracted,
and importantly these would include H+ ions. Thus, the microenvironment would also be enriched
in H+ ions, so the pH surrounding the enzyme would be lower than the pH of the bulk phase.
Consequently, the enzyme would also exhibit an altered pH profile compared with that of its
soluble counterpart.

In addition, the immobilisation matrix might act as a barrier to the diffusion of substrates, products
and other molecules. For example, if a high enzyme loading was put into a gel particle and this
was then immersed in substrate solution, the substrate would diffuse into the gel and rapidly be
converted into product. Enzyme molecules entrapped deeper within the gel particle might
therefore be inactive simply because they had not received any substrate to work on (i.e. all of
the substrate was converted to product in the outer layers of the particle).

Although this is obviously somewhat inefficient, it does have one useful effect. When over time
the enzyme within the system denatures, the loss of activity of the enzyme in the outer part of the
particle means that substrate will now diffuse deeper into the particle to reach the previously
unused core enzyme molecules. In effect this inner reserve of enzyme will offset the loss of
enzyme activity through denaturation, so the system will show little or no overall loss of activity.
This explains the observation that immobilised systems often have a longer operational lifetime
than their soluble equivalents.

In addition, it is of interest that enzymes bound to natural cell membranes (phospholipid bilayers)
within living cells will also probably demonstrate these effects, and immobilised systems thus
provide useful models for the study of such membrane-bound proteins in living cells.

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Immobilised Enzymes at Work
The major industrial processes that utilize immobilised enzymes are listed in Table 7. Sales of
immobilised enzymes peaked in 1990, when they accounted for about 20% of all industrial
enzyme sales, almost entirely due to the use of glucose isomerase for the production of
sweetening agents. Other commercial applications utilize penicillin acylase, fumarase, β-
galactosidase and amino acid acylase. Since 2000, although there has been consistent growth in
enzyme markets, few new processes employing immobilised enzymes have been introduced.

The following three examples highlight many of the biochemical, technological and economic
considerations relating to the use of immobilised enzymes on a commercial and industrial scale.

Production of High-Fructose Syrup

Undoubtedly the most significant large-scale application of immobilised enzymes involves the
production of high-fructose corn syrup (HFCS). Although most of the general public believe that
sucrose is responsible for the ‘sweetness’ of food and drinks, there have been significant efforts
to replace sucrose with alternative, and often cheaper, soluble caloric sweetening agents.

HFCS is a soluble sweetener that has been used in many carbonated soft drinks since the 1980s,
including brand-name colas such as Coca-Cola and Pepsi-Cola. HFCS is produced by the
enzymatic digestion of starch derived from corn (maize). Developments in HFCS production have
been most prominent in countries such as the U.S.A., which have a high capacity to produce
starch in the form of corn, but which do not cultivate significant amounts of sugar cane or sugar
beet, and must therefore import either the raw products (for processing) or the refined sugar
(sucrose) itself.

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 Enzyme Stabilization
It is a fact that enzymes function in industrial solvents with enhanced technological utility. Enzyme-
catalyzed reactions in organic solvents and supercritical fluids have found numerous potential
applications, some of which have been successfully commercialized. Research over the past
decades has shown that in such solvents, enzymes can catalyze reactions which are seemingly
impossible in water (Klibanov 2001).

A priori, it can be of concern that when an enzyme is exposed to an organic solvent, it can
denature, but enzymes were instead discovered to be much stable and to retain “molecular
memory” in such solvents. The reason behind is that reactions such as deamidation of
thermolabile residues and hydrolysis of peptide bonds do not ensue in solvents in the absence of
water.

The stability of enzymes in organic solvents is also due to their conformational rigidity in the
dehydrated state. Furthermore, proteolytic degradation does not occur in the absence of water
they are insoluble in organic solvents (Klibanov 2001).

Irrespective of the aforesaid, large-scale usage of organic solvents provided with certain
limitations, such as increased polarity of organic solvents, typically results in reduced catalytic
activity. Recent advances in nanotechnology have promised solution in this booming area of
enzyme stabilisation, suggesting the usage of inorganic nonaqueous solvents or the room
temperature ionic liquids (RTILs) for reaction medium engineering or by means of nanostructures
with large surface area for the immobilisation of enzyme molecules (Kim et al. 2006a). Ionic liquids
have proved to be a better reaction media for enzymes since they have essentially zero vapor
pressure, are highly stable to the range of operating conditions in bioreactors, are stable to air
and water, and enable the choice of a wide range of substrates that is insoluble in most organic
solvents. For example, Erbeldinger and colleagues used 1-butyl-3-methylimidazolium
hexafluorophosphate as the solvent for thermolysin- catalyzed synthesis of Z-aspartame. Along
similar lines, lipase-catalysed transamination of carboxylic acids with ammonia has been
established in 1-butyl-3-methylimidazolium tetrafluoroborate (Kim et al. 2006b).

Other than room temperature ionic liquids, nanostructures like nanoparticles, nanofibers,
mesoporous silica, and nanoparticles prepared via sol–gel encapsulation have been instrumental
in enzyme stabilization.

More recent development in this area is the “single enzyme nanoparticles (SENs)” which is a
new enzyme composite of nanometer scale by Kim and Grate Nanostructures are better carriers
for immobilisation of enzymes since their reduced size can generally improve the efficiency of
immobilised enzymes due to larger surface area per unit mass, pore sizes tailored to protein
molecule dimensions, functionalised surfaces, multiple sites for interaction or attachment, and
reduced mass-transfer limitations (Kim et al. 2006a)

Each enzyme molecule is surrounded with a porous composite organic/ inorganic network of less
than a few nanometers thick. Converting free enzymes to SENs can result in significantly more
stable catalytic activity, while the nanoscale structure of the SEN does not impose a serious mass-
transfer limitation on substrates (Kim et al. 2006a).

Interestingly another breakthrough in nanoenzymology was reported in November 2006 when it

was discovered that silica-spun FMS pores which are hexagons of approximately 30 nm in
diameter and mimic the crowding of cells can be used for refolding of enzymes and upon refolding

96
the enzyme gets reactivated and becomes capable of catalysing thousands of reactions a second
(PNNL 2006).

A report using magnetic nanoparticles for the enzyme immobilisation was demonstrated with
covalently attached lipase on the magnetic γ-Fe2O3 nanoparticles and showed stability for a
month (Dyal et al. 2003). One disadvantage in using nanoparticles is that their dispersion in
reaction solutions and the subsequent recovery for reuse are often found to be a daunting task.
Thus, a replacement of nano-particles can be the electrospun nanofibers which provide a large
surface area for the attachment or entrapment of enzymes and the enzyme reaction.

Improved Bioseparation Techniques: Downstream

Processing of Enzymes
Downstream processing of enzymes holds an important position in industries as the bulk of the
product cost is determined by the steps leading to purification of enzymes. Thus, biotechnology
companies are prospecting for new and improved processing methods. New improved methods
over the conventional chromatography and membrane filtration techniques are the cloud point
extraction (CPE) and field-assisted separation technologies like MAGSEP (magnetic separator)
and ELECSEP (electrophoretic separator) for the quantitative separation of enzymes are gaining
more importance.

Figure: Cloud Point Extraction

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Cloud point extraction technique has been used to separate proteinase 3 (Pr3) from neutrophil
azurophilic granules, tyrosinase from mushroom pileus by Garcia-Carmona and co-workers,
cholesterol oxidase extracted from Nocardia rhodochrous by Kula and co-workers, and
hexokinase and lactate dehydrogenase from aqueous solutions using Triton X-114 as the
surfactant. The technique in brief can be described as surface- active agents (surfactants,
detergents) can aggregate in aqueous solution to form colloidal- sized clusters referred to as
micelles (normal micelles). The minimum concentration of surfactant required for this
phenomenon to occur is called the critical micelle concentration (CMC).

Upon heating, aqueous solutions of many nonionic surfactants become turbid at a temperature
known as the cloud point (or the lower consulate temperature), above which there is a separation
of the solution into two phases (Minuth et al. 1996).

Affinity chromatography techniques are the most applied tools available for downstream
processing. However, porous affinity supports can only work in a later stage of purification in clear
solutions and not in early stages when fouling compounds are present in the system. Nonporous
support particles can replace such a system if and only if the size of nonporous support particles
is in the range of 0.1 to 1 μm. Magnetic separation seems to be the only feasible method for the
recovery of such small particles from the biological debris. For separating enzymes, an
appropriate affinity ligand is usually immobilized on a magnetic carrier, such as silanized
magnetite, or on polymeric magnetic (chitin) or magneThisable particles.

The electrophoretic separation of proteins without gels has been an innovative goal in separation
research. Electrophoretic separations are influenced by factors such as size (or weight), shape,
secondary structure, and charge of the macromolecule or cell. Ohmic
heating hinders the scale-up of electrophoresis. The heat generated is equal to the product of the
current and voltage, and this heat can cause free convection and mixing within the system. Too
much heat can denature the labile biomolecules or cells.

To overcome such problems, the multistage electrophoretic method was developed which is a
combination of free electrophoresis and mulThistage extraction. The apparatus used is known as
advanced separation apparatus (ADSEP). This ADSEP was modified to act as an ELECSEP
by replacing the chamber bottoms with metal cover plates. In these systems electrodes are kept
over these cover plates with gaskets in between them. Each cavity has a height of few millimeters
ensuring the fluid within it to remain isothermal during the application of an electric field, which
transfers the molecules from the bottom to the top cavity. As each molecule is transferred to the
new cavity, it is either drawn into the upper cavity by the electric field or left in the lower cavity,
depending on its electrophoretic mobility (Karumanchi et al. 2002).

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Zakpaa Domakyaara Hilary (Ph.D.)

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