Immobilization of enzymes

Enzyme Immobilization
CARRIER BINDING METHOD
• Binding of biocatalyst to water insoluble carriers through ionic interaction,
covalent bonding
• polysaccharides- cellulose, dextran, proteins (gelatin and albumin),synthetic
polymers (polyurethane) and inorganic materials (brick,sand ,clay
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• Properties of carrier
1. possessing adequate functional group for immobilization
2. mechanical strength
3. Physical, chemical and biological stability
4. non-toxic
Covalent binding
• Amino acid residues not involved in active site are used for covalent binding
with carriers
• hydroxyl groups of serine, mercapto group of cysteine, phenolic group of
tyrosine etc reacts with a rweactive group in carrier
• advantages
1. no leakage of biocatalyst from carrier
2. stability increases because of strong interaction with carrier
disadvantages
The activity yield is likely to be low owing to exposure of enzymes to toxic
agents
 Ionic binding method
• Ionic interaction with the carrier
• DEAE Cellulose, CM CELLULOSE
• Binding of the biocatalyst on the carrier is affected by buffer used,
pH, ionic strength and temperature
Physical adsorption
• physical interaction between biocatalyst and carrier –hydrogen
bonding, hydrophobic interaction
• enzymes adsorbed into suitable carriers –adsorbents by direct
physical interaction
• glass, silica, nylon, cellulose
Entrapment method
Based on the localization of an enzyme within the lattice of a polymer matrix or
membrane
Gel entrapment: The enzymes are entrapped within gel beads .
Gel materials used : Polyacrylamide , alginate, carrageen, agar ,agarose.
Sodium alginate is a common gel used. The gel allows the substrate to enter and
the product to leave.
 enzyme becomes enclosed in a fine network
 trapping due to steric hindrance – mesh must be narrow enough to prevent
movement of enzymes from the net -to retain protein while allowing penetration
of substrate.
• Classified into lattice and micro capsule, encapsulation, membrane types
Example : calcium alginate gel entrapment
• Immobilization of laccase in calcium alginate gel
• Steps
• 1.Enzyme solution is mixed with sodium alginate solution
• 2.droplets of this solution are added to a solution of calcium chloride
• 3. droplets turns into a bead containing the enzyme
• Encapsulation
• Enzymes are entrapped in a semi permeable membrane
• liposomal encapsulation –enzyme molecules are encapsulated within
the vesicular structure – involves encapsulation of enzymes within
the amphipathic phospholipids like phosphatidyl choline , cholesterol
etc.

• Enzymes are immobilized without a chemical or structural

modification.
• Disadvantage
• High molecular weight substrates have limited diffusivity, and
cannot be treated with entrapped enzymes.

Alcohol dehydrogenase, hexokinase, glucosse oxidase ,glucose isomerase

Membrane entrapment
• Enzymes encapsulated within ultra filtration membrane
• Spherical semipermeable membrane of cellulose or nylon
• Ex: catalase , urease immobilized by this method
 Microcapsule-Type entrapping involves enclosing the enzymes within semi permeable
polymer membranes. The preparation of enzyme micro capsules requires extremely
well-controlled conditions and the procedures for micro capsulation of enzymes can be
classified as:

Interfacial Polymerization Method: enzymes are enclosed in semi -permeable membranes

of polymers.
An aqueous mixture of the enzyme and hydrophilic monomer are emulsified in a water-
immiscible organic solvent. Then the same hydrophilic monomer is added to the organic
solvent by stirring. Polymerization of the monomers then occurs at the interface between the
aqueous and organic solvent phases in the emulsion. The result is that the enzyme in the
aqueous phase is enclosed in a membrane of polymer.
Lattice-Type entrapment involves entrapping enzymes within the interstitial spaces of a
cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide,
polyvinylalcohol, etc... and natural polymer (starch) have been used to immobilize enzymes
using this technique.
Immobilization procedure:
Enzyme + polymer solution → polymerization → extrusion/shape the particles

13
Cross linking method
• Bi-or multifunctional agents used as cross linkers – intermolecular
crosslinking of biocatalyst .
• Cross linked enzymes becomes insoluble .
• Ex; glutaraldehyde, toluene diisocyanate , hexamethylene
diisocyanate .
• Activity is reduced due to interaction of active site amino acid
residues .
•
INORGANIC SUPPORTS
Silica and Inorganic Oxides
• one of the most frequently used inorganic support materials for enzyme immobilization.
• high thermal and chemical resistance and good mechanical properties.
• Silica offers good sorption properties due to its high surface area and porous structure. These
properties allow effective enzyme attachment and reduce diffusional limitations .
• Moreover, the presence of many hydroxyl groups on the surface of silica facilitates enzyme
attachment and favours its functionalization with surface modifying agents such as
• glutaraldehyde or 3-aminopropyltriethoxysilane .
• biocatalytic systems obtained demonstrate high catalytic activity retention and good thermal and pH
• resistance.
• For example, lipases immobilized on a silica gel matrix and on mesoporous silica retained
• respectively 91% and 96% of the activity of the free enzyme
Mineral Materials
• The minerals used as supports for enzyme immobilization are mainly clay
materials such as bentonite, halloysite, kaolinite, montmorillonite and
sepiolite
• abundant in nature, are easily available, offer high biocompatibility and can
be used as obtained without further advanced treatment and purification,
which makes them relatively cheap
• Enzymes immobilized on minerals are used mainly in environmental
engineering for waste and wastewater treatment as well as in biosensors
• For example, according to Chrisnasari et al., glucose oxidase immobilized on
bentonite modified by tetramethylammonium hydroxide retains over 50%
of its initial activity after five repeated catalytic cycles
Carbon-Based Materials
• activated carbons and unmodified and modified charcoals
• High adsorption capacity, the abundance of many functional groups
and minimal release of fine particulate matter make carbon-based
materials suitable carriers for the adsorption immobilization of
various enzymes
• For example, unmodified charcoal support was used for the
immobilization of amyloglucosidase. The immobilized enzyme
• when used for starch hydrolysis without any additional treatment
retained over 90% of the free enzyme catalytic activity.
LIMITATIONS O INORGANIC MATERIAL
AS SUPPORT
• Inorganic carriers have certain limitations, such as limited
biocompatibility, lower
• affinity to biomolecules and reduced possibilities to create various
geometrical shapes. Moreover,
• a cross-linking agent such as glutaraldehyde is usually required to
create covalent bond between
• the enzyme and an inorganic support
Organic Materials
• organic support materials can be divided into two groups: (i) synthetic materials
(mainly polymers) and (ii) renewable materials obtained from natural sources
(biopolymers)
• The greatest advantage of synthetic polymers as support materials is that : the
monomers that build the polymeric chain can be selected according to the
requirements of the enzyme and process in which the product of immobilization
will be used
• by using polymeric supports, control of the length of the matrix–enzyme spacers
has been achieved.
• Longer spacers allow the enzyme to retain higher conformational flexibility, while
shorter spacers can protect the biomolecules against thermal inactivation and
reduce leaching of the enzyme
• Various polymer materials can be used as effective supports and
improve properties of the immobilized enzyme such as thermal
stability and reusability.
• The polymer layers play a very important role in protecting the active
sites of the enzyme from negative effects of the ingredients of the
reaction mixture and the process conditions.
• However, it should be noted that synthesis of a polymer with the
desired properties and functional groups is usually a time-consuming
and costly process.
 examples
• , a-amylase was covalently immobilized on polyaniline via –NH groups,
• tyrosinase was immobilized via –NH and C=O groups on polyamide 66 (Nylon 66) without any
• linkers .
• In another study, commercial lipase was immobilized by covalent binding on strongly
• hydrophobic polystyrene microspheres activated by epoxy groups.
• In a hydrophobic environment, lipase exhibits extremely high catalytic properties which are related to the
phenomenon called interfacial activation.
• polyurethane foam has been used for covalent immobilization of inulinase .
• Bai et al. used polyvinyl alcohol modified by glutaraldehyde as a support for the immobilization of laccase
via –OH groups. After immobilization, as a result of the strong interactions, the product was characterized
by good storage stability and reusability which make it suitable for use in biosensors to detect bisphenol
A.
• Glucose oxidase, was immobilized on amino- and carboxyl-plasma-activated polypropylene film. The
introduction of these groups enhanced the affinity of the polymer to the enzyme
• Commercially available ion exchange resins—for example Amberlite and Sepabeads—have also been
used, respectively, for the immobilization of enzymes such as a-amylase and alcohol dehydrogenase
 Biopolymers
• carbohydrates , proteins such as albumin and gelatin
• Materials such as collagen, cellulose, keratins and carrageenan as
• well as chitin, chitosan and alginate are examples of biopolymers used for
immobilization
• possess a unique set of properties, from biodegradability to harmless
products, biocompatibility and non-toxicity, to an outstanding affinity to
proteins, which make them suitable supports for enzymes
• the availability of reactive functional groups in their structure—mainly
• hydroxyl but also amine and carbonyl moieties—enables direct reaction
between the enzyme and matrix and facilitates modification of their surface
•
MAGNETIC PARTICLES
• attachment of the enzyme molecules to magnetic iron oxide
nanoparticles (MNPs) and simple separation of the biocatalytic
system with the use of an external magnetic field .
• MNPs are known for their large surface area and the abundance of
hydroxyl groups on their surface which enables their easy
modification and strong (covalent) binding of the enzyme.
• . High mechanical stability and low porosity, however, which minimize
steric hindrances, are also relevant for the creation of a stable
enzyme–matrix
• Enzymatic Fuel Cells: Materials and Applications
Immobilization yield (%) = (added enzyme −unbound enzyme)/added enzyme × 100.
Recovered activity (%) = immobilized enzyme/(added enzyme − unbound enzyme) ×
100

Note: unbound enzyme measured as enzyme remaining in solution after the

immobilization

Effectiveness factor :
Mass transfer resisitance
 Properties of Immobilized Enzymes
The properties of an enzyme can be modified by
suitable choice of the immobilisation protocol, whereas the same method may have
appreciably different effects on different enzymes.
These changes may be due to conformational alterations within the enzyme due to the
immobilisation procedure, or the presence and nature of the immobilisation support.
A decrease in specific activity of an enzyme upon insolubilization, can be attributed to
denaturation of the enzymes caused by the coupling process.

Once an enzyme has been insolubilized, it finds itself in a microenvironment that may be
drastically different from that existing in free solution.

The new microenvironment may be a result of the physical and chemical character of the
support matrix alone, or it may result from interactions of the matrix with substrates or
products involved in the enzymatic reaction.

Changes can be observed in the stability of enzymes and in their kinetic properties
because of the microenvironment imposed upon them by the supporting matrix and by the
products of their own action.
 HIGH

Immobilized I L
F AN
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Enzyme TR
REACTION E
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A R
A-P ER
TR SF
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Kinetics of immobilized enzymes

• • The kinetics of immobilized enzyme reactions are influenced by a variety of factors:

• • Mass transfer resistances
• • Possible reactions between support and the enzyme.
• • Micro-environmental conditions
• • Effect of Mass transfer resistances on Immobilized Enzyme Kinetics:
• • Mass transfer resistances play a very important role in determining the immobilized enzyme
• kinetics.
• The substrate has to cross the film above the solid support and then diffuse through the matrix
• to the enzyme molecule embedded in the matrix.
• • The rate at which substrate passes over the insoluble particle affects the thickness of the
• diffusion film, which in turn determines the concentration of substrate in the vicinity of the
• enzyme and hence the rate of reaction.
• • The diffusion of substrate from the bulk solution to the micro-environment of an immobilized
• enzyme can limit the rate of the enzyme reaction.
• • So, the enzyme will have to encounter two mass transfer resistances :
• External Mass transfer Resistance encountered while passing through the film.
• • Internal Mass transfer Resistance encountered while diffusing through the matrix
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials
To determine the significant effect of external diffusion
resistance on the rate of enzyme catalytic reaction rate:
Damköhler numbers (Da)

maximum rate of reaction Vm '

Da  
maximum rate of diffusion k L [ Sb ]
Vm ' is the maximum reaction rate per unit of
external surface area (e.g. g/cm2-s)
kL is the liquid mass transfer coefficient (cm/s)

[ Sb ] Is the substrate concentration in bulk solution (g/cm3)

• When Da >> 1, the external diffusion rate is limiting;
• Da << 1, the reaction rate is limiting;
• Da ≈ 1, the external diffusion and reaction resistances are
comparable.
Applications : production of High-fructose corn syrup (HFCS)

• processing corn starch to yield glucose, and then processing the glucose to produce a high
percentage of fructose.
• First, cornstarch is treated with alpha-amylase to produce shorter chains of sugars .
• Next, an enzyme called glucoamylase breaks the sugar chains down even further to yield the
simple sugar glucose.
•  
• The third enzyme, glucose-isomerase, converts glucose to a mixture of about 42 percent fructose
and 50-52 percent glucose with some other sugars mixed in.
• While alpha-amylase and glucoamylase are added directly to the slurry, glucose-isomerase is
packed into columns and the sugar mixture is then passed over it.
• This 42-43% fructose glucose mixture is then subjected to a liquid chromatography step where the
fructose is enriched to approximately 90%.
• The 90% fructose is than back-blended with 42% fructose to achieve a 55% fructose final product.
Numerous ion-exchange and evaporation steps are also part of the overall process
 Corn
GLUCOSE ISOMERASES Syrup
• Catalyzes isomeric rearrangement of glucose to
fructose Gives a sweeter product than corn
syrup
• Enzymes are immobilized in large columns where
the reaction takes place – can reuse them
Glu isomerase
pH 7
50-60C

42% (HFCS) 52%

Effect of solute partition on the kinetics of
immobilised enzymes
Charges are always present on the surface of immobilised enzyme particles due to the amphoteric
nature of enzymes.
Where these positive and negative charges are not equally balanced, the net charge on the surface
exerts a considerable effect over the properties of the microenvironment. This surface charge,
(produced by the use of ion-exchange or similar charged matrices for the immobilization), repels
molecules of similar charge whilst attracting those possessing opposite charge.
A partitioning of charged molecules (e.g. substrates and products) occurs between the bulk
solution and the microenvironment; molecules of opposite charge to the immobilised enzyme
surface being partitioned into the microenvironment, whereas molecules possessing similar
charge to the immobilised enzyme surface are expelled, with equal effect, into the bulk
solution. The solute partition may be quantified by introduction of the electrostatic partition
coefficient

• where [C0n+] and [A0n-] represent each cation and anion bulk concentration, [Cn+]
and [An-] represent their concentration within the microenvironment and n is the
number of charges on each ion