L 7 Immobilized Enzymes Reactors

Immobilized Enzymes
Reactors
The methods for the heterogenisation
(or localization) of enzymes
by coupling them to insoluble supports or

by entrapment.
The types of reactors used for

immobilized enzymes are
summarized in Figure given bellow.
Reactors for immobilized enzymes. (ac)
(a) Batch reactors with complete

backmixing;
(b) Stirred-tank reactor;
(c) Fixed-bed reactor;
Fluidized-bed reactor. (df)
are
Continuously operated
reactors
with
(gh)complete back mixing. operated
are the Continuously
reactors with plug-flow behavior.
(i) Reactor with the enzyme
immobilized in or on a membrane
that may at the same time separate
two phases such as water and
organic solvent.
(j) reactor with physically separated

enzyme and organic solvent in order
to prevent denaturation of the
protein
The principles developed for general
heterogeneous catalysis in synthetic
chemistry are valid, resulting in well-known
reactor configurations.
Differences between enzyme catalysis and

other systems result from the nature of the
biocatalyst and reaction medium.
For example, soft particles containing the

biocatalyst, such as alginate beads, may
limit the pressure drop in fixed-bed
reactors.
The decision as to specific reactor
design will be based on a careful
analysis of the kinetic properties of
the reaction system.
For example, if the enzyme shows a

strong substrate-surplus inhibition, a
continuously operated reactor with
complete backmixing working at high
conversion is advantageaous.
A reaction with strong product inhibition may
utilize a batch reactor or a plug flow reactor to
achieve higher volume and catalyst specific
productivities.
An extractive bioreactor may be used if

substrates and products show different
solubilities.
By using this reactor configuration, the

destabilizing effect of organic solvents may also
be overcome, because the enzyme is separated
from the organic phase, which is used to extract
the insoluble product .
The aqueous phase containing the enzyme will be

saturated until the maximum solubility of with the
substrate is reached.
Reactions using biocatalysts are normally
performed in aqueous solution at temperatures
between 10 and 80 C and at ambient pressure.
Due to the inhibition of some enzymes by heavy

metals, the materials of construction must not
release these elements.
Reactors are operated under conditions that

prevent microbial contamination.
The reactor itself as well as the substrate may be

sterilized prior to reaction by using chemical
agents (ethanol, formaldehyde, ethylenoxide,
Velcorin) or steam.
Ultraviolet rays may be used to
sterilize the immobilized enzyme on
its support .
Alternatively, the immobilization may

be performed under sterile
conditions.
Antibacterial agents may be added

to the reaction mixture to prevent
microbial growth while the reactor is
running.
In some cases, the reactants may act as
sterilants or inhibitors of microbial growth,
such as ketones or alcohols.
At higher concentrations (more than 500

mmol/L), solutions may become
autosterile because of osmotic pressure
effects.
Ndustrial processes are often performed at

elevated temperatures, above 55 C,
reducing the danger of microbial
contamination.
For a constant product quality and
reproducibility of downstream processing,
the reactor should be operated at constant
conversion.
To overcome the deactivation per unit of

time that shows all biocatalysts as a result
of denaturation processes, either the
residence time has to be increased or
fresh enzyme has to be supplied.
The latter is especially easy for soluble

enzymes. For carrier-fixed enzymes, a
combination of both methods is used, as
discussed later.
Immobilized Enzyme
Reactors
cycle packed column reactor:

llow the reactor to operate at high fluid velocities.
uidized Bed Reactor:
a high viscosity substrate solution
a gaseous substrate or product in a continuous reaction syst
care must be taken to avoid the destruction and
decomposition of immobilized enzymes
- An immobilized enzyme tends to decompose
upon physical stirring.
- The batch system is generally suitable for the production
of rather small amounts of chemicals.
The immobilization of enzymes onto particulate
carriers that may be packed into a column (the
packed-bed reactor), such as a typical HPLC
column, facilitates repetitive use of the enzyme
and also allows the automation of enzymatic
assays.
Open-tubular reactors have also been constructed

by covalently immobilizing an enzyme onto the
inner wall of a nylon or polyethylene tube.
Immobilized enzyme reactors are used in

conjunction with a pump, to force a buffer, or
mobile phase, through the reactor at a steady rate,
an injector located between the pump and the
reactor to allow the introduction of substrate
solutions, and a detector located close to the
column exit.
The mobile phase contains all required cosubstrates and
activators required for the enzymatic reaction, but does not
contain the analyte substrate.
A typical packed-bed system may use a 25-cm long reactor

with a 5-mm inner-diameter, packed with the carrier-
enzyme solid phase at high pressures.
Flow rates of 0.52 mL/min and sample injection volumes of

10100 mL are common.
Detection involves the same principles used in

homogeneous enzymatic assays, and flow-through optical
absorbance and fluorescence detectors, and amperometric
and potentiometric electrochemical detectors may be
employed, with detector volumes of the order of tens of
microliters being standard.
Enzyme reactor systems may be of the
continuous flow or the stopped-flow variety.
Continuous flow systems are further categorized
as open or closed systems.
The open system, shown in Figure , continuously

pumps fresh buffer through the injector, reactor
and detector, ultimately into a waste reservoir for
discarding.
This arrangement is preferred for the testing of

enzyme reactors, since unreacted substrate,
cofactors and the products of the enzymatic
reactions will not be reexposed to the column.
Diagram of an open enzyme reactor
system
Closed systems may be employed
when buffer recycling is possible,
that is when the buffer contains high
concentrations of all necessary
cosubstrates, when complete
consumption of injected substrate
occurs within the reactor, and when
products of the enzymatic reaction
do not inhibit the immobilized
enzyme.
A closed system for immobilized
oxidase enzymes is shown in Figure
Diagram of a closed enzyme reactor
system.
Both open and closed continuous
flow systems rely on the fixed time,
or endpoint method for the
determination of substrate
concentrations.
At a fixed and constant flow rate, the

injected volume of substrate will
spend a fixed time on the column,
and this time is related to the volume
of the column (that volume not
occupied by stationary phase) and
the mobile-phase flow rate.
Indicator reactions that are chemical
in nature may be introduced either
into the mobile phase or at the end
of the column by the method of
postcolumn reagent addition.
Postcolumn addition of reagents

dilutes the column eluent, so that,
when possible, the addition of
indicator reagents to the mobile
phase is preferable.
The conditions under which chemical
indicator reactions are used often
necessitates the use of postcolumn
addition, however.
Figure given below shows an experimental

setup for urea assays using an
immobilized urease reactor.30 The
postcolumn addition of sodium hydroxide
allows the NH4 produced by the reactor
to be detected as NH3 at an ammonia gas-
sensing electrode placed in a flow cell.
Enzyme reactor system for urea based on
immobilized urease and potentiometric detection.
Stopped-flow enzyme reactor systems
have been designed for automated kinetic
assays.
A diagram of a stopped-flow reactor that

uses a postcolumn chemical indicator
reaction is shown in Figure below.
In this system, the flow rate of themobile

phase through the reactor dictates the
residence time of the analyte on the
column.
Stopped-flow enzyme reactor with
absorbance detection
THEORETICAL TREATMENT OF PACKED-BED
ENZYME REACTORS
Packed-bed enzyme reactors, those employing enzymes
immobilized onto a particulate phase that is subsequently
packed into a column, may be characterized by their column
capacity, C, and the degree of reaction P.
The parameter C is defined by the equation.
where k is the decomposition rate constant for the enzyme

substrate complex (either k2 or kcat),
Et is the total number of moles of enzyme immobilized, and the

value of is a constant for a given reactor, and is equal to the
ratio of reactor void volume to total reactor volume (i.e., is
always less then unity).
The degree of reaction, P, varies between zero (no product

formed) and unity (complete conversion of substrate).
An equation equivalent to the MichaelisMenten equation has
been derived for immobilized enzymes in packed-bed reactor
systems, and is given in Eq.
where Q is the volume flow rate of the mobile phase. In

general, this equation predicts that for a given column
capacity, the degree of reaction, P, is inversely related to the
mobile-phase flow rate, Q. That is, the faster the analyte plug
flows through the reactor, the less likely will be its complete
conversion into product.

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L 7 Immobilized Enzymes Reactors

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Immobilized Enzymes

by coupling them to insoluble supports or

The types of reactors used for

(a) Batch reactors with complete

(j) reactor with physically separated

Differences between enzyme catalysis and

For example, soft particles containing the

For example, if the enzyme shows a

An extractive bioreactor may be used if

By using this reactor configuration, the

The aqueous phase containing the enzyme will be

Due to the inhibition of some enzymes by heavy

Reactors are operated under conditions that

The reactor itself as well as the substrate may be

Alternatively, the immobilization may

Antibacterial agents may be added

At higher concentrations (more than 500

Ndustrial processes are often performed at

To overcome the deactivation per unit of

The latter is especially easy for soluble

cycle packed column reactor:

Open-tubular reactors have also been constructed

Immobilized enzyme reactors are used in

A typical packed-bed system may use a 25-cm long reactor

Flow rates of 0.52 mL/min and sample injection volumes of

Detection involves the same principles used in

The open system, shown in Figure , continuously

This arrangement is preferred for the testing of

At a fixed and constant flow rate, the

Postcolumn addition of reagents

Figure given below shows an experimental

A diagram of a stopped-flow reactor that

In this system, the flow rate of themobile

where k is the decomposition rate constant for the enzyme

Et is the total number of moles of enzyme immobilized, and the

The degree of reaction, P, varies between zero (no product

where Q is the volume flow rate of the mobile phase. In

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