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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. (a–c)
(a) Batch reactors with complete backmixing;
(b) Stirred-tank reactor;
(c) Fixed-bed reactor;
Fluidized-bed reactor. (d–f) are
Continuously operated reactors
with complete back mixing.
(g–h) are the Continuously operated 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
Recycle packed column reactor:
- allow the reactor to operate at high fluid velocities.
Fluidized Bed Reactor:
- a high viscosity substrate solution
- a gaseous substrate or product in a continuous reaction system
- 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 0.5–2 mL/min and sample injection volumes of 10–100 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 below.
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 mobilephase 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 NHþ4 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
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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 β is a
constant for a given reactor, and is equal to the ratio of reactor void volume to
total reactor volume (i.e., β 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 Michaelis–Menten 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.