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.

Recycle packed column reactor: - allow the reactor to operate at high fluid velocities.

Immobilized Enzyme Reactors

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 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 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-BEDENZYME 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 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.