Enzyme immobilization examples

Rasor and Tischer (1998) have brought out the advantages of enzyme immobilization. Examples of penicillin-G to 6-APA, hydrolysis of cephalospwrin C into 7-ACA, hydrolysis of isosorbide diacetate and hydrolysis of 5-(4-hydroxy phenyl) hydantom are cited. De Vroom (1998) has reported covalent attachment of penicillin acylase (EC 3.51.11) from E.Coli in a gelatine-based carrier to give a water insoluble catalyst assemblase which can be recycled many times, and is suitable for the production of semi-synthetic antibiotics in an aqueous environment. The enzyme can be applied both in a hydrolytic fashion and a synthetic fashion. 6-APA was produced from penicillin-G similarly, 7-ADCA was produced from desa acetoxycephalosporin G, a ring expansion product of penicillin G. 

Substances other than enzymes can be immobilized. Examples include the fixing of heparin on polytetrafluoroethylene with the aid of PEI (424), the controUed release of pesticides which are bound to PEI (425), and the inhibition of herbicide suspensions by addition of PEI (426). The uptake of anionic dyes by fabric or paper is improved if the paper is first catonized with PEI (427). In addition, PEI is able to absorb odorizing substances such as fatty acids and aldehydes. Because of its high molecular weight, PEI can be used in cosmetics and body care products, as weU as in industrial elimination of odors, such as the improvement of ambient air quaHty in sewage treatment plants (428). 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

It is not only the activity that can be altered by incorporation of noncoded amino acids. Introduction of structures possessing certain chemical functions leads to the possibility of highly regioselective modification of enzymes. For example, selective enzymatic modification of cystein residues with compounds containing azide groups has led to the preparation of enzymes that could be selectively immobilized using click chemistry methods [99]. 

Biocatalysts also operate in ionic liquids [28]. The ones that have been most widely investigated are the lipase family of enzymes. For example, Candida Antarctica lipase B immobilized in [bmim][BF4] or [bmim][PFe] under anhydrous conditions is able to catalyse transesterifications at rates comparable to those observed in other solvents. Certain lipase mediated enantioselective acylations have even resulted in considerable improvements in enantiomeric excesses... 

In a second example, Storey et al. demonstrated that one could covalently immobilize amyloglucosidase using hydrophilic prepolymers. A 5-mg/ml solution of the enzyme was mixed with an equal volume of prepolymer. The method was judged superior as a support for enzyme immobilization. The percent activity inuno-bili/ed in the polyurethane foams was 25 1.5%. 

One solution to the above problems would be to purify either the substrate or the enzyme. For example, in the case of ECB, the substrate of the enzyme could be extracted from the cells and then purified by chromatography to remove any impurities that would interfere with the bioconversion processes. The ECB could be left as a solution in a solvent or dried to a powder form. ECB deacylase could also be purified to either a concentrated enzyme solution or solid form. Another option is to immobilize the enzyme or substrate on a suitable support. In the case of ECB deacylase, the advantage would be that the enzyme could be reused and the savings gained would obviously have to be compared with the extra costs of immobilization. The next section looks in more detail at the novel approach of immobilizing the ECB substrate. 

The gas-sensing electrodes also are used for the potentiometric measurement of biologically important species. An enzyme is immobilized at or near the gas probe. The gas sensor measures the amount of characteristic gas produced by the reaction of the analyzed substance with the enzyme. For example, an enzyme electrode for urea [NH2C(0)NH2] determination is constructed by the immobilization of urease onto the surface of an ammonia-selective electrode. When the electrode is inserted into a solution that contains urea, the enzyme catalyzes its conversion to ammonia ... 

Another enzyme that was studied extensively in microreactors to determine kinetic parameters is the model enzyme alkaline phosphatase. Many reports have appeared that differ mainly on the types of enzyme immobilization, such as on glass [413], PDMS [393], beads [414] and in hydrogels [415]. Kerby et al. [414], for example, evaluated the difference between mass-transfer effects and reduced effidendes of the immobilized enzyme in a packed bead glass microreactor. In the absence of mass-transfer resistance, the Michaelis-Menten kinetic parameters were shown to be flow-independent and could be appropriately predicted using low substrate conversion data. 

Another example in which biocatalysis is combined with analysis is the system reported by Honda et al. [436]. A microreaction system, consisting of an enzyme-immobilized microreactor, for optical resolution of racemic amino acids was devel-... 

The amino group can be activated with bifunctional reagents. A commonly applied procedure is the introduction of an aldehyde function by the bifunctional reagent glutardialdehyde. The activated carrier can be used directly for the covalent bonding of the enzyme as shown in Fig. 7. Table 1 summarizes various examples of enzyme immobilization. 

To date, only a few examples of laboratory preparative-scale processes based on purified enzyme have been reported. Several studies have focused on the small-scale implementation of processes associating a new co-factor regenerating system, enzyme immobilization, membrane reactor, continuous substrate feeding, or resin-based SFPR with various results [110], Using the outstanding stabihty of PAMO, a 200 ml biotransformation of 5g/l phenyl cyclohexanone by an engineered mutant under two-Hquid phase conditions using methyl tert-butyl ether as solvent was described [102]. 

Other examples of enzyme immobilization have also been reported. For example, the recombinant human al,3/l,4-fucosyltransferase were immobilized on Ni -agarose through a 6His tag and exhibited a remarkable stability. It was exploited in the synthesis of Le and Le trisaccharides (184). 

A special case arises when the "skin" (membrane) layer of a normal composite membrane element is immobilized with a catalyst and not intended for separating reaction species. Consider the example of an enzyme, invertase, for the reaction of sucrose inversion. Enzyme is immobilized within a two<layer alumina membrane element by filtering an invertase solution from the porous support side. After enzyme immobilization, the sucrose solution is pumped to the skin or the support side of the membrane element in a crossflow fashion. By the action of an applied pressure difference across the element, the sucrose solution is forced to flow through the composite porous structure. Nakajima et al. [1988] found that the permeate direction of the sucrose solution has pronounced effects on the reaction rate and the degree of conversion. Higher reaction rates and conversions occur when the sucrose solution is supplied from the skin side. The effect on the reaction rate is consistently shown in Figure 11.6 for two different membrane elements membrane A is immobilized by filtering the enzyme solution from the support layer side while membrane B from the skin layer side. 

Many examples of polymer-embedded enzyme bioelectrocatalytic electrodes exist [91-93]. The electrical contacting of enzymes immobilized in polymer matrices is achieved either by virtue of a conducting polymer, or by incorporating electron relay groups within the polymer, providing electron hopping between the enzyme and the electrode support. 

Enzyme immobilization methods other than using the hydrogels (A) Since appUcations of the redox hydrogels (A) to glucose sensors was first reported by Pishko et al. in 1991, various other methods have been reported to immobilize redox polymers to construct biosensors as indicated (see Section 3.3.3.2). Other recent examples are as follows. 

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