Immobilization, enzyme stabilization method

This reaction was exploited by Tsukatani and Matsumoto (2000) in a stopped-flow FIA method. An immobilized D-malate dehydrogenase enzyme reactor was employed and the reduced enzymatic cofactor NADH that was formed was monitored fluorometrically (Xex = 340 nm = 460 nm). Due to the slow reaction rate, the flow was stopped with the sample in the reactor to increase reaction time. The intrinsic sample fluorescence was also assessed using a parallel blank reactor without immobilized enzyme. The method was validated through the analysis of red and white wine samples. The enzyme reactor stability was also evaluated and it was found that the sensitivity (evaluated as amplitude of response at a constant concentration of the analyte) gradually decreased to 60% within a week but then remained stable for a month. As D-malate cannot be present in naturally fermented wines (except for fraudulent addition), the interference of this primary substrate can be considered negligible. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

It is possible to bind enzymes to an insoluble matrix by a variety of methods and still retain their catalytic activity. The reusable nature of immobilized enzymes can significantly reduce costs and provides a convenient source of enzymes for performing substrate assays. Such preparations often show a greater stability and reduced inhibition effects than do soluble enzymes, although occasionally optimum pH values may be altered slightly. 

In order to improve the usability of enzymes, immobilization matrices have been proposed with both environmental decontamination as well as personal detoxification in mind. Effective immobilization methods allow for the preparation of an immobilized enzyme that retains most of its native activity, maintains high operational stability as well as high storage stability. Recent advances in material synthesis using enzymes have allowed the preparation of a variety of bioplastics and enzyme-polymer composites, which involve the incorporation of the enzyme material directly into the polymer. Enzymes stabilized in this way maintain considerable stability under normally denaturing conditions [21]. A number of methods have been used to prepare bioplastic or enzyme-polymer composite materials with OP-degrading enzymes. Drevon Russel described the incorporation... 

Adsorption on solid matrices represents a quite simple and inexpensive method for enzyme immobilization. Enzyme dispersion is improved, reducing the diffusion limitations and favoring the accessibility of substrate to the enzyme [12]. On the other hand, because of the weak binding, the system can suffer from catalyst leaching, and there is little stabilization of the enzyme. The most common carriers... 

The stability of covalent bonds formed in method A have been tested by washing off the immobilized enzyme with detergents.40 This study indicated that glutaraldehyde reacts quickly with the APTS surface with the formation of stable bonds. The enzymes adsorb to the activated surface, with the majority being adsorbed within the first minute. Stable covalent bonds are formed between the glutaraldehyde and the enzyme. 

Each method of immobilization has specific limitations37, and it is necessary to find an appropriate procedure for any particular enzyme and an application that is simple, not too expensive, and leads to an immobilized enzyme with good retention of activity and good stability. 

No matter which method is selected for immobilization, two essential needs should be balanced, the catalytic needs (expressed as productivity, STY, stability and selectivity) and the noncatalytic needs (e.g., separation, control, down-streaming process) that are required by a given application. When both needs are satisfied, the immobilized enzyme can be labeled as robust [88]. 

The following new trends in enzymatic synthesis can be delineated the development of new enzymatic reactions enzyme immobilization and stabilization the use of organic solvents and two phase systems site-directed mutagenesis chemical modification of enzymes antibody catalysis catalysis by RNA and DNA de novo design ofbiocatalists employment of recombinant DNA for production of enzymes and use computational and combinatorial methods... 

When constructing biosensors, which are to be used continuously in vivo or in situ, maintaining sensor efficiency while increasing sensor lifetime are major issues to be addressed. Researchers have attempted various methods to prevent enzyme inactivation and maintain a high density of redox mediators at the sensor surface. Use of hydrogels, sol-gel systems, PEI and carbon paste matrices to stabilize enzymes and redox polymers was mentioned in previous sections. Another alternative is to use conductive polymers such as polypyrrole [123-127], polythiophene [78,79] or polyaniline [128] to immobilize enzymes and mediators through either covalent bonding or entrapment in the polymer matrix. Application to various enzyme biosensors has been tested. 

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme s catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification. 

Another method of following the rate of urea hydrolysis is based on a specific-ion electrode for ammonium ions (see Section 21D). Here, the production of NH4 is monitored potentiometrically and is used to obtain the reaction rate. In yet another approach, the urease can be immobilized on the surface of a pH electrode and the rate of change of pH monitored. Many enzymes have now been immobilized onto supports such as gels, membranes, tubing walls, glass beads, polymers, and thin films. Immobilized enzymes often show enhanced stability over their soluble counterparts. In addition, they can be reused often for hundreds or thousands of analyses. 

Due to their limited stability, the use of immobilized enzymes might be problematic for monitoring enzyme production (e. g., urease for arginase monitoring). Optical methods which do not need immobilized biocompounds should be more profitable in long-term procedures. Nevertheless, special dyes or substrates with varying optical characteristics after an enzymatic conversion must have at one s disposal. 

A variety of physical and chemical methods have evolved for immobilizing enzymes on or within solid supports. Kennedy and Cabral employed a variation of the scheme in Fig. 1 to classify techniques for immobilization of enzymes.f Judicious choice of the support is essential not only for the stability of immobilized enzymes, but also for the operational characteristics of the device containing the immobilized enzyme and the economic viability of the intended application. The discussion below and the information in Table 1 indicate some of the criteria employed in selecting a mode of immobilization. 

As far as manufacturing costs are concerned the yield of immobilized enzyme activity is mostly determined by the immobilization method and the amoimt of soluble enzyme used. Under process conditions, the resulting activity may be further reduced by mass transfer effects. More precisely, the yield of enzyme activity after immobilization depends not only on losses caused by the binding procedure but may be further reduced as a result of diminished availability of enzyme molecules within pores or from slowly diffusing substrate molecules. Such limitations, summarized as mass transfer effects, lead to lowered efficiency. On the other hand, improved stability under working conditions may compensate for such drawbacks, resulting in an overall benefit. Altogether, these interactions are a measure of productivity or of enzyme consumption, for example, expressed as enzyme units per kg of product. If we replace enzyme units by enzyme costs we obtain the essential product related costs, for example, in US per kg of product. 

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