Enzymes immobilized, catalyzing

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

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). 

Some non-silica sol-gel materials have also been developed to immobilize bioactive molecules for the construction of biosensors and to synthesize new catalysts for the functional devices. Liu et al. [33] proved that alumina sol-gel was a suitable matrix to improve the immobilization of tyrosinase for detection of trace phenols. Titania is another kind of non-silica material easily obtained from the sol-gel process [34, 35], Luckarift et al. [36] introduced a new method for enzyme immobilization in a bio-mimetic silica support. In this biosilicification process precipitation was catalyzed by the R5 peptide, the repeat unit of the silaffin, which was identified from the diatom Cylindrotheca fusiformis. During the enzyme immobilization in biosilicification the reaction mixture consisted of silicic acid (hydrolyzed tetramethyl orthosilicate) and R5 peptide and enzyme. In the process of precipitation the reaction enzyme was entrapped and nm-sized biosilica-immobilized spheres were formed. Carturan et al. [11] developed a biosil method for the encapsulation of plant and animal cells. 

The potentialities offered by chemical processes catalyzed by enzymes immobilized in a polymeric matrix are obvious and are now successfully utilized in various ways [6, 13, 16, 17, 40, 43,44, 50, 58, 61], This idea was introduced into electroanalytical chemistry by Clark and Lyons [9], who proposed a glucose electrode, with glucose oxidase immobilized between cuprophane membranes and with amperometric determination of the hydrogen peroxide formed by the reaction... 

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Enzymatic Modification of Lipids. The value of enzymes as biosynthetic agents has been recognized for many years, particularly in the field of lipids. Because of the highly selective mode of action and the ability of specific enzymes to catalyze reactions in organic—aqueous interfaces, enzymes are useful in synthetic organic chemistry. To enhance stability and the rates of reaction, immobilized enzymes are often used. 

We conclude that a commercial immobilized lipase from C. antarctica (Novozym 435) was stable in SCC02 for all experimental conditions investigated. Based on the results obtained here and comparison of them with the results obtained by other investigators, it can be concluded that the magnitude of pressure, temperature, decompression rate, and exposure time needed to inactivate the enzyme strongly depends on the nature and the source of enzyme and, primarily, whether the enzyme is in its native or immobilized form. For the purpose of using this enzyme to catalyze the transesterification reaction of vegetable oils in order to produce esters, the results obtained herein are relevant, because the immobilized lipase can be used with low activity loss at typical conditions of temperature and pressure employed in many biotransformations of raw materials. 

In enzyme-catalyzed reactions, parameters such as concentration of water, reaction temperature, pH, enzyme immobilizing agents, and the nature of any solvent may be of great importance. A brief description of each of these factors is given below. 

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