Polyacrylamide immobilized enzyme

Immobilized enzymes are becoming increasingly important in commercial processes. In this experiment, students will trap molecules of the enzyme horseradish peroxidase within a polyacrylamide gel matrix. The reaction kinetics and thermal stability of the immobilized enzyme will be measured. This experiment introduces students to the use of enzymes in biotechnology. 

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 ... 

Fumarase. The development and use of this immobilized enzyme by Tanabe Seiyaku for production of L-malic acid is very similar to that of aspartase ( 3). Lysed Brevibacterium ammoniagenes or B. flavin cells are treated with bile acid to destroy enzymatic activity which converts fumarate to succinate. As with aspartase, the cells can be immobilized in polyacrylamide or k-carrageenan gels. Using a substrate stream of 1 M sodium fumarate at pH 7.0 and 37°C, L-malic acid of high purity has been produced since 1974 by a continuous, automated process (3,39) for example, using a 1000-L fixed-bed bioreactor, 42.2 kg L-malic acid per hour was produced continuously for 6 months. 

Examples of the use of immobilized enzymes in food processing and analysis have been listed by Olson and Richardson (1974) and Hultin (1983). L-aspartic acid and L-malic acid are produced by using enzymes contained in whole microorganisms that are immobilized in a polyacrylamide gel. The enzyme aspartase from Escherichia coli is used for the production of aspartic acid. Fumarase from Brevibacterium ammoni-agenes is used for L-malic acid production. 

Fig. 6. Kinetics of immobilization of glutaryl-7-ACA-acylase on epoxy-activated polymethacrylate. The Gl-7-ACA-acylase was incubated with the epoxy-activated carrier. At definite times aliquots were taken from the reaction suspension. Supernatant and carrier-fixed enzyme were separated by centrifugation. The carrier-fixed enzyme was washed with water to remove non-covalently linked enzyme. The activities of the immobilized enzyme and supernatant were determined (5 mM potassium phosphate buffer pH 8,37°C, 2% glutaryl-7-amino cepha-losporanic acid, pH-stat 8.0). Simultaneously, an aliquot of carrier-fixed enzyme was boiled in sodium dodecylsulfate (SDS)/glycine buffer and the supernatant was subjected to SDS-polyacrylamide electrophoresis (see insert from left to right lane 1 Carrier-fixed enzyme, 2 h lane 2 Carrier-fixed enzyme, 4 h lane 3 Carrier-fixed enzyme, 6 h lane 4 Carrier-fixed enzyme, 21 h lane 5 Carrier-fixed enzyme, 69 h lane 6 Dialyzed enzyme lane 7 Supernatant, 2 h lane 8 Supernatant, 21 h lane 9 Supernatant, 69 h lane 10 Molecular weight calibration markers)...

Transformations with immobilized enzymes or cells Often the stability of the biocatalyst can be increased by immobilization and many different enzymes and cells have been immobilized by a variety of different methods. The most popular method for the fixation of whole cells is entrapment or encapsulation with calcium alginate. Other natural gels e.g., carrageenan, collagen, chemically-modified natural polymers e.g., cellulose acetate and synthetic gels and polymers e.g., polyacrylamide or polyhydroxyethylmethacrylate can also be used for this type of immobilization. 

Immobilization can be achieved by adsorption or covalent fixation of the biocatalyst to a solid support (e.g. surface-modified polymer or glass beads), by entrapment or by encapsulation in gel beads (e.g., agarose, polyacrylamide, alginate, etc.). Hundreds of immobilization methods have been described and reviewed in the literature [83-89], but only a limited set of methods has found real technical applications. The first large-scale applications of immobilized enzymes were established for the enantioseparation of D- and L-amino acids by Chib-ata, Tosa and co-workers at Tanabe Seiyaku Company. The Japanese achievements in the large-scale application of immobilized systems are very well documented in an excellent multi-author publication edited by Tanaka, Tosa and Kobayashi [90] (see also section 7). Some enzyme suppliers sell important industrial enzymes not only in the free form (solution or powder) but also immobilized on solid supports. 

In 1969, we [1, 2] succeeded in the industrial application of immobilized enzyme, l.e. immobilized amlnoacylase, for the continuous production of L-amino acids from acetyl-DL-amino acids. This is the first industrial application of immobilized enzymes in the world. Since then we [3, 4, 5, 6, T also carried out the Industrial applications of Immobilized microbial cells for the continuous productions of L-aspartic acid and L-malic acid using immobilized microbial cells with polyacrylamide gel. 

The influence of colloidal forces on reactions involving Immobilized enzymes acting on Insoluble substrates has received less attention, yet it appears to offer some clear examples of fundamental phenomena important in enzyme kinetics. Datta examined lysis of Micrococcus Ivsodelktlcus by soluble and (polyacrylamide) immobilized lysozyme. He noted that the decrease in soluble enzyme activity with decreasing ionic strength (Table 1) paralleled the measured decrease in cell lysis measured in flow through a packed bed reactor of Immobilized enzyme. 

Enzyme activity has a certain dependence on temperature, and the general enzyme has an optimal temperature, and immobilized enzyme is no exception. Compared to the solution enzyme, the optimum temperature of immobilized enzyme shows ups and downs. The study found that the aminoacylase was bound to DEAE-cellulose and DEAE-dextran using ion-binding, or embedded in crosslinked polyacrylamide gel. Thus, the immobihzed enzyme was prepared, and its optimum temperature was somewhat higher than that before the immobilization. When aminoacylase was immobilized by iodine acetyl cellulose with covalent binding, its optimum temperature was somewhat lower than that before the immobilization. When the glucose isomerase was boimd to porous resin with covalent binding. 

The use of polymers for the immobilization of enzymes and other bio-logically-active molecules has been discussed. The advantages of polymeric support materials and rules for their selection according to the type of use have been discussed. A review of various types of polymers which can serve as support matrices has been given. They are, e.g., polymeric carbohydrate derivatives, poly(allyl carbonate) and poly(allyl alcohol), polymers of acrylamidosalicylic acids, polyacrylamide derivatives, etc. Some examples of the use of immobilized enzymes and other biologically-active molecules were mentioned. 

Entrapment in polyacrylamide gel Active immobilized enzyme study of the characteristics and overall reaction rate of unbuffered gel-immobilized urease particles 817... 

Entrapment in polyacrylamide gel Active immobilized enzyme theoretical treatment of and experimental results for the use of the immobilized enzyme in a packed-bed differential recycle reactor 818... 

Gluconobacter melanogenus cells Entrapment in a polyacrylamide gel Active immobilized enzyme for the 279 ... 

Monophenol mono-oxygenase 1.14.18.1 Entrapment in polyacrylamide gels during polymeriza- Active immobilized enzyme 368... 

Nitrate reductase 1.7.99.4 Entrapment in polyacrylamide gels cast around Active immobilized enzyme used 369... 

Other applications of the technique include the use of immobilized glucose isomerase for the enrichment in fructose of corn starch hydrolysates and the use of immobilized pectinases for the clarification of fruit juice and wine. Immobilized enzymes have also been used as sensors for particular substrates. For example, a glucose-specific electrode has been developed which consists of glucose oxidase (page 94) entrapped in a polyacrylamide gel which is layered over a conventional polarographic oxygen electrode. 

Archaebacterial (l-galactosidases include that from Caldariella acidophila (i.e. Sulfolobus solfataricus) with an optimum pH of 5.0 [274]. The stability properties of the enzyme were retained on immobilization of the organism in polyacrylamide gel [275]. In a recent paper [276] describing the properties of a more highly purified preparation of the enzyme, however, the pH optimum of the enzyme was determined to be pH 6.5 (in sodium phosphate buffer, pH measured at room temperature) and its thermostability was apparently some twenty times lower at 85 °C than the more crude preparation or the immobilized enzyme. Two other archaebacterial ]3-galactosidases, one from the Desulfurococcus strain Tok 12 S.l and the other from Thermococcus strain ANl have been shown to be extremely thermostable the former being a possible two component system and the latter a single enzyme [277]. 

A model for the amperometric enzyme electrode, obtained by digital simulation, has been applied to D-glucose oxidase immobilized within polyacrylamide gel. D-Glucose oxidase was selected for screening a number of inorganic supports as matrices for immobilization. The activity of the immobilized enzyme on matrices of silica-alumina (with or without nickel), or silicon carbide, or Kieselguhr depended on the size and nature of the supporting particles. 

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