Enzyme membrane, preparation

Enzyme containing Nation membranes prepared according to the proposed protocol have shown high specific activity and stability of immobilized glucose oxidase. As expected, the simplicity of preparation provided high reproducibility. When the same casting solution is used, the maximum deviation in membrane activity is <2%. This, however, is also the precision limit for kinetic investigations. 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Perform the necessary controls, i.e., withhold the substrate in the reaction mixture, boil the membrane preparation, and then carry out the enzyme reaction. Adapted in part from Morre et al. (22). 

Enzyme membranes can be prepared by adsorbing the enzyme on the surface of a suitable native or synthetic membrane, or, in the case of membranes with large pores, by impregnating the whole membrane with enzyme. The resulting enzyme membrane can be stabilized by covalently cross-linking the adsorbed protein with a suitable bifunctional reagent (8 ). 

In spite of the theoretical interest in enzyme membranes, they have so far not been used in industry and their use in the clinic and in the laboratory is rather limited. However, as techniques for the preparation and stabilization of immobilized complex enzyme systems develop, one can expect to see an increase in the number of cases in which permeable and impermeable enzyme membranes will be used advantageously. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

PVA/acrylamide blend membranes prepared on cheese cloth support by y-irradiation induced free radical polymerization can be used for urease entrapment. The enzyme urease is entrapped in the membrane during polymerization process and using glutaraldehyde as cross-linking agent. The main advantage of this blend to this process is that it can be reused a number of times without significant loss of urease activity [292],... 

Figure 2. Electrophoretic profiles of glucan synthase fractions purified from celery. Proteins were transferred to nitrocellulose and stained by colloidal gold. Symbols PM, plasma membranes SOL, CHAPS-solubilized RC, reconstituted glucan synthase preparations. Plasma membranes were isolated by two-phase partitioning. Specific activities of the three membrane preparations were 398, 1355, and 626 nmol/min/mg, respectively. The low specific activity of RC relative to SOL may be a reflection of enzyme instability following the gel filtration step.

The gram-scale preparation of rare sugars by E. coli transketolase was demonstrated successfully for (S)-erythrulose from glycolaldehyde and hydroxypyruvate in an enzyme membrane reactor which allowed the continuous production of (S)-erythrulose with high conversion and a space-time yield of 45 g L" d was reached [12]. 

The continuous high-pressure enzyme membrane reactor [30] is shown in Figure 9.2-4. The membrane with 35 mm diameter is placed between two sintered plates and fitted in the reactor. A certain amount of the catalyst (hydrated enzyme preparation) is put in the reactor which is electrically heated, with a heating jacket, to constant temperature. The substrates and the gas are pumped into the membrane reactor with the high-pressure pump. The products and unreacted reactants are collected in the separator. The catalyst remains in the reactor (behind the membrane). 

By reversing the ratio of acceptor/donor, namely by using D-arabinose in a 25-fold excess, the percentage of KDO, the product of the inverted enzyme stereoselectivity, could be increased up to 83% [48]. The reason for that is still unclear, but this interesting result was exploited in the preparation of KDO 2, which was achieved using the enzyme membrane reactor technique [48]. Compound 2 could be separated from its epimer according to the procedure previously described [16]. 

The ADSA Committee on Milk Protein Nomenclature (Eigel et al. 1984) presented a tentative nomenclature for the new enzyme membrane proteins. While the primary structures of these proteins have not been established, sufficient information exists to obtain an operational definition. The total protein complement of the membrane as observed is dependent upon the past history of the membrane from its formation to its analysis. Both the temperature and the time of storage before analysis can alter the membrane composition and physical state (Wooding 1971). In addition, plasmin has been shown to be associated with preparations of the membrane, and proteolytic products of the membrane protein have been observed in milk (Hoffman et al. 1979 Kanno and Yamauchi 1979). Therefore, one should use fresh warm raw milk for the study of the native MFGM protein. 

Synthetic ribitol phosphate polymer, unlike teichoic acid in a wall, is readily extracted from the particulate enzyme or membrane preparation by treatment with phenol.18 Similarly, teichoic acid synthesized by intact cells in the presence of penicillin is only loosely attached to the wall,111 and it may be significant that, in each case, synthesis of teichoic acid has occurred without the simultaneous synthesis of glycosaminopeptide. It is now known that, in the normal wall, teichoic acid and glycosaminopeptide are attached to each other, and it has been suggested that the low activity of cell-free synthetase is due to the absence of suitable acceptor molecules of glycosaminopeptide. This possibility could account for the ease of removal of teichoic acid formed when simultaneous synthesis of glycosaminopeptide was not possible. 

The situation is complicated by the fact that the efficiency of cellulose synthesis in the cell-free system is low, and the same enzyme preparation catalyzes the incorporation of D-glucosyl groups from UDP-Glc into alkali-soluble (1 - 2)-/3-D-glucans.370 A similar process was reported to occur with a membrane preparation of Rhizobium meliloti,371... 

It has been discovered that the enzymic synthesis of cellulose is specifically activated by guanosine 5 -triphosphate in the presence of a protein factor and poly(ethyleneglycol)372 or calcium ions.373 This activation results in a dramatic increase in the rate of synthesis of the polymer. The enzyme, solubilized by treatment of membrane preparation with digitonin,373 retains its regulatory properties, and does not show any requirements in lipids for its activity. 

Initially, the use of urea, dilute alkali, or anionic detergent was suggested415 for solubilization of the enzyme from S. aureus membrane. Nonionic detergents were effective for this purpose with the enzyme from the same micro-organism and from Micrococcus luteus.416,417 Repeated freezing and thawing of a membrane preparation was used in the case of Escherichia co/i.418... 

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