Preparation of immobilized enzyme

Figure 13. Preparation of immobilized enzymes with different solubilities in aqueous solutions and organic solvents. Procedure A mixture of an enzyme (3 mg) and the polymer (10 mg) was incubated at pH 7.5 for 20 min. Ammonium phosphate (0.1 M, pH 7, 1 mL) was then added to react with the remaining active ester. After 20 min, the solution was ready for use, or lyophilization to give the immobilized enzyme as a powder to be used for reaction in organic solvents. Each gram of the polymer contains approximately 0.7 mmol of the active ester.

Although the unique properties of enzymes have long been known, the history of bioelectrocatalysis is rather short the first research into the use of enzymes as catalysts dates back to the 1970s. Until quite recently enzymes had not been used in electrocatalysis for a number of reasons the absence of pure enzyme preparations, instability of enzymes, impossibility of multiple application. It has become possible to overcome these difficulties due to improvements in methods for isolation and purification of enzymes, as well as preparation of immobilized enzymes. By way of immobilization it is possible to raise the stability of protein macromolecules, converting an enzyme into a heterogeneous state while its activity is either fully preserved or reduced insignificantly. 

Many preparations of immobilized enzymes and of enzymes coupled to other macromolecules to give water-soluble or -insoluble forms have been reported during the past year. These derivatives and their uses are summarized in Table y 736-825 Immobilized enzymes are referred to in this Table, whether they are enzymatically active or not. 

Kondo, A. and Teshima, T., Preparation of immobilized enzyme with high-activity using affinity tag based on protein-a and protein-G, BiotechnoL Bioeng., 46, 421, 1995. 

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. 

Scheme 2.74 Rapid preparation of GABA analogs using a combination of immobilized enzymes and supported reagents.

Table VII gives a list of nucleotide-sugars prepared with immobilized enzymes.

Gal-(1— 3)-D-GalNAc. By using 0.7 U of STB, as a soluble preparation readily obtained from 300 g of porcine liver, the sialylation of /J-D-Gal-(1 — 3)-d-G1cNAc was performed on a one-mmol scale and sialylated trisaccharide 65 was obtained in 21 % isolated yield. In this respect, the purification of reaction mixtures is still troublesome, especially because of the presence of Triton X-100 from our experience, the use of immobilized enzymes, eliminating the need for detergent, greatly facilitates the purification procedure. 

Carbohydrates are used increasingly for the synthesis of antibiotics and their analogues. This subject was discussed by J. G. Moffat and S. Umezawa in London (1978) and by C.-H. Wong in Hamburg (2000). Enzymes came into more extensive use S. David discussed the use of immobilized enzymes in preparative carbohydrate chemistry (Ithaca 1986) and C.-H. Wong further developed this subject in Paris (1992). The mechanism of the action of enzymes was discussed by S. G. Withers in his Whistler Award lecture in Cairns (2002). 

This study on the immobilization of glucose oxidase and the characterization of its activity has demonstrated that a bioactive interface material may be prepared from derivatized plasma polymerized films. UV/Visible spectrophotometric analysis indicated that washed GOx-PPNVP/PEUU (2.4 cm2) had activity approximately equivalent to that of 13.4 nM GOx in 50 mM sodium acetate with a specific activity of 32.0 U/mg at pH 5.1 and room temperature. A sandwich-type thin-layer electrochemical cell was also used to qualitatively demonstrate the activity of 13.4 nM glucose oxidase under the same conditions. A quantitatively low specific activity value of 4.34 U/mg was obtained for the same enzyme solution by monitoring the hydrogen peroxide oxidation current using cyclic voltammetry. Incorporation of GOx-PPNVP/PEUU into the thin-layer allowed for the detection of immobilized enzyme activity in 0.2 M sodium phosphate (pH 5.2) at room temperature. 

For various reasons, it was only in the 1970s that enzymes began to be used as bioelectrochemical catalysts. Some of the reasons were difficulty in preparation of pure enzymes, their instability, and the lack of multiple applications. These problems have been largely overcome, and better purification methods and enzyme immobilization methods on electrode surfaces have been developed. 

The three enzyme membrane deposition methods can be adapted for the preparation of different enzyme-immobilized membranes on a single FET chip simply by repeating the cycle of coating, irradiation, and development procedures in the cases of the first and third methods, and by the injection of a different enzyme-immobilizing solution into a different micropool in the case of the second method. 

Protein-coated micro-crystals (PCMC) are straightforwardly prepared forms of immobilized enzymes. An aqueous solution of the enzyme and a salt such as potassium sulfate is made and a water miscible solvent is added. The salt precipitates and the enzyme forms a layer on top of the micro-crystals. It is thus readily accessible. Given the direct interaction of the protein with the growing crystal of... 

The immobilized enzyme was removed from the tube and rinsed with D.I. water, followed sequentially by 2N urea solution, 2N NaCl solution and tris buffer, pH 7.5, containing 0.2% sodium azide and 20 mM CaClg, then stored at 4 C. About 0.18-0.2 g of immobilized enzyme beads were used to slurry pack stainless steel 10 cm x 2.1 mm column reactors. Complete details of enzyme preparation and assay for activity are described elsewhere [12],... 

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