MEMBRANE IMMOBILIZED GLUCOSE ISOMERASE.

摘要

High molecular weight copolymers of acrylonitrile with m-aminostyrene, p-aminostyrene of 4-vinylpyridine were synthesized by slurry polymerization so as to contain about 5 mole percent of amine or pyridine monomer. The monomer content was determined by ultraviolet absorption and non-aqueous titration of the acid copolymer salt in dimethylformamide. Unsupported ultrafiltration membranes were cast with an average pore radius of 40 to 80 nm. (alpha)-Chymotrypsin (CT), glucose isomerase (GI) or lactate dehydrogenase were immobilized to the membranes after appropriate chemical activation. The most frequently used activation methods were: trichloro-s-triazine on the aryl amine membrane for the coupling of CT; diazotization of the aryl amine membrane for the coupling of GI. The weight loading for CT was 6 to 12 percent with 40 to 65 percent retention of enzymatic specific activity. A purified GI from Streptomyces albus was covalently immobilized with 10 percent weight loading and 50 to 70 percent retention of enzymatic specific activity. Activity of membrane-bound GI was assayed at 70(DEGREES)C in pH 6.8 maleate buffer containing 0.3 M glucose, 7 mM MgSO(,4), and 3 mM CoSO(,4). Fructose formation was determined via cysteine-carbazole assay. A GI reactor containing a single 47 mm diameter membrane disk was operated in a single pass flow mode at a flow rate of 0.5 ml/min and resulted in 10% conversion; up to 20% conversion was observed with higher flow rates. A Co('+2) concentration of 0.4 and 3.0 mM was necessary for optimal thermal stability of soluble and immobilized GI, respectively. The GI-membrane reactor had a half-life of about 150 hours. The K(,m) and the V(,max) for the immobilized GI were 0.25 M and 15 (mu)mole/min-mg, respectively; these are slightly lower than and comparable to, respectively, those for soluble GI. When the flow rate was increased, a maximum level of activity was observed, at which diffusion control of the rate of reaction no longer existed. Diffusional limitations were shown to be insignificant at flow rates greater than or equal to 3 ml/min with a substrate concentration of 0.3 M glucose.