Immobilization of enzymes on poly(vinyl alcohol)‐coated perfluorocarbon supports: comparison of techniques for the immobilization of trypsin and α‐amylase on poly(vinyl alcohol)‐coated solid and liquid perfluorocarbons

Abstract

An investigation of different methods for the immobilization of α‐amylase and trypsin on solid and liquid poly(vinyl alcohol) (PVA)‐coated perfluorocarbon supports has been made. It is found that very reactive PVA‐coated perfluorocarbon supports are produced only via the reactions between the hydroxy groups of PVA adsorbed on perfluorocarbons and 2, 2, 2‐trifluethanesulphonyl chloride (tresyl chloride), 2, 4, 6‐trichloro‐1, 3, 5‐triazine (cyanuric chloride) or p‐β‐sulphate‐(ethyl sulphonide)‐aniline (SESA), a preactivating reagent for diazotization reactions. The amounts of immobilized trypsin (specific activity and percentage recovery of enzyme activity toward casein) on solid PVA‐coated perfluorocarbon activated by tresyl chloride, SESA or cyanuric chloride were 58 mg/g (1200 units/g, 5.8%), 25 mg/g (1100 units/g, 12.4%) or 44 mg/g (1128 units/g, 7.2%) respectively. The amounts of immobilized α‐amylase (specific activity and the recovery of enzyme activity towards starch) on the same supports were 32 mg/g (4000 units/g, 7.2%), 20 mg/g (4940 units/g, 14.2%) or 29 mg/g (2725 units/g, 5.4%) respectively. Most other methods, i.e. epoxy activation (with epichlorohydrin or 1, 4‐butanediol diglycidyl ether), periodate oxidation, glutaraldehyde coupling, 1, 1'‐carbonyldi‐imidazole chemistry, 1, 1′‐carbonyldi‐imidazole chemistry in an oriented fashion, isothiocyanate chemistry and carbodiimide activation, all resulted in lower degrees of activation of PVA‐coated perfluorocarbon supports and thus correspondingly lower specific activities of the immobilized enzymes. Only the methods with SESA or cyanuric chloride are suitable for enzyme immobilization on PVA‐coated liquid perfluorocarbon emulsions. The amounts of immobilized enzymes and the specific activities of the enzymes on liquid PVA‐coated perfluorocarbon emulsion were, however, lower than those on solid PVA‐coated perfluorocarbon owing to the lower surface area of this carrier. The method with SESA seems to be the best for all applications. The addition of (NH4)2SO4 to the immobilized reactions significantly enhanced the activity recovery and the specific activity of the α‐amylase. The immobilized enzymes were stable during storage. The study demonstrates that solid PVA‐coated perfluorocarbon supports are promising carriers for enzyme immobilization.