Immobilization of alpha-amylase on poly(vinyl alcohol)-coated perfluoropolymer supports for use in enzyme reactors.

Abstract

The suitability and potential for the use of poly(vinyl alcohol) (PVA)-coated solid perfluoropolymers in immobilized-enzyme engineering have been evaluated by using alpha-amylase from Bacillus licheniformis for the hydrolysis of starch. alpha-Amylase was covalently immobilized on PVA-coated poly(tetrafluoroethylene-hexafluoropropylene) (PVA-FEP) by covalent coupling with the use of p-beta-sulphate-(ethyl sulphonide)-aniline, 2,4,6-trichloro-1,3,5-triazine, 1.1'-carbonyldi-imidazole and 2,2, 2-trifluoroethanesulphonyl chloride activation procedures, and also for comparison with cyanogen bromide-activated Sepharose 4B. In all cases, immobilization greatly improved the thermostability of the alpha-amylase and its resistance to inactivation by 6 M urea. Also the enhancements of enzymic activities with increased temperature were higher for the immobilized enzymes than for the soluble enzyme, and the immobilized alpha-amylases were well suited to the continuous hydrolysis of starch conducted at elevated temperatures. Although the specific activities of the enzymes immobilized on PVA-FEP were lower than for that immobilized to Sepharose 4B, these novel supports showed far superior strength. The enzymes immobilized on PVA-FEP were able to be readily recovered from stirred batch bioreactors for repeated reuse, whereas the enzymes immobilized to Sepharose were fractured and fragmented under similar conditions of stirring. A conventional fixed-bed bioreactor was found to be unsuitable for continuous starch hydrolysis owing to an unacceptable build-up of pressure drop across the bed. However, an expanded bed reactor containing alpha-amylase immobilized on solid PVA-coated perfluorocarbon showed great potential for the continuous hydrolysis of starch. Only 20% of the enzyme activity was lost after use for 3 weeks at 72 degrees C. It is concluded that PVA-coated solid perfluorocarbon is a highly promising support for use in immobilized enzyme engineering.