Immobilization of α‐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 α‐amylase from Bacillus licheniformis for the hydrolysis of starch. α‐Amylase was covalently immobilized on PVA‐coated poly(tetrafluoroethylene‐hexafluoropropylene) (PVA‐FEP) by covalent coupling with the use of p‐β‐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 α‐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 α‐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 α‐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 °C. It is concluded that PVA‐coated solid perfluorocarbon is a highly promising support for use in immobilized enzyme engineering.