Immobilization of α-Amylase to Temperature-Responsive Polymers by Single or Multiple Point Attachments

Abstract

Temperature-responsive N-isopropylacrylamide (NIPAAm) polymer (PNIPAAm) with a free carboxyl functional end group and a copolymer (NIPNAS) of NIPAAm and N-acryloxysuccinimide (NAS) were synthesized and used for immobilization of α-amylase. The enzyme forms covalent bonds with the former polymer by single point attachment and with the latter polymer by multiple point attachment. Such a difference influences the enzyme activity and properties of the immobilized enzymes. The polymers are temperature-sensitive with lower critical solution temperatures (LCST) of 34·7 and 36·0°C for NIPNAS and PNIPAAm, respectively. The immobilized enzyme exhibited an LCST of 35·5°C for NIPNAS-amylase and 37·1°C for PNIPAAm-amylase. They precipitated and flocculated in aqueous solution above the LCST and redissolved when cooled below that temperature. The activity of the immobilized enzyme depended on the pH of the coupling buffer, with 8·0 being the optimum value. The specific activities of the immobilized enzymes were 87% and 108% compared with that of free enzyme with soluble starch as the substrate for NIPNAS-amylase and PNIPAAm-amylase, respectively. By characterizing the properties of the immobilized enzymes and comparing with those of free enzyme, no diffusion limitation of substrate was found for the immobilized enzymes and they are more thermal stable than the free enzyme. Within the two immobilized enzymes, NIPNAS-amylase showed better thermal stability and reusability. Repeated batch hydrolysis of soluble starch can be carried out efficiently with the immobilized enzymes by intermittent thermal precipitation and recycle of the enzyme. © 1997 SCI.