Preparations and properties of temperature-sensitive poly( N-isopropylacrylamide) -chymotrypsin conjugates

A copolymer of N-isopropylacrylamide (NIPAAm) and N-acryloxysuccinimide (NAS) was synthesized and used as a support for the immobilization of α-chymotrypsin. The copolymer is temperature-sensitive with a lower critical solution temperature (LCST) of 33.5°C when synthesized with an initial molar ratio NIPAAm:NAS = 39. The immobilized enzyme exhibited a LCST of 35.5°C, precipitated and flocculated in aqueous solution above the LCST and redissolved when cooled below that temperature. The coupling yields and specific activity of the immobilized enzyme depended on the pH of the coupling buffer and the enzyme to polymer ratio. Specific activities of the immobilized enzyme were 83.5%, 67.1%, and 63.4% compared with those of free enzyme with Suc-Phe-4-NA, casein, and hemoglobin as the substrate, respectively. From kinetic analysis the K_m value and thus the affinity between enzyme and substrate changed after enzyme immobilization, and could be related to the hydrophobicity/hydrophilicity of the substrate and the copolymer. The immobilized enzyme showed enhanced thermal stability compared to free enzyme and had a similar activation energy. It could be recycled with repeated precipitation/dissolution cycles with high enzyme activity.