Synthesis and characterization of thermally reversible bioconjugates composed of α-chymotrypsin and poly(N-isopropylacrylamide-co-acrylamido-2-deoxy-D-glucose)

The enzyme α-chymotrypsin was chemically conjugated to a series of temperature-sensitive copolymers composed of N-isopropylacrylamide and acrylamido-2-deoxy-D-glucose in different molar ratios. The C-C bond of two adjacent hydroxyl groups in a glucose ring moiety in the copolymer backbone was oxidized to yield two aldehyde groups, to which primary amine groups present in α-chymotrypsin were reacted via a Schiff-base linkage that was subsequently reduced. The polymer-enzyme conjugates were purified by dialysis and ultrafiltration. The conjugates dissolved in aqueous solution exhibited an array of lower critical solution temperatures depending on the amount of acrylamido-2-deoxy-D-glucose in the copolymer backbone. The enzyme conjugates were characterized with respect to survival activity after conjugation, the extent of conjugation, K(m) values, and thermal bioseparation efficiency. A stepwise thermal cycling operation below and above the lower critical solution temperatures induced reversible precipitation-solubilization behaviors of the enzyme conjugates in the aqueous solution. These thermally precipitative enzyme-polymer adducts could potentially be reused for many batch cycles of enzyme reactions by a simple one-step bioseparation process based on a cyclic operation of heating and cooling. Copyright (C) 1999 Elsevier Science Inc.