COLLOIDS AND B SURFACES ELSEVIER Colloids and Surfaces B: Biointerfaces 4 (1995) 267-274 Enzyme immobilization on thermosensitive hydrogel microspheres Toshifumi Shiroya, Nobuko Tamura, Mamoru Yasui, Keiji Fujimoto, Haruma Kawaguchi * Department of Applied Chemistry Faculty of Science and Technology Keio University 3-14-I Hiyoshi, Kouhoku-ku, Yokohama 223, Japan Received 28 June 1994; accepted 18 November 1994 Abstract Precipitation polymerization of N-isopropylacrylamide, acrylamide, and methylenebisacrylamide in water at 70°C resulted in thermosensitive hydrogel microspheres. Carboxyl groups on the microspheres were introduced by hydrolysis, and amino groups by the Hofmann reaction of amide units on the microspheres. Trypsin was immobilized on the carboxylated microspheres using carbodiimide. Phase transitions were detected using a hydrophobic fluorescence probe. The temperatures at which a phase transition occurred were increased by immobilizing enzymes. The enzymatic activity of the immobilized enzymes decreased above the transition temperature. This was attributed to (i) a decrease in the diffusion of substrate; and (ii) entrapment of enzyme in the surface layer of the microspheres. In an attempt to overcome the entrapment, enzymes were immobilized via a hydrophilic spacer (ct-(carboxymethyl)-~o-(carboxymethoxy)-poly(oxy-l,2-ethanediyl), PEO acid) to the microspheres. These enzyme-carrying hydrogel microspheres were found to show an enzymatic activity independent of temperature, even though these conjugates show a phase transition at the lower critical solution temperature. Keywords: Enzyme activity; Lower critical solution temperature; Thermosensitive hydrogel microsphere 1. Introduction Recently, polymers which recognize and respond to external stimuli such as light [1,2], an electric field [3], pH [4] or temperature [-5], have attracted interest. These polymers have been used as supports for stimuli-responsive immobi- lized proteins [5]. Poly(N-isopropylacrylamide) (poly-NIPAM) in an aqueous medium has its lower critical solution temperature (LCST) around 32°C and exhibits a coil-globule trans- ition around this LCST I-6]. Therefore, poly- NIPAM has been investigated as an enzyme * Corresponding author. 0927-7765/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0927-7765(94)01177-X support for the control of enzymatic activity and precipitation separation. Dong and Hoffman used a cross-linked copolymer gel of NIPAM and acrylamide (AAm) with entrapped asparaginase I-7,8]. They have shown that the enzymatic activity was "shut off" when the temperature of the gel was raised above its LCST. For the immobilization of trypsin, microspheres which were prepared by cross-linking polyallylamine- graft-poly-NIPAM with glutaraldehyde in a water-in-oil emulsion have been used by Kitano et al. [9]. The activity of trypsin immobilized onto these microspheres showed a discontinuous temperature dependence caused by the shrinkage of the graft chains above the LCST.