PREPARATION OF A NOVEL THERMORESPONSIVE POLYMER AND ITS USE AS A CARRIER FOR IMMOBILIZATION OF THERMOLYSIN

A copolymer (GNM) composed of three monomers, glycidyl methacrylate (G MA), N-isopropyl acrylamide (NIPAm), and methacrylamide (MAm), was pre pared as a new thermo-responsive polymer for immobilization of an enzy me which has a maximal activity at high temperature. The polymer (GNM- 75) prepared at the mole percentage ratio of GMA:NIPAm:MAm=15:77.5:7.5 has a critical temperature (CT) of around 42 degrees C, at which its solubility exhibits a sharp change. The CT of GNM-75 is 10 degrees C h igher than those of poly(NIPAm) and poly(GMA/NIPAm) reported previousl y [Hoshino K. et al.: J. Chem. Eng. Jpn., 20, 569-574, 1992]. Thermoly sin was covalently coupled with GNM-75 at pH 10.5. The thermolysin imm obilized on GNM-75 showed a good solubility response: the immobilized thermolysin in the buffer solution (pH 8.0) became soluble upon decrea se of the temperature of the buffer solution to below 44 degrees C, wa s completely insoluble about 54 degrees C and could be recovered effic iently by centrifugation. Moreover, the CT of the immobilized thermoly sin was decreased by about 4.6 degrees C for each 20 g/l increase in t he ammonium sulfate concentration of the mixture. The specific activit y of the immobilized thermolysin at 40 degrees C was higher than that of thermolysin immobilized on a conventional thermo-responsive polymer (GMA-NIPAm). For its repeated use in hydrolysis of alpha-casein, the immobilized thermolysin was recovered from the reaction mixture, by tw o methods. When the reaction was finished, the immobilized thermolysin was insolubilized either by elevation of the temperature of the mixtu re from 40 degrees C to 55 degrees C, or by adjustment of the ammonium sulfate concentration of the mixture from 0 g/l to 60 g/l at 40 degre es C. The thermo-responsive polymer possessing a relatively high CT wa s found to be useful as a carrier for immobilization of an enzyme whic h has a maximal activity at high temperature such as thermolysin.