Preparation of a novel thermo-responsive polymer and its use as a carrier for immobilization of thermolysin

A copolymer (GNM) composed of three monomers, glycidyl methacrylate (GMA), N-isopropyl acrylamide (NIPAm), and methacrylamide (MAm), was prepared as a new thermo-responsive polymer for immobilization of an enzyme 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 higher than those of poly(NIPAm) and poly(GMA/NIPAm) reported previously [Hoshino K. et al.: J. Chem. Eng. Jpn., 20, 569-574, 1992]. Thermolysin was covalently coupled with GNM-75 at pH 10.5. The thermolysin immobilized on GNM-75 showed a good solubility response: the immobilized thermolysin in the buffer solution (pH 8.0) became soluble upon decrease of the temperature of the buffer solution to below 44 degrees C, was completely insoluble about 54 degrees C and could be recovered efficiently by centrifugation. Moreover, the CT of the immobilized thermolysin was decreased by about 4.6 degrees C for each 20 g/l increase in the ammonium sulfate concentration of the mixture. The specific activity 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 two methods. When the reaction was finished, the immobilized thermolysin was insolubilized either by elevation of the temperature of the mixture 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 degrees C. The thermo-responsive polymer possessing a relatively high CT was found to be useful as a carrier for immobilization of an enzyme which has a maximal activity at high temperature such as thermolysin.