Toshisuke Sasakura

Preparation of a new thermo‐responsive adsorbent with maltose as a ligand and its application to affinity precipitation

A thermo‐responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation. Among the thermo‐responsive polymers synthesized as carriers for adsorbent, poly(N‐acryloylpiperidine)–cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4°C, at which its solubility exhibits a sharp change. Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine–borane as a reducing reagent. This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4°C and was completely insoluble above 8°C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4°C, precipitation of the complex at 10°C, desorption by adding the desorption reagent at 4°C, and recovery of a target protein at 10°C. In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl‐α‐D‐mannopyranoside as a desorption reagent. In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance. Moreover, when pAPM was used for the purification of thermolabile α‐glucosidase from the cell‐free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206‐fold higher than that of the cell‐free extract without thermal deactivation of the enzyme.

Hydrolysis of starchy materials by repeated use of an amylase immobilized on a novel thermo-responsive polymer

Abstract A copolymer of methacrylic acid (MAA) and N -isopropyl acrylamide (NIPAM) was used as a novel, reversibly soluble-insoluble support whose solubility changes depending on the temperature of the solution. Amylase (Dabiase K-27) immobilized covalently on the thermo-responsive polymer showed good solubility response: the immobilized enzyme (D-MN) was in a soluble state below 32°C, but insoluble above 42°C. D-MN in a soluble state has a high specific activity for the hydrolysis of soluble or uncooked starch. The solubility response of D-NM to changes in the temperature of the solution was more sensitive when 0.5% NaCl was added to a buffer solution (pH 4.5) with D-MN than in the buffer solution without NaCl. D-MN was used successively for repeated hydrolysis reactions of soluble and uncooked starches, in which D-MN was insolubilized either by changing the temperature of the reaction mixture from 30°C to 36°C with 0.5% NaCl or by adjusting the NaCl concentration of the reaction mixture from 0% to 1% at 30°C. In the repeated hydrolysis, glucose was produced successively from the soluble and uncooked starches, and D-MN could be repeatedly used after being recovered from the reaction product by centrifugation at the end of each batchwise hydrolysis.

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

Abstract 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°C, at which its solubility exhibits a sharp change. The CT of GNM-75 is 10°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°C, was completely insoluble above 54°C and could be recovered efficiently by centrifugation. Moreover, the CT of the immobilized thermolysin was decreased by about 4.6°C for each 20 g/l increase in the ammonium sulfate concentration of the mixture. The specific activity of the immobilized thermolysin at 40°C was higher than that of thermolysin immobilized on a conventional thermo-responsive polymer (GMA-NIPAm). For its repeated use in hydrolysis of α-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°C to 55°C, or by adjustment of the ammonium sulfate concentration of the mixture from 0 g/l to 60 g/l at 40°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.

Repeated utilization of β-glucosidase immobilized on a reversibly soluble-insoluble polymer for hydrolysis of phloridzin as a model reaction producing a water-insoluble product

Abstract β-Glucosidase immobilized on a reversibly soluble-insoluble polymer, G-AS, was utilized for the hydrolysis of phloridzin. The solubility of the immobilized enzyme was dependent on pH and temperature of the solution: its responsive ranges was between pH 4.0 and 5.0 at 50°C and between pH 3.5 and 4.5 at 4°C. Soluble G-AS had a higher specific activity toward phloridzin than that of the conventional enzyme immobilized on a water-insoluble carrier. For the hydrolysis of phloridzin at a concentration of 5 g/ l (the reaction produces a water-insoluble product from a water-soluble substrate), G-AS was used repeatedly and was easily separated by centrifugation from the reaction mixture containing a water-insoluble product. In addition, for the hydrolysis of phloridzin at a concentration of 50 g/ l (the reaction produces a water-insoluble product from a slightly water-soluble substrate), G-AS was also used repeatedly and was separated for reuse from the mixture of unreacted phloridzin, glucose, and phloretin. The reaction method using such a reversibly soluble-insoluble enzyme in a heterogeneous reaction with a water-insoluble product is a potential procedure for repeatedly utilizing the enzyme and separating the water-insoluble product from the suspended mixture.