融合蛋白CR2-GDH固定化及不对称还原制备(S)-4-氯-3-羟基丁酸乙酯

Immobilization of fusion enzyme CR2-GDH and bioreduction of ethyl(S)-4-chloro-3-hydroxybutyrate

比较介孔分子筛材料SBA-15、MCM-41、海藻酸钙、改性二氧化硅4种载体固定化融合蛋白CR2-GDH其酶固载量和酶活回收率,选择SBA-15为固定化载体。研究固定化条件对固定化融合酶量的影响以及固定化酶的稳定性,固定化酶在双相体系催化不对称还原反应。结果表明,在p H值为5.5、酶浓度为1.4mg/m L、反应1h条件下,固定化酶量为27.7mg/g。加入25mmol/L的Ca2+,固定化酶的酶活回收率由58.6%提高到78.1%。与游离酶相比,固定化酶的热稳定性显著提高,40℃条件下酶活回收率提高19.1%。固定化酶水相中反复使用7批次后,剩余活性仍超过30%,具有较好的操作稳定性。与游离酶相比,固定化酶更耐受烷烃类有机溶剂。在水/有机溶剂双相反应体系中,Ca2+/SBA-15固定化酶和游离酶催化相比,产物得率提高23.8%。

Mesoporous molecular sieves SBA-15 was the best one among 4 kinds of carriers in terms of both carrier content and recovery of enzyme activity. The remaining carriers were MCM-41,modified silica,and calcium alginate. Immobilization of fusion enzyme CR2-GDH on SBA-15 was realized,and effect of immobilization conditions on immobilization content and activity,stabilization of immobilized enzyme,asymmetric reduction in biphasic system was investigated. The results showed that adsorption capacity was 27.7mg/g carrier under the condition of pH5.5,enzyme concentration 1.4mg/mL,reaction time of one hour. The recovery of immobilized enzyme activity was increased by near 20% adding metal ion Ca2＋ of 25mmol/L. It was also found that the thermal and operational stabilities of the immobilized enzyme were higher than those of free enzyme. The recovery was increased by 19.1% at the temperature of 40℃. Immobilized enzyme was not more sensitive to organic solvents especially alkanes than free enzyme. In biphasic system,the production yield obtained by Ca2＋/SBA-15 immobilized enzyme was increased by 23.8%.