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

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pH值为5.5、酶浓度为1.4mg/mL、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%。

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

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%.