羰基还原酶基因工程菌产酶和不对称合成(R)-苯乙醇的条件优化

Optimization of enzyme production and asymmetric synthesis of (R)-phenylethyl alcohol by carbonyl reductase genetic engineering bacteria

以羰基还原酶基因工程菌全细胞为催化剂,探讨不对称合成(R)-苯乙醇的各种影响因素,优化其产酶和转化条件。结果表明,优化后的发酵培养基为甘油10 g/L、蛋白胨10 g/L、酵母粉5 g/L,(NH_4)_2SO_4 5 g/L、NaCl 10 g/L,氨苄青霉素50μg/mL,pH 7.2;诱导剂IPTG的浓度为0.25 mmol/L,诱导时间为20 h,诱导温度为18℃,此条件下测得基因工程菌粗酶活最高为6.65 U/mL。建立的最佳转化条件:羰基还原酶基因工程菌发酵20 h后的细胞浓度为0.3 g/mL,转化体系初始pH为7.0,温度为37℃,辅助底物异丙醇浓度为10%,底物终浓度为60 mmol/L,转化时间为24 h。此时底物转化率最高可达98.88%,产物对映体过量值(e.e.值)为99.43%。反应体系扩增至3 000 mL后,产物e.e.值仍保持在99.0%左右,底物转化率可为92%以上,(R)-苯乙醇的产量也可达到6.67 g/L。

The carbonyl reductase genetic engineering bacteria whole cells were used as catalysts to investigate various influencing factors of asymmetric synthesis of (R)-phenylethyl alcohol in order to establish optimal enzyme production and transformation conditions. The results showed that the optimized fermentation medium was glycerol 10 g/L, peptone 10 g/L, yeast powder 5 g/L,(NH4)2SO4 5 g/L, NaCl 10 g/L, ampicillin 50 μg/mL, pH 7.2, and the concentration of IPTG was 0.25 mmol/L, the induction time was 20 h, the induction temperature was 18 ℃. Under these conditions, the crude enzyme activity of carbonyl reductase genetic engineering bacteria was up to 6.65 U/mL. The optimal transformation conditions were as follows: the concentration of carbonyl reductase genetic engineering bacteria cells fermented for 20 h was 0.3 g/mL, the initial pH was 7.0, the reaction temperature was 37 ℃, the isopropanol concentration as the auxiliary substrate was 10%, the final concentration of the substrate was 60 mmol/L, and the conversion time was 24 hours. At this time, the conversion could reach 98.88% with enantiomeric excess value of produce 99.43%. After the reaction system was amplified to 3 000 mL, the e.e. value of product remained at about 99.0%, the conversion was over 92%, and the yield of (R)-phenylethyl alcohol reached 6.67 g/L.