作者：郝军莉, 章英, 杨天兵, 王婷, 杨宇, 王丹
作者单位：成都医学院生物科学与技术学院, 四川 成都 610065
关键词：羰基还原酶;基因工程菌;不对称合成;产酶条件;转化条件
摘要：
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，（NH4）2SO4 5 g/L、NaCl 10 g/L，氨苄青霉素50 μg/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。
Optimization of enzyme production and asymmetric synthesis of (R)-phenylethyl alcohol by carbonyl reductase genetic engineering bacteria
HAO Junli, ZHANG Ying, YANG Tianbing, WANG Ting, YANG Yu, WANG Dan
College of Bioscience and Technology, Chengdu Medical College, Chendu 610065, China
Abstract: 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.
Keywords: carbonyl reductase;genetic engineering bacteria;asymmetric synthesis;enzyme production conditions;transformation conditions
2019, 45(4):73-79 收稿日期: 2018-12-30;收到修改稿日期: 2019-01-27
基金项目: 四川省科技厅科技支撑项目（No.2016GZ0364）；四川省教育厅科研项目（16ZA0287，17ZA0102）；四川省卫生厅项目（18PJ586，16PJ103）；成都医学院科研创新团队项目（CYTD16-04）
作者简介: 郝军莉(1985-),女,河南开封市人,副教授,博士,研究方向为分子生物学
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