羰基还原酶突变体高选择性催化不对称还原大位阻羰基化合物

doi:10.7503/cjcu20180214

高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (11): 2438.doi: 10.7503/cjcu20180214

羰基还原酶突变体高选择性催化不对称还原大位阻羰基化合物

梁晨, 聂尧(), 徐岩

江南大学生物工程学院, 工业生物技术教育部重点实验室, 无锡 214122

收稿日期:2018-03-19 出版日期:2018-11-10 发布日期:2018-10-09
作者简介:联系人简介: 聂尧, 男, 博士, 教授, 博士生导师, 主要从事酶工程领域研究. E-mail: ynie@jiangnan.edu.cn
基金资助:
国家自然科学基金(批准号: 21336009, 21676120)、江苏省自然科学基金(批准号: BK20151124)、江苏省六大人才高峰计划项目(批准号: 2015-NY-007)和江苏高校优势学科建设工程项目资助.

Highly Stereoselective Reduction of Bulky Carbonyl Compounds by Carbonyl Reductase Variant^†

LIANG Chen, NIE Yao^*(), XU Yan

School of Biotechnology, Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China

Received:2018-03-19 Online:2018-11-10 Published:2018-10-09
Contact: NIE Yao E-mail:ynie@jiangnan.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.21336009, 21676120), the Natural Science Foundation of Jiangsu Province, China(No.BK20151124), the Program for Advanced Talents within Six Industries of Jiangsu Province, China(No.2015-NY-007) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.

摘要/Abstract

摘要：

以立体选择性羰基还原酶(RCR)的双突变酶RCR-F285A/W286A为对象, 考察了该突变酶对酮酯类和杂环酮类底物的催化性能(包括酶活力和动力学参数)及对映体选择性, 发现其对2-氧代-4-苯基丁酸乙酯(OPBE)、苯甲酰基乙酸乙酯(EBA)和1-苄基-3-吡咯烷酮均具有酶活性, 并且以高光学纯度生成相应手性醇(e.e.>99%). 考察了反应条件对产率最高的OPBE的不对称转化反应的影响, 结果表明, 当pH=7.0, 反应温度30 ℃, 3.5%(体积分数)的异丙醇作助溶剂, 加入1 g/L底物时, 产率达到80.3%. 进一步通过分批补料减弱底物抑制作用, 在10 g/L的底物浓度下, 时空产率为0.062 g·L^-1·h^-1, 为酶法不对称合成(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]的工业应用提供了实验基础.

关键词: 羰基还原酶, 2-氧代-4-苯基丁酸乙酯, 苯甲酰基乙酸乙酯, 1-苄基-3-吡咯烷酮, 不对称转化

Abstract:

RCR-F285A/W286A, the variant of the (R)-specific carbonyl reductase(RCR), was used as the biocatalyst for asymmetric reduction of carbonyl compounds with bulky groups. In this study, ketoesters and heterocyclic ketone, including ethyl 2-oxo-4-phenylbutyrate(OPBE), ethyl benzoylacetate(EBA) and 1-benzyl-3-pyrrolidone, were employed. Catalytic property(including enzyme activity and kinetic parameters) and enantioselectivity of RCR-F285A/W286A towards tested bulky substrates were measured. RCR-F285A/W286A exhibited enzymatic activity towards all of the tested substrates. The corresponding chiral alcohols of the tested substrates acting as important pharmaceutical intermediates were prepared with high optical purity(e.e.>99%). Moreover, the effects of reaction conditions, including pH, temperature, substrate concentration, cosolvents and ratio of the optimum cosolvent, on the asymmetric reduction of OPBE with the highest yield were investigated. The results showed that the yield of 80.3% was obtained at pH=7.0, 30 ℃, 1 g/L substrate and isopropanol with volume fraction of 3.5%. Furthermore, fed-batch strategy was adopted to weaken substrate inhibition with the space-time yield 0.062 g·L^-1·h^-1 at 10 g/L OPBE. This result would provide the experimental basis for the industrial application of the enzyme-mediated asymmetric synthesis of ethyl (R)-2-hydroxy-4-phenylbutanoate.

Key words: Carbonyl reductase, Ethyl 2-oxo-4-phenylbutyrate, Ethyl benzoylacetate, 1-Benzyl-3-pyrrolidone, Asymmetric transformation

TrendMD:

梁晨, 聂尧, 徐岩. 羰基还原酶突变体高选择性催化不对称还原大位阻羰基化合物. 高等学校化学学报, 2018, 39(11): 2438.

LIANG Chen, NIE Yao, XU Yan. Highly Stereoselective Reduction of Bulky Carbonyl Compounds by Carbonyl Reductase Variant^†. Chem. J. Chinese Universities, 2018, 39(11): 2438.

图/表 7

Table 1 Catalytic property of RCR-F285A/W286A towards bulky substrates

Specific activity/ (U·mg^-1)	K_m/ (mmol·L^-1)	k_cat/s^-1	k_cat/K_m/ (L·mmol^-1·s^-1)
1.33±0.1	3.38	0.43	0.13
1.2±0.09	3.89	1.04	0.27
0.36±0.06	8.76	4.35	0.50

Fig.1 Effect of pH on asymmetric reduction of OPBEThe reactions were carried out in 2 mL PBS buffer(0.1 mmol/L, pH=6.0—8.0) or Tris-HCl buffer(0.1 mmol/L, pH=8.5) with addition of 5 mmol/L NADH, 1 g/L OPBE, and 0.5%(volume fraction) isopropanol at 30 ℃, 12 h.

Fig.2 Effect of temperature on asymmetric reduction of OPBEReaction conditions: 5 mmol/L NADH, 1 g/L OPBE, and 0.5%(volume fraction) isopropanol in 2 mL PBS buffer(0.1 mmol/L, pH=7.0), 15—40 ℃, 12 h.

Fig.3 Effect of substrate concentration on asymmetric reduction of OPBEReaction conditions: 5 mmol/L NADH, OPBE(0.5—3 g/L) and 0.5%(volume fraction) isopropanol in 2 mL PBS buffer(0.1 mmol/L, pH=7.0), 30 ℃, 12 h.

Table 2 Effect of cosolvents on asymmetric reduction of OPBE*

Cosolvent	Ethanol	Isopropanol	N-Hexane	Ethyl acetate	DMSO	THF	Toluene	Blank
lgP	-0.24	0.38	3.50	0.68	-1.30	0.49	2.50	—
Yield(%)	15.7	64.79	0.22	3.94	3.54	0.48	0.41	0.21

Fig.4 Effect of isopropanol ratio on asymmetric reduction of OPBEReaction condition: 5 mmol/L NADH, 1 g/L OPBE and isopropanol(0.5%—5.5%, volume fraction) in 2 mL PBS buffer(0.1 mmol/L, pH=7.0), 30 ℃, 12 h.

Fig.5 Time course of asymmetric reduction of OPBE in fed-batch by crude enzymeReaction conditions: 5 mmol/L NADH, 1 g/L OPBE and isopropanol(0.35%, volume fraction) in 100 mL PBS buffer(0.1 mmol/L, pH=7.0), 30 ℃. 1 g/L OPBE dissolved in 350 μL isopropanol was added every 12 h and freeze-dried powder of crude enzyme was added every 30 h.

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羰基还原酶突变体高选择性催化不对称还原大位阻羰基化合物

Highly Stereoselective Reduction of Bulky Carbonyl Compounds by Carbonyl Reductase Variant^†

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羰基还原酶突变体高选择性催化不对称还原大位阻羰基化合物

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