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过程工程学报 ›› 2016, Vol. 16 ›› Issue (3): 488-493.DOI: 10.12034/j.issn.1009-606X.215392

• 生化工程专栏 • 上一篇    下一篇

源于红豆的羰基还原酶在(R)-苯基乙二醇制备中的应用

彭益强 鞠翰雪   

  1. 华侨大学工业生物技术福建省高校重点实验室 华侨大学工业生物技术福建省高校重点实验室
  • 收稿日期:2015-11-25 修回日期:2016-01-04 出版日期:2016-06-20 发布日期:2017-04-28
  • 通讯作者: 彭益强

Application of Red Bean Originated Carbonyl Reductase in the Preparation of (R)-Phenylethanediol

PENG Yi-qiang JU Han-xue   

  1. Provincial Key Laboratory of Industrial Biotechnology, Huaqiao University Provincial Key Laboratory of Industrial Biotechnology, Huaqiao University
  • Received:2015-11-25 Revised:2016-01-04 Online:2016-06-20 Published:2017-04-28
  • Contact: PENG Yi-qiang

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摘要： 采取研磨、离心分离、多层过滤和丙酮沉析等步骤，从红豆中提取具有不对称还原芳香酮能力的羰基还原酶(CR)，经快速分离纯化获得了纯化倍数为5.8倍的红豆源CRrb酶液，考察了其酶学特性，与微生物源CR的酶学特性进行比较，将CRrb与甲酸脱氢酶(FDH)耦合构建CRrb/FDH双酶体系连续催化b-羟基苯乙酮制备(R)-苯基乙二醇. 结果表明，源于红豆的CRrb最适反应pH值为6.0，最适反应温度为45℃，在40~60℃范围内耐热性好于一般微生物源CR. CRrb的米氏常数Km=5.68 mmol/L，最大反应速率Vmax=20.21 μmol/(min×mL)，对底物的亲和力和催化效率比微生物源CR好. 底物较佳耐受浓度为60 mmol/L，较微生物源的CR高. CRrb/FDH酶活比为1:1.5时耦合体系反应效率最佳，批次反应转化1 mol辅酶可获得产物的量由266 mol提高到365 mol.

关键词: 红豆, 羰基还原酶, 甲酸脱氢酶, 辅酶再生, (R)-苯基乙二醇

Abstract: Carbonyl reductasese (CR) with the ability of aromatic ketone asymmetry reduction was extracted from red bean (CRrb) by grinding, centrifigation, filtration and acetone precipitation. CRrb liquid with 5.8 fold increase in purity was achieved by rapid purification, and its enzymatic properties were examined and compared with the microbe origin CR. CRrb was coupled with formate dehydrogenase (FDH) to construct a bi-enzyme system to continuously catalyse the reaction of b-hydroxyacetophenone to (R)-phenylethanediol. The results showed that the optimal reaction pH value of CRrb was 6.0, and the optimal reaction temperature of CRrb was 45℃. It showed higher thermostability between 40~60℃ than the microbe origin CR. The kinetics parameters (Michaelis constant Km and maximum reaction velocity Vmax) of CRrb were 5.68 mmol/L and 20.21 mmol/(min×mL) respectively, which indicate its affinity with the substrate and catalysis efficiency were higher than CR from microbe origin. Under the optimization reaction conditions of coupling system, the substrate tolerance concentration reached 60 mmol/L which was higher than the microbe origin CR. The reaction efficiency of the coupling system was the highest when the enzymatic activity rate of CRrb/FDH was optimized as 1:1.5, and the acquired production mol number when 1 mol cofactor was turned of coupling system batch reaction was improved from 266 mol to 365 mol.

Key words: red bean, carbonyl reductases, formate dehydrogenase, cofactor regeneration, (R)-phenylethanediol

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