前手性酮全细胞转化体系中甲酸脱氢酶C-端无序结构与转化效率的关系研究

中国生物工程杂志

2013, Vol. 33

Issue (8): 1-10

研究报告

前手性酮全细胞转化体系中甲酸脱氢酶C-端无序结构与转化效率的关系研究

李炳娟^1,2, 李玉霞¹, 李北平¹, 凌焱¹, 周围¹, 刘刚¹, 张景海², 岳俊杰¹, 陈惠鹏¹

1. 军事医学科学院生物工程研究所北京 100071;
2. 沈阳药科大学沈阳 110016

Study of the Relationship between the Disordered C Terminusin of Formate Dehydrogenase and the Conversion Rate in the Whole-cell Catalysis of Prochiral Acetone

LI Bing-juan^1,2, LI Yu-xia¹, LI Bei-ping¹, LING Yan¹, ZHOU Wei¹, LIU Gang¹, ZHANG Jing-hai², YUE Jun-jie¹, CHEN Hui-peng¹

1. Beijing Institute of Biotechnology, Beijing 100071, China;
2. Department of Life Science, ShenYang Pharmaceutical University, Shenyang 110016, China

全文: PDF(1772 KB) HTML

摘要：

目的: 构建前手性酮类化合物的重组全细胞催化体系,并研究CbFDH C-端无序的11个氨基酸与酶活性的关系。方法:利用基因工程方法构建野生型LbADH ,CbFDH及C-端11个氨基酸缺失的CbFDH截短突变体的表达菌株;SDS-PAGE分析其表达方式及表达量,利用分光光度计法测定全菌破碎上清中的酶活性;将含野生型LbADH及CbFDHΔ^354-364的菌株与底物混合孵育,考察双菌全细胞体系的催化效果;并将含野生型CbFDH及CbFDHΔ的菌液分别与含野生型LbADH的菌株混合催化苯乙酮的转化,比较两种菌的协助转化效率。结果: 构建了重组表达载体pETDuet-1-adh,pETDuet-1-fdh及pETDuet-1-fdhΔ^354-364,并实现了在大肠杆菌中的异源表达,含野生型LbADH 及CbFDH的两种菌株能够实现苯乙酮的协同转化,转化率为24.4%,产物对映体纯度为96.79% ;CbFDH及CbFDHΔ^354-364分别占上清总蛋白的28.54%及37.72%,C-端的缺失未影响CbFDH的可溶性表达,但CbFDHΔ^354-364全菌上清催化NAD⁺转化为还原型NADH的效率降低,为CbFDH粗酶液的29.82%;且重组菌株Rossetta(DE3)-pETDuet-1-fdhΔ^354-364与Rossetta(DE3)-pETDuet-1-adh协同转化苯乙酮的转化率降低至8%。结论: 重组菌株Rossetta(DE3)-pETDuet-1-adh与Rossetta(DE3)-pETDuet-1-fdh能够协同催化苯乙酮转化,转化效率为24.4%,产物对映体纯度为96.79%;重组菌株Rossetta(DE3)-pETDuet-1-fdhΔ^354-364与Rossetta(DE3)-pETDuet-1-adh协同转化苯乙酮的效率为Rossetta(DE3)-pETDuet-1-fdh与Rossetta(DE3)-pETDuet-1-adh协同转化效率的32.79%,且Rossetta(DE3)-pETDuet-1-fdhΔ^354-364全菌上清催化还原型辅酶再生的效率降低,提示C-端无序的11个氨基酸残基对CbFDH酶活性的发挥起着重要的作用。

关键词： 博伊丁假丝酵母; 甲酸脱氢酶; 辅酶再生; 全细胞催化; 结构改造

Abstract:

Objective: Construct the whole cell biocatalysis system for the reduction of prochiral carbonyal compounds. And valuate the function of the disordered C terminus in the catalysis of formate dehydrogenase from Candida boidinii. Methods: The recombinant strains for the expression of LbADH ,CbFDH and CbFDHΔ^354-364 were constructed by the genetic engineering methods. The expression of recombinant proteins were analyzed by SDS-PAGE, and the activity of the whole soluble proteins was determined photometrically at 340 nm. Rossetta(DE3)-pETDuet-1-adh and Rossetta(DE3)-pETDuet-1-fdh were mixed and incubated with acetophenone, the products were analyzed by high performance liquid chromatography (HPLC). To compare the NADH regeneration rate of Rossetta(DE3)-pETDuet-1-fdhΔ^354-364 and Rossetta(DE3)-pETDuet-1-fdh, the two strains were mixed with Rossetta(DE3)-pETDuet-1-adh separately, and incubated with acetophenone, the products were detected timely. Results: The recombinant plasmid pETDuet-1-adh, pETDuet-1-fdh and pETDuet-1-fdhΔ^354-364 were constructed correctly. The recombinant proteins LbADH, CbFDH and CbFDHΔ^354-364 could be expressed solublely. The two recombinant strains harboring LbADH and CbFDH separately can be used coupled for the asymmetric reduction of acetophenone, the conversion rate reached 24.4% and enantiomeric excess reached 96.79%; the soluble expression of CbFDHΔ^354-364 has no significant difference compared with CbFDH, but when using the whole soluble proteins of Rossetta(DE3)-pETDuet-1-fdhΔ^354-364 catalyzed the regeneration of NADH, the conversion rate was sharply decreased to 8% when compared with Rossetta(DE3)-pETDuet-1-fdh. Conclusions: The recombinant strains Rossetta(DE3)-pETDuet-1-fdhΔ^354-364 and Rossetta(DE3)-pETDuet-1-adh could be used coupled for the reduction of acetophenone, resulted in a chemical yield of 24.4% and an enantiomeric excess of 96.79%. Rossetta(DE3)-pETDuet-1-adh and Rossetta(DE3)-pETDuet-1-fdh were mixed and incubated with acetophenone, the conversion rate was decreased 32.79% compared with Rossetta(DE3)-pETDuet-1-fdh mixed with Rossetta(DE3)-pETDuet-1-adh. The disordered residues in C terminus of CbFDH may play an important role in the catalysis process of CbFDH.

Key words: Candida boidinii Formate dehydrogenase Cofactor regeneration Whole cell biocatalysis Structure alteration

收稿日期: 2013-05-08 出版日期: 2013-08-25

ZTFLH:

Q819

基金资助:

国家自然科学基金(81000763);国家科技重大专项(2012ZX09301003);国家"973"计划(2009CB52604)资助项目

通讯作者: 陈惠鹏,E-mail:chenhp0909@163.com;凌焱,E-mail:lingyan@bmi.ac.cn E-mail: chenhp0909@163.com;lingyan@bmi.ac.cn

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引用本文:

李炳娟, 李玉霞, 李北平, 凌焱, 周围, 刘刚, 张景海, 岳俊杰, 陈惠鹏. 前手性酮全细胞转化体系中甲酸脱氢酶C-端无序结构与转化效率的关系研究[J]. 中国生物工程杂志, 2013, 33(8): 1-10.

LI Bing-juan, LI Yu-xia, LI Bei-ping, LING Yan, ZHOU Wei, LIU Gang, ZHANG Jing-hai, YUE Jun-jie, CHEN Hui-peng. Study of the Relationship between the Disordered C Terminusin of Formate Dehydrogenase and the Conversion Rate in the Whole-cell Catalysis of Prochiral Acetone. China Biotechnology, 2013, 33(8): 1-10.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/1

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