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

2013, Vol. 33

Issue (8): 1-10 DOI:

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

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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

Received: 08 May 2013 Published: 25 August 2013

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	LI Bing-juan
	LI Yu-xia
	LI Bei-ping
	LING Yan
	ZHOU Wei
	LIU Gang
	ZHANG Jing-hai
	YUE Jun-jie
	CHEN Hui-peng

Cite this article:

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.

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

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