Overexpression of a nitrile hydratase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

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• 作者：Fa-Mou Guo ; Jian-Ping Wu ; Li-Rong Yang ; Gang Xu
• 关键词：nitrile hydratase ; recombinant expression ; Klebsiella oxytoca KCTC 1686 ; biotransformation ; enantioselective
• 刊名：Biotechnology and Bioprocess Engineering
• 出版年：2015
• 出版时间：November 2015
• 年：2015
• 卷：20
• 期：6
• 页码：995-1004
• 全文大小：509 KB
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• 作者单位：Fa-Mou Guo (1)
  Jian-Ping Wu (1)
  Li-Rong Yang (1)
  Gang Xu (1)
  
  1. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310-027, China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  
• 出版者：The Korean Society for Biotechnology and Bioengineering
• ISSN：1976-3816

文摘

The putative Co-type nitrile hydratase (NHaseK, consisting of α- and β-subunits) genes and the putative activator (17K) gene adjacent to the β subunit region were cloned from Klebsiella oxytoca KCTC 1686. 17K is essential for the functional expression of recombinant NHaseK in Escherichia coli; however, the expression level of 17K was very low when the 17K gene and NHaseK structural genes were expressed as a gene cluster in E. coli BL21(DE3). To improve the 17K expression level and NHaseK activity, the expression cassette was redesigned by placing the 17K gene and NHaseK structural genes under the control of different promoters in the pETDuet-1 expression vector, co-expressing the 17K gene with the gene cluster in a double plasmid or a single plasmid with a double promoter, and introducing an efficient Shine- Dalgarno sequence 5' to the17K gene. The specific activity of NHaseK was improved when 17K was co-expressed with the gene cluster, whereas the production of NHaseK protein decreased. The maximum activity was achieved when an efficient Shine-Dalgarno sequence was introduced 5' to the 17K gene: the expression level of 17K was significantly improved and the expression level of NHaseK did not decrease significantly. The maximum activity was about 63,480 ± 1915.6 U/L broth towards 3-Cyanopyridine. Recombinant NHaseK could hydrolyze a wide range of aliphatic, aromatic, and heterocyclic nitriles, and convert racemic nitriles to the corresponding S-amides, with E values ranging from 9 to 17. The enzyme had a temperature optimum of 35°C and exhibited remarkably stability below 35°C. Keywords nitrile hydratase recombinant expression Klebsiella oxytoca KCTC 1686 biotransformation enantioselective