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A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A novel amidase gene (bami) was cloned from Brevibacterium epidermidis ZJB-07021 by combination of degenerate PCR and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). The deduced amino acid sequence showed low identity (≤55 %) with other reported amidases. The bami gene was overexpressed in Escherichia coli, and the resultant inclusion bodies were refolded and purified to homogeneity with a recovery of 22.6 %. Bami exhibited a broad substrate spectrum towards aliphatic, aromatic and heterocyclic amides, and showed the highest acyl transfer activity towards butyramide with specific activity of 1331.0 ± 24.0 U mg−1. Kinetic analysis demonstrated that purified Bami exhibited high catalytic efficiency (414.9 mM−1 s−1) for acyl transfer of butyramide, with turnover number (K cat) of 3569.0 s−1. Key parameters including pH, substrate/co-substrate concentration, reaction temperature and catalyst loading were investigated and the Bami showed maximum acyl transfer activity at 50 °C, pH 7.5. Enzymatic catalysis of 200 mM butyramide with 15 μg mL−1 purified Bami was completed in 15 min with a BHA yield of 88.1 % under optimized conditions. The results demonstrated the great potential of Bami for the production of a variety of hydroxamic acids.

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Acknowledgments

This work was supported by the National Nature Science Foundation of China (No. 21202150), National Science Foundation of Zhejiang (Y4080334, LY13B060004) and National High Technology Research and Development Program of China (No. 2012AA022201B).

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Li-Tao Ruan, Ren-Chao Zheng & Yu-Guo Zheng

  2. Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Li-Tao Ruan, Ren-Chao Zheng & Yu-Guo Zheng

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  1. Li-Tao Ruan
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  2. Ren-Chao Zheng
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  3. Yu-Guo Zheng
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Correspondence to Yu-Guo Zheng.

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Ruan, LT., Zheng, RC. & Zheng, YG. A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis. J Ind Microbiol Biotechnol 43, 1071–1083 (2016). https://doi.org/10.1007/s10295-016-1786-y

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