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A Novel Amidase From Brevibacterium Epidermidis ZJB-07021: Gene Cloning, Refolding and Application in Butyrylhydroxamic Acid Synthesis

A Novel Amidase From Brevibacterium Epidermidis ZJB-07021: Gene Cloning, Refolding and Application in Butyrylhydroxamic Acid Synthesis

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

J Ind Microbiol Biotechnol. 2016 Aug;43(8):1071-83.

PMID: 27276936

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP4312918	Butyrylhydroxamic acid		4312-91-8	Price

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