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

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±24.0U mg(-1). Kinetic analysis demonstrated that purified Bami exhibited high catalytic efficiency (414.9mM(-1) s(-1)) for acyl transfer of butyramide, with turnover number (K cat) of 3569.0s(-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 200mM butyramide with 15μgmL(-1) purified Bami was completed in 15min 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.