A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis.
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.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Li-Tao
LT
Ruan
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Ren-Chao
RC
Zheng
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.
Yu-Guo
YG
Zheng
D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D001951:Brevibacterium / Q000201:enzymology*; D003001:Cloning, Molecular; D006877:Hydroxamic Acids / Q000378:metabolism*; D007700:Kinetics; D058849:Protein Refolding; D013379:Substrate Specificity; D013696:Temperature
D000577:Amides; D006877:Hydroxamic Acids; C006866:butyramide; D000581:Amidohydrolases
Acyl transfer activity; Amidase; Brevibacterium epidermidis; Butyrylhydroxamic acid; Refolding