title: Mining and characterization of two amidase signature family amidases from Brevibacterium epidermidis ZJB-07021 by an efficient genome mining approach.
abstract: Amidases have received increasing attention for their significant potential in the production of valuable carboxylic acids. In this study, two amidases belonging to amidase signature family (BeAmi2 and BeAmi4) were identified and mined from genomic DNA of Brevibacterium epidermidis ZJB-07021 by an efficient strategy combining comparative analysis of genomes and identification of unknown region by high-efficiency thermal asymmetric interlaced PCR (HiTAIL-PCR). The deduced amino acid sequences of BeAmi2 and BeAmi4 showed low identity (< 40%) with other reported amidases. The two amidases displayed optimum activity toward a wide spectrum of substrates at a mild alkaline pH and 45 °C. Both of them were remarkably inactivated by serine-directed inhibitor and sulfhydryl-reducing agent. Kinetic analysis revealed that nicotinamide was the preferable substrate for both amidases and the chlorine substitutions on the pyridine ring had a negative effect on activity. The bioprocesses for hydrolysis of 100 mM nicotinamide, isonicotinamide, 2-chloronicotinamide and 5-chloronicotinamide with purified BeAmi2 (6 U mL(-1)) were complete in 60 min with full conversion except 2-chloronicotinamide. These results indicated BeAmi2 was an effective catalyst for hydrolysis of several nicotinamide derivatives.
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Li-Tao
authors.initials: LT
authors.lastname: Ruan
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
authors.forename: Ren-Chao
authors.initials: RC
authors.lastname: Zheng
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China. Electronic address: zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001951:Brevibacterium / Q000201:enzymology / Q000235:genetics; D057225:Data Mining; D016680:Genome, Bacterial / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D000581:Amidohydrolases; C019665:amidase
keywords: Amidase; Amidase signature family; Brevibacterium epidermidis; Genome mining; HiTAIL-PCR