title: Exploitation and characterization of three versatile amidase super family members from Delftia tsuruhatensis ZJB-05174.
abstract: Amidases can be assigned into two families according to their amino acid sequences. Three amidases (Dt-Amis) were mined and identified from genome of Delftia tsuruhatensis. Homology analysis demonstrated that Dt-Ami 2 and Dt-Ami 6 belonged to amidase signature (AS) family, while Dt-Ami 7 belonged to nitrilase superfamily. AS amidases were shown to hydrolyze a wide spectrum of amides. Kinetic analysis demonstrated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover numbers (kcat) were high with linear aliphatic amides as substrates. Dt-Ami 2 showed maximum activity near a quite alkaline pH (11.0) and exhibited opposite enantioselectivity to Dt-Ami 6. Furthermore, a novel bioprocess for hydrolysis of 1-cyanocyclohexaneacetamide was developed using Dt-Ami 6 as biocatalyst, resulting in >99% conversion within 1.5h at a substrate loading of 100g/L by 0.5g/L of Escherichia coli cells. On the other hand, nitrilase superfamily amidase only hydrolyzed aliphatic amides. The Km values of Dt-Ami 7 were considerably increased with the extension of chain length of aliphatic amides. The characterized enzymes from different families showed distinct biochemical characteristics and catalytic properties, leading to a better understanding of the two super amidase family members.
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: Zhe-Ming
authors.initials: ZM
authors.lastname: Wu
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: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000581:Amidohydrolases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D042742:Delftia / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D007700:Kinetics; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000577:Amides; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000581:Amidohydrolases
keywords: Amidase; Amidase signature family; Delftia tsuruhatensis; Gene mining; Kinetic resolution; Nitrilase superfamily