title: Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase.
abstract: Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.
authors.affiliation: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China.
authors.forename: Zhi-Jian
authors.initials: ZJ
authors.lastname: Lin
authors.forename: Ren-Chao
authors.initials: RC
authors.lastname: Zheng
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Lei
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
authors.forename: Yin-Chu
authors.initials: YC
authors.lastname: Shen
mesh_terms: D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000032:analysis*; D000596:Amino Acids / Q000378:metabolism; D003124:Colorimetry / Q000379:methods; D003486:Cyanides / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism*; D006836:Hydro-Lyases / Q000032:analysis*; D007477:Ions / Q000378:metabolism; D008403:Mass Screening / Q000379:methods*; D009570:Nitriles / Q000378:metabolism
chemical_list: D000577:Amides; D000596:Amino Acids; D003486:Cyanides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D007477:Ions; D009570:Nitriles; D000581:Amidohydrolases; C019665:amidase; D006836:Hydro-Lyases; C048831:nitrile hydratase