title: Nitrile-synthesizing enzyme: Screening, purification and characterization.
abstract: Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show β-cyano-L-alanine (β-CNAla) synthetic activity. β-CNAla synthase is known to catalyze nitrile synthesis: the formation of β-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and β-chloro-DL-alanine. In addition, we purified β-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5-9.0 at an optimal temperature of 40-50°C. The enzyme is specific for O-acetyl-L-serine and β-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 μmol/min/mg.
authors.affiliation: Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, University of Tsukuba.
authors.forename: Takuto
authors.initials: T
authors.lastname: Kumano
authors.forename: Takahisa
authors.initials: T
authors.lastname: Suzuki
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D003470:Culture Media / Q000737:chemistry; D003486:Cyanides / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D051050:Serine O-Acetyltransferase / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003470:Culture Media; D003486:Cyanides; D009570:Nitriles; D012694:Serine; C004631:3-cyanoalanine; D051050:Serine O-Acetyltransferase; D008190:Lyases; C019955:beta-cyanoalanine synthase; D003545:Cysteine; D000409:Alanine