title: An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid.
abstract: Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V max and K m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg(-1) min(-1) and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml (-) resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Bhatia
authors.forename: P K
authors.initials: PK
authors.lastname: Mehta
authors.forename: R K
authors.initials: RK
authors.lastname: Bhatia
authors.forename: T C
authors.initials: TC
authors.lastname: Bhalla
mesh_terms: D000421:Alcaligenes / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D000581:Amidohydrolases / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006836:Hydro-Lyases / Q000096:biosynthesis*; D006885:Hydroxybutyrates / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology*; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D009570:Nitriles; C022246:isobutyronitrile; C008039:2-hydroxyisobutyric acid; D000581:Amidohydrolases; D006836:Hydro-Lyases; C048831:nitrile hydratase