title: Spectrophotometric assay of D-isoleucine using an artificially created D-amino acid dehydrogenase.
abstract: D-isoleucine (D-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for D-Ile determination using an artificially created NADP(+)-dependent D-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of D-Ile to (3R)-2-oxo-3-methyl valerate by DAADH in which NADP(+) is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. D-Ile was determined from 1 to 50 µM, and the assay was unaffected by the presence of any of three other isomers (100 µM), alcohol and organic acids.
authors: Applied Molecular Microbiology and Biomass Chemistry Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan, h-akita@aist.go.jp.
authors: Hironaga
authors: H
authors: Akita
authors: Yoshifumi
authors: Y
authors: Imaizumi
authors: Hirokazu
authors: H
authors: Suzuki
authors: Katsumi
authors: K
authors: Doi
authors: Toshihisa
authors: T
authors: Ohshima
mesh_terms: D000438:Alcohols / Q000737:chemistry; D000818:Animals; D001709:Biotechnology / Q000379:methods; D001842:Bone and Bones / Q000737:chemistry; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D006736:Horses; D007202:Indicators and Reagents / Q000737:chemistry; D007532:Isoleucine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*; D013778:Tetrazolium Salts / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D000438:Alcohols; D007202:Indicators and Reagents; D008055:Lipids; D013778:Tetrazolium Salts; D007532:Isoleucine; D009249:NADP; D014508:Urea; D003605:D-Amino-Acid Oxidase