TY - JOUR
T1 - Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase
AU - Akita, Hironaga
AU - Imaizumi, Yoshifumi
AU - Suzuki, Hirokazu
AU - Doi, Katsumi
AU - Ohshima, Toshihisa
N1 - Funding Information:
Acknowledgments We thank Professor Tadao Oikawa for his kind advice. This work was supported by Grants of KAKENHI (25 4653) to H. Akita, Promotion of Basic Research Activities for Innovate Bioscience from the Bio-oriented Technology Research Advancement Institution (BRAIN) and NPO Geo Biotechnology Development Organization to T. Ohshima.
PY - 2014/11
Y1 - 2014/11
N2 - 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.
AB - 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.
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U2 - 10.1007/s10529-014-1597-z
DO - 10.1007/s10529-014-1597-z
M3 - Article
C2 - 24966047
AN - SCOPUS:84920707979
VL - 36
SP - 2245
EP - 2248
JO - Biotechnology Letters
JF - Biotechnology Letters
SN - 0141-5492
IS - 11
ER -