Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase

Hironaga Akita; Yoshifumi Imaizumi; Hirokazu Suzuki; Katsumi Doi; Toshihisa Ohshima

doi:10.1007/s10529-014-1597-z

Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase

Hironaga Akita, Yoshifumi Imaizumi, Hirokazu Suzuki, Katsumi Doi, Toshihisa Ohshima

Systems Biology

Research output: Contribution to journal › Article

5 Citations (Scopus)

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.

Original language	English
Pages (from-to)	2245-2248
Number of pages	4
Journal	Biotechnology letters
Volume	36
Issue number	11
DOIs	https://doi.org/10.1007/s10529-014-1597-z
Publication status	Published - Jan 1 2014

All Science Journal Classification (ASJC) codes

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology

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Akita, H., Imaizumi, Y., Suzuki, H., Doi, K., & Ohshima, T. (2014). Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase. Biotechnology letters, 36(11), 2245-2248. https://doi.org/10.1007/s10529-014-1597-z

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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.",

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AU - Akita, Hironaga

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AU - Suzuki, Hirokazu

AU - Doi, Katsumi

AU - Ohshima, Toshihisa

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