Sorbitol dehydrogenase from Pseudomonas sp.: Purification, characterization and application to quantitative determination of sorbitol
1991; Elsevier BV; Volume: 13; Issue: 4 Linguagem: Inglês
10.1016/0141-0229(91)90153-2
ISSN1879-0909
AutoresKarl‐Heinz Schneider, Friedrich Giffhorn,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoSorbitol dehydrogenase (l-iditol: NAD+ oxidoreductase, EC 1.1.1.14) was purified from Pseudomonas sp. to apparent homogeneity by (NH4)2SO4 precipitation, chromatography on Q-Sepharose, affinity chromatography on Procion-blue, and chromatography on hydroxylapatite. The relative molecular mass (Mr) of the native sorbitol dehydrogenase was determined to be 65,800 and its isoelectric point was pH 4.7. SDS-PAGE resulted in one single band representing a polypeptide with a Mr of 31,300, indicating that the native enzyme is a dimer. Sorbitol dehydrogenase was specific for NAD and catalysed the interconversion of d-glucitol to d-fructose, and of galactitol to d-tagatose, The pH optimum of substrate oxidation was pH 11.0, and that of substrate reduction was pH 6.7. The determined KM values were for NAD, 0.24 mm; d-glucitol, 9.1 mm; galactitol, 3.1 mm; d-fructose, 175.5 mm; d-tagatose, 10.0 mm; and NADH. 0.09 mm. The equilibrium constants for d-glucitol and galactitol interconversion were 3.2 and 1.3 nm, respectively. Due to the favorable kinetic properties of sorbitol dehydrogenase, an assay for substrate transformation was elaborated.
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