Purificatio of a specific d-apiitol dehydrogenase from a Micrococcus isolated from the surface of germinating parsley seeds

Abstract

The branched-chain sugar d-apiose was oxidized to CO 2 by both Lemma minor and a bacterium which was isolated from the surface of germinating parsley seeds. An inducible dehydrogenase which catalyzed the interconvension of d-apiose and d-apiitol was detected in extracts of this microorganism. The enzyme which was purified about 200-fold was specific for d-apiose and d-apiitol. It oxidized myo- inositol and meso- erythritol slowly, but it was completely inactive with all of the other sugars and polyols tested. The enzyme was specific for NAD + and NADH as electron acceptor and donor, respectively. NADP +, NADPH, ascorbate, FAD, FADH 2, cytochrome c and ferricyanide were inactive. The K m for D-apiitol was 1.16·10 -2 M, d-apiose was 7.14 · 10 −2 M, NAD + was 3.5 · 10 -4 M and NADH was 1.5–15 -5 M. At high concentrations NADH inhibited the reaction. The molecular weight of the dehydrogenase determined by chromatography on Sephadex 200 and sucrose density centrifugal was 110 000. The products of the reaction were characterized by paper chromatography, periodate oxidation and gas chromatography of acetylated derivatives. A colorimetric method for the quantitative determination of small amounts of d-apiose was also developed during the course of this study.