Crystallization and properties of NADPH-dependent L-sorbose reductase from Gluconobacter melanogenus IFO 3294

被引:19
|
作者
Adachi, O [1 ]
Ano, Y
Moonmangmee, D
Shinagawa, E
Toyama, H
Theeragool, G
Lotong, N
Matsushita, K
机构
[1] Yamaguchi Univ, Fac Agr, Dept Biol Chem, Yamaguchi 7538515, Japan
[2] Ube Natl Coll Technol, Dept Chem & Biol Engn, Ube, Yamaguchi 7558555, Japan
[3] Kasetsart Univ, Fac Sci, Dept Microbiol, Bangkok 10900, Thailand
关键词
acetic acid bacteria; NADP-dependent D-sorbitol dehydrogenase; Gluconobacter melanogenus; L-sorbose reductase;
D O I
10.1271/bbb.63.2137
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
NADPH-Dependent L-sorbose reductase (SORD, synonimously NADP-dependent D-sorbitol dehydrogenase) was purified and crystallized for the first time from the cytosolic fraction of Gluconobacter melanogenus IFO 3294. The enzyme catalyzed oxidoreduction between D-sorbitol and L-sorbose in the presence of NADP or NADPH. Affinity chromatography by a Blue-dextran Sepharose 4B column was effective for purifying the enzyme giving about 770-fold purification with an overall yield of more than 50%. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.8 s. Gel filtration on a Sephadex G-75 column gave the molecular mass of 60 kDa to the enzyme, which dissociated into 30kDa subunit on SDS-PAGE, indicating that the enzyme is composed of 2 identical subunits. Reduction of L-sorbose to D-sorbitol predominated in the presence of NADPH with the optimum pH of 5.0-7.0. Oxidation of D-sorbitol to L-sorbose was observed in the presence of NADP at the optimum pH of 7.0-9.0. The relative rate of L-sorbose reduction was more than seven times higher to that of D-sorbitol oxidation. NAD and NADH were inert for both reactions. D-Fructose reduction in the presence of NADPH did not occur with SORD. Since the reaction rate in L-sorbose reduction highly predominated over D-sorbitol oxidation over a wide pH range, the enzyme could be available for direct enzymatic measurement of L-sorbose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the L-sorbose reduced. Judging from the enzymatic properties, SORD would contribute to the intracellular assimilation of L-sorbose incorporated from outside the cells where L-sorbose is accumulated in huge amounts in the culture medium.
引用
收藏
页码:2137 / 2143
页数:7
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