Purification and Characterization of a Glycolic Acid(GA)Oxidase Active toward Diglycolic Acid(DGA)Produced by DGA-Utilizing Rhodococcus sp. No. 432

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概要

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• Diglycolic acid(DGA)oxidizing activity was found in crude extracts of Rhdococcus sp. no. 432 grown in DGA. Glycolic acid(GA)oxidase was purified approximately 80 times by treatment with streptomycin sulfate, precipitation with(NH_4)_2SO_4,chromatographies with DEAE-cellulose, DEAE-Toyopearl and Butyl-Toyopearl, and gel filtration on Toyopearl HW-55. The purified GA oxidase was almost homogeneous on sodium dodecyl sulfate(SDS)polyacrylamide gel electrophoresis. The purity was calculated to be more than 95%. The molecular weight of the enzyme, which appeared to consist of three identical units, was 158,000. Each subunit of GA oxidase included one molecule of FAD as a cofactor. The isoelectric point of the enzyme was around 5.3. GA oxidase was stable below 30℃ and at the pH range of 6.0-8.5. The optimum pH and temperature were around 7.5 and 40℃, respectively. Oxygen, cytochrome c, ferricyanide and 2,6-dichlorophenol in dophenol(DCIP)acted as an electron acceptor. The activity of GA oxidase was strongly inhibited by potassium cyanide, quinine, quinacrine, monoiodoacetate, 1,4-benzoquinone and some heavy metal ions. GA oxidase also had activity in DGA, GA, glyoxylic acid(GOA), methoxy acetate, ethoxy acetate and L-malate. Alcohols and other organic acids were not oxidized by the enzyme. THe apparent K_m values for DGA, GA and GOA were about 26.7,0.5 and 4.4mM, respectively. THe reaction products from DGA were supposed to be GOA and GA by the enzymatic assays. The reaction mechanism of GA oxidase in oxidation of DGA was supposed to be as followe : HOOC-H_2C-O-CH_2-COOH+H_2O-acceptor→HOOC-CHO+HOOC-CH_2OH+reduced acceptor.

• 社団法人日本生物工学会の論文
• 1991-02-25

著者

• KAWAI Fusako

  Department of Biology, Kobe University of Commerce

• YAMANAKA Hiroshi

  Department of Electrical Engineering, Nagaoka University of Technology

• Kawai Fusako

  Department Of Biology Kobe University Of Commerece

• Kawai Fusako

  Department Of Agricultural Chemistry College Of Agriculture Kyoto University

• Yamanaka Hiroshi

  Department Of Biology Kobe University Of Commerece:(present Address)research Laboratory Hakutsuru Sake Brewing Co. Ltd.

• Yamanaka Hiroshi

  Department Of Agricultural Chemistry Faculty Of Agriculture

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