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Home , Sorbose

[Agr. Biol. Chem., Vol. 30, No. 1, p. 97~98, 1966]

L-Sorbose Oxidase from Trametes sanguinea

Sir: enzyme preparation obtained with DEAE- In the previous papers, we reported on the column chromatography was about two kinds of new enzymes concerning the 30-fold of that of the crude extracts. formation and reduction of 5-keto-D- (5- This preparation showed an intensive ac­ keto-L-sorbose); i.e. D-fructose dehydrogenase1’ tivity toward L-sorbose and the oxidation pro­ and 5-ketofructose reductase,2’ both of which duct was identified as 5-keto-D-fructose. In were found in acetic acid bacteria. contrast, D-fructose was completely inactive While studying on the metabolism of as substrate. This enzyme specificity is Basidiomycetes, we have found that L-sorbose noticeable, considering from the fact that d - was actively oxidized by the cell-free extracts fructose dehydrogenase obtained from acetic of Trametes sanguinea, which, however, could acid bacteria forms 5-keto-D-fructose from d - not assimilate the for growth. fructose but not from L-sorbose.11 Beside l -

The present communication concerns with sorbose, D-, D-, D- and Downloaded from https://academic.oup.com/bbb/article/30/1/97/5976797 by guest on 30 September 2021 the purification and some properties of the were oxidized by this partially puri­ aerodehydrogenase obtained from the fungus fied enzyme preparation. (Table I) However, which catalyzes the following reaction: it is obscure whether or not this preparation L-Sorbose + 0 2 —> 5-Keto-D-fructose + H 20 2 is a mixture of several enzymes, since sugar- Trametes sanguinea IFO 4923 was recipro­ oxidizing enzymes of separate entity, to date, cati vely shaken at 27 °C for 48 hours in the are not definitely clarified as yet. Sakaguchi flasks containing 100 ml of medium The optimal pH was 6.0 and the range was composed of koji juice and 0.3 per cent corn rather broad. Michaelis constant for l - steep liquor. At the end of the cultivation sorbose was 2.2x 10”2m/1 at pH 6.0 and 30°G small pellets were collected, washed, twice with cold saline, and suspended in m / 1 0 phos­ T a b l e I. Su b str a te Sp e c if ic it y phate buffer, pH 6.0, for the sonication for Substrates Activity % 15 minutes. The sonicated cells were centri­ L-Sorbose 100 fuged for 20 minutes at 3,5000 xg. The super­ D-Fructose 0 natant fluid was then ultracentrifuged for one D-Glucose 155 D- 0 hour at 105,000 X g and to the resulting super­ D-Galactose 71 natant ammonium sulfate was added. The D-Xylose 42 precipitate formed with 37~57 per cent satu­ D- 0 ration was collected and the procedure was L-Arabinose 0 repeated once again. This (ammonium sulfate Sorbitol 0 precipitated) fraction was dissolved in m / 1 0 Mannitol 0 phosphate buffer, pH 6.0, and the enzyme Erythritol 0 Sodium D-gluconate 0 solution was dialyzed overnight against m / 1 0 0 of the same buffer. The activity of the Glucurono-f-lactone 1 0 1) Y. Yamada, K. Aida and T. Uemura, This Journal, 30, Maltose 15 95 (1966); Y. Yamada, K. Aida and T. Uemura, Symposia Each reaction mixture (3.0 ml) contained 100 /¿moles of on Enzyme Chemistry, Tokushima, 17th, 113 (1965). substrate, 250/mmoles of phosphate buffer (pH 6.0), 0.2ml of 2) K. Aida and Y. Yamada, This Journal, 28, 74 (1964); 1.0x 10-8 m/ 1 of 2,6-di-chlorophenol indophenol, and 0.5 ml of K. Aida, Y. Yamada, and T. Uemura, Symposia on Enzyme the enzyme solution and half-decolorization time was Chemistry, Tokyo, 16th, 327 (1964). measured. in air. When catalase was added to the dehydrogenase from Aspergillus oryza^'lQ) were reaction mixture, the oxygen consumption inactive toward L-sorbose. was one half of that without catalase, in As for the chemical structure of dicarbonyl­ which the red-violet color was observed at formed from D-fructose by acetic acid the detection with KI- reaction, showing bacteria, two different ones, i.e. 6-aldo-D- the presence of hydrogen peroxide. Also the fructose111 and 5-keto-D-fructose12 ’ were in­ enzyme could reduce 2,6-dichlorophenol in- dependently proposed. Our present report dophenol, in the presence of which (6.7 x 10“4 which showed that dicarbonylhexose was m / 1) Michaelis constant of the enzyme showed formed only from L-sorbose but not from d- a rather higher value, 1.0 x 10_ 1 m /1 under the fructose may be another proof of the validity same conditions. that it is 5-keto-D-fructose as confirmed by The enzyme was not inhibited by IO”4 m /1 some authors.13,141 ÿ-chloromercuribenzoate, but silver and mer­ Yuzo Y amada curic ions ( 10“ 4 m /1, respectively) abolished the enzyme activity. Inhibition by atebrine was Kazuaki I izuka not observed. Ko A id a Recently, some of sugar oxidases such as Tei jiro U emura hexose oxidase,3’ galctose oxidase4,51 and The Institute of Applied Microbiology Downloaded from https://academic.oup.com/bbb/article/30/1/97/5976797 by guest on 30 September 2021 xylose oxidase6' have been precisely research­ The University o f Tokyo, Tokyo ed, but there is no description on the enzyme which oxidizes L-sorbose to 5-keto-D-fructose. Received December 4, 1965 Keilin and Hartree7’ reported that L-sorbose 9) S. Boku and R. Sato, Symposia on Enzyme Chemistry, was not oxidized by their glucose oxidase Osaka, 15th, 180 (1963): Personal Communication. 10 D. Ogura ond M. Nagahisa, Botanical Magazine, Tokyo, (notatin) from Pénicillium. Both de­ Si, 599 (1937). hydrogenase from Pseudomonas8) and glucose 11) R. Weidenhagen and G. Bernsee, Chem. Ber., 93, 2924 (1960). 3) R. C. Bean, G.G. Porter and B. M. Steinberg, /. Biol. 12) 0. Terada, S. Suzuki and S. Kinoshita, This Journal, Chem., 2 36, 1235 (1961). 25, 871 (1961): O. Terada, K. Tomizawa, and S. Kinoshita, 4) J. A. D. Cooper, W. Smith, M. Bacila and H. Medina, /. Agr. Chem. Soc. Japan, 35, 127 (1961); O. Terada K. /. Biol. Chem., 2 34, 445 (1959). Tomizawa, S. Suzuki and S. Kinoshita, ibid., 131; O. Terada, 5) G. Avigad, D. Amaral, C. Asensio and B. L. Horecker, S. Suzuki and S. Kinoshita, ibid., 178. ibid., 237, 2736 (1962). 13) J. G. Carr, R. A. Coggins and G. C. Whiting, Chemistry 6) H. Lyr, Enzymologia, 24, 69 (1962). and Industry, p, 1279 (1963). 7) D. Keilin and E. F. Hartree, Biochem. J., 42, 221 (1948). 14) G. Avigad and S. Englard, /. Biol. Chem,, 240, 2290 8) Y. Nishizuka and O. Hayaishi, /. BioL Chem., 237, (1965); S. Englard and G. Avigad, ibid., 2297; S. Englard, G. 2721 (1962). and L. Prosky, ibid., 2302.