Celluomonas sp. AP-7이 생산하는 Ascorbic Acid Phosphorylating Enzyme의 정제 및 특성

  • 이상협 (고려대학교 자연과학대학 농화학과) ;
  • 최현일 (고려대학교 자연과학대학 농화학과) ;
  • 방원기 (고려대학교 자연과학대학 농화학과)
  • Published : 1997.06.01


An ascorbic acid phosphorylating enzyme, which catalyzes the formation of ascorbic acid-2-phosphate from ascorbic acid and pyrophosphate, was purified 32.7-folds to homogeneity from a cell-free extract of Cellulomonas sp. AP-7. The combination of DEAE- Sephacel ion exchange chromatography and Sephacryl S-200 get filtration was used for their purification. The molecular weight of the native protein was estimated to be 96.lkDa on high performance gel filtration chromatography. The SDS-PAGE analysis indicated that the protein consisted of four identical subunits of 24.6 kDa. The purified enzyme showed the optimal tempeature of 40$\circ$C and optimal pH of 4.5. The Km for ascorbic acid and pyrophosphate were 119 mM and 11.9 mM, respectively. The addition of 5,5'-dithiobis-(2-nitrobenzoic acid) into the reaction mixture resulted in the reduction of the enzyme activity at 51%. The enzyme also had a phosphatase activity at weakly acidic pH and the Km for ascorbic acid-2-phosphate in phosphatase activity was 7.9 mM.



  1. Vitamins Friedrich, W.
  2. Chem. Pharm. Bull. v.25 Effects of metal ions and flavonoids on the oxidation of ascorbic acid Takamura, K.;M. Ito
  3. Chem. Pharm. Bull. v.17 Studies on L-ascorbicd acid derivatives. Ⅱ. L-ascorbic acid 3-phosphate and 3-pyrophosphate Nomura, H.;T. Ishiguro;S. Morimoto
  4. Chem. Pharm. Bull. v.14 Synthesis of L-ascorbic acyl derivatives stabilizied against oxidation Nomura, H.;K. Sugimoto
  5. Chem. Pharm. Bull. v.19 Studies on L-ascorbicd acid derivatives. Ⅵ. phosphorylation of L-ascorbic acid and its isopropylidene derivatives Nomura, H.;M. Shimomura;S. Morimoto
  6. J. Chem. Soc. Perkin Trans. v.1 Synthesis and stability of L-ascorbate 2-sulfate Seib, P. A.;Y. T. Liang;C. H. Lee;R. C. Hoseney;C. W. Deyo
  7. Agric. Biol. Chem. v.54 Formation of a stable ascorbic acid 2-glucoside by specific transglucosylation with rice sees α-glucosidase Muto, N.;S. Suga;K. Fujii;K. Goto;I. Yamamoto
  8. Am. J. Clin. Nutr. v.32 Antiscobutic activity of ascorbic acid phosphate in the rhesus monkey and the Guinea pig Machlin, L. J.;F. Garcia;W. Kuenzig;M. Brin
  9. Jap. J. Pharmacol. v.17 The Antiscorbutic activity of L-ascorbic acid phosphate given orally and percutaneously in Guinea Pigs Imai, Y.;Y.Usui;T. Matsuzaki;H. Yokotani;H. Mima;Y. Aramaki
  10. Vatamins(Japan) v.41 Chemistry and application of ascorbic acid phosphate Mima, H.;H. Nomura;Y. Imai;H. Takashima
  11. J. Assoc. Off. Anal. Chem. v.71 Liquid chromatographic determination of L-ascorbate 2-phosphate in fish feeds by enzymatic release of L-ascorbate Wang, X. Y.;M. L. Liao;T. H. Hung;P. A. Seib
  12. J. Nutr. v.121 L-Ascorbyl 2-monophosphate has Equal antiscobutic activity as L-ascorbic acid but L-ascorbyl-2-sulfate is inferior to L-ascorbic acid for channel catfish El Naggar, G. O.;R. T. Lovell
  13. J. Food Sci. v.54 Control of enzymatic browning in apple with ascorbic acid derivatives, polyphenol oxidase inhibitors, and complexing agents Sapers, G. M.;K. B. Hicks;J. G. Phillips;L. Garzarella;D. L. Pondish;R. M. Matulaitis;T. J. McCormack;S. M. Sondey;P. A. Seib;Y. S. El-Atawy
  14. J. Food Sci. v.56 Uptake and fate of acorbic acid-2-phosphate in infiltrated fruit and vegetable tissue Sapers, G. M.;R. L. Miller;F. W. Douglas;K. B. Hicks
  15. Am. Perf. Cosmet. v.86 Ascorbic acid esters and skin pigmentation Takashim, H.;H. Nomura;Y. Imai;H. Mima
  16. Eur. Pat. Appl. EP319130 Enzymic preparation of ascorbic acid-2-phosphate Fujio, T.;A. Maruyama;S. Koizumi
  17. Agric. Biol. Chem. v.54 Enzymatic production of ascorbic acid-1-phosphate Maruyama, A.;S. Koizumi;T. Fujio
  18. 日本公開持許公報 平2-283283 酵素を固定化した樹脂組成物およぴ該樹脂組成物を用ぃるアスコルピン酸-2-リン酸の製造法 弓場善雄;田村圭史郞;橋本幸生;藤尾達郞
  19. 한국산업미생물학회지 v.24 Ascorbic acid와 pyrophosphate로부터 ascorbic acid-2- phosphate의 효소적 생산 최현일;이상협;방원기
  20. Anal. Biochem. v.150 Measurement of protein using bicinchoninic acid Smith, P. K.;R. I. Krohn;G. T. Hermanson;A. K. Mallia;F. H. Gartner;M. D. Provenzano;E. K. Fujimoto;N. M. Goeke;B. J. Olson;D. C. Klenk
  21. Anal. Biochem. v.171 A Malachite green procedure for orthophosphate determination and Its Use in alkaline phohphatase-based enzyme immunoassay Baykov, A. A.;O. A. Evtushenko;S. M. Avaeva
  22. Nature v.227 Cleavage of structural proteins during the asembly of the head of bacteriophage T₄ Laemmli, U. K.
  23. J. Biol. Chem. v.246 Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system Nevile, D. M.
  24. Agric. Biol. Chem. v.54 Purification and charaterization of ascorbic acid phosphorylating enzyme from Pseudomonas azotocolligans Koizumi, S.;A. Maruyama;T. Fujio
  25. Biochem. Biophys. Res. Commun. v.28 Crystalline acid phosphatase having pyridoxine-phosphating activity Tani, Y.;Tochikura T;Yamada H.;K. Ogata
  26. Biochem. J. v.70 The phosphotransferase activity of phosphotase Morton, R. L.
  27. Agric. Biol. Chem. v.29 Microbial phosphorylation of pyridoxine Ogata, K.;Y. Tani;S. Yamamot
  28. Agric. Biol. Chem. v.36 Distribution and properties of NAD phosphorylating reaction Kuwahara, M.;Tachiki, T.;T. Tochikura
  29. Agric. Biol. Chem. v.36 Distribution and properties of NAD phosphorylating reaction Kuwahara, M.;Tachiki, T.;Tochikura T.;K. Ogata