Production and Characterization of Cholesterol Oxidase from Streptomyces sp. No.4

방선균으로부터 Cholesterol Oxidase의 생산 및 특성

  • 김현수 (계명대학교 자연과학대학 미생물학과) ;
  • 고희선 (계명대학교 자연과학대학 미생물학과)
  • Published : 1999.04.01

Abstract

An actinomycetes strain No.4 which produce the cholesterol oxidase(EC 1.1.3.6), was isolated from soil and identified as Streptomyces sp. based on taxonomic studies. The conditions of cholesterol oxidase production and enzymatic properties were investigated. The optimum composition of medium for production of the enzyme was 1% soluble starch, 2% corn steep liquor, 0.1% $KH_2PO_4$, 0.1% $NaNO_3$ and 0.05% $MgSO_4$ (pH 7.0). The optimum pH and temperature of the cholesterol oxidase were pH 6.0~7.5 and $37^{\circ}C$, respectively. The enzyme was stable in the range of pH 6.0~9.0. The isoelectric point determined by multichambered electrofocusing unit was in the range of pH 6.0~6.5.

Cholesterol oxidase(EC.1.1.3.6)는 cholesterol을 산화 또는 이성화시키는 효소로, 혈중 cholesterol이 측정 등의 임상진단용 시약에 이용되고 있으며, 여려종류의 미생물에서 분리, 연구되어 왔다. 현재에는 농업 및 식품 등에도 응용이 기대되고 있는 매우 유용한 효소이다. 본 실힘은 토양에서 분리한 방선균으로부터 cho1esterol oxidase생산능이 우수한 균주를 선발하여 속의 동정, 효소의 생산조건 및 효소학적 특성을 조사하였다 . 본 분리균주는 배양학적, 형태학적 생리학적 특성 및 현미경 검정을 통하여 Streptomyces sp으로 동정하였다. 본 효소의 생산조건을 조사한 결괴 탄소원으로 1% soluble starch, 질소원으로 2% corn steep liquor가 효소생산에 우수하였으며, 이들 탄소원, 질소원이의 0.1% $NaNO_3$, 0.1% $KH_2PO_4$, 0.05% $MgSO_4$가 함유된 배지를 생산 최적 배지로 사용하였다. 본 효소의 특성을 검토한 결과, 최적온도와 최적pH는 각각 $37^{\circ}C$, pH 6.5~7.5이었으며. 효소와 안정성은 온도 $30-40^{\circ}C$에사 80% 이상 활성이 유지되었으며, pH 6.0~9.0의 범위까지 안정한 것으로 나타났다. 본 효소의 등전점은 multichambered electrofocusing unit를 사용하여 측정한 결과 pH 6.0-6.5인 것으로 추정되었다.

Keywords

References

  1. Biochem. J. v.38 The microbiological degradation of steroids: 2. Oxidation of cholesterol by Proactinomyces spp. Turfitt, G. E.
  2. Clin. Chem. v.19 Preparation and properties of bacterial cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum Richmond, W.
  3. Ann. Clin. Biochem. v.10 An investigation of the determination of serum cholesterol by enzymatic method Flegg, H. M.
  4. Clin. Chem. v.20 Enzymatic determination of total serum cholesterol Allain, C. C.;L. S. Poon;C. S. G. Chan;W. Richmond;P. C. Fu
  5. Appl. Microbial. Biotechnol. v.31 Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of strain Lee, S. Y.;H. I. Rhee;W. C. Tae;J. C. Shin;B. K. Park
  6. Am. J. Med. v.56 Accidental hypothermia in an alcoholic population Weyman, A. E.
  7. Chem. Pham. Bull v.21 A method to screen anticholesterol substance produced by microbes and a new cholesterol oxidase produced by Streptomyces violascens Fukuda, H.;Y. Kawakami;S. Nagamura
  8. Agric. Biol. Chem. v.37 Isolation and crystallization of extracelular 3β-hydroxysteroid oxidase of Brevibacterium sterolicum nov. sp. Uwajima, T.;H. Yagi;S. Nagamura;O. Terada
  9. J. Ferment. Technol. v.55 Purification and some properties an extracellular 3β-hydroxysteroid oxidase produced by Corynebacterium cholesterolicum Shirokane, Y.;K. Nakamura;K. Mizusawa
  10. Chem. Pharm. Bull. v.26 Purification of 3β-hydroxysteroid oxidse of Streptomyces violascens origin by affinity chromatography on cholesterol Kamei, T.;Y. Takiguchi;M. Matsuzaki;S. Nakamura
  11. Appl. Environ. Microbial. v.52 Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ 702 Murooka, Y.;T. Ishizaki;O. Nimi;N. Maekawa
  12. Kor. J. Appl. Microbiol. Biotechnol. v.20 Purification and Characterization of Choleterol Oxidase Produced by Soil Microorganism HSL613 Lee, H. S.;S. C. Lee;T. J. Kwon;T. W. Chung
  13. Korean J. Biotechnol. Bioeng. v.13 Purification and characterization of cholesterol oxidase produced by Rhodococcus sp. 3T6-5MJ isolated from Changram-jeot Park, S. H.;H. S. Kim;Y. S. Lee;I. B. Kwon;U. H. Chun
  14. J. Clin. Lab. Anal. v.10 Optimization studies of components in enzymatic cholesterol regents containing cholesterol oxidase from Nocardia erythropolis, Streptomyces sp. or Pseudomonas fluorescens Lolekha, P. H.;Y. Teerajetkul
  15. J. Ferment. Bioeng. v.76 Nueletide sequence analysis of a region upstream of the cholesterol oxidase - cytochrome P450 operon of Streptomyces sp. SA-COO revealing repeating unit coding for putative transmembrane and DNA-binding proteins Molnar, I.;Y. Murooka
  16. Appl. Environ. Microbiol. v.60 Cloning of an insecticide cholesterol oxidase gene and its expression in bacteria and in plant protoplasts Corbin, D. R.;J. T. Greenplate;E. Y. Wong;J. P. Purcell
  17. Bioscience and Industry v.54 Cholesterol oxidase Murooka Y.
  18. J. Ferment. Technol. v.65 Humic acid - vitamin agar, a new medium for the selective isolation of soil actinomycetes Hayakawa, M.;H. Nonomura
  19. 放線菌の同定實驗法 淸野昭雄
  20. Bergey's manual of systematic Bacteriology v.4 Williams, S. T.;M. E. Sharpe;J. G. Holt
  21. Chem. Pharm. Bull. v.30 Purification and characterization of extracellular 3β-hydroxysteroid oxidase produced by Streptoverticillium cholesterolicum Inouye, Y.;K. Taguchi;A. Fujii;K. Ishimaru;S. Nakamura;R. Nomi
  22. Agric. Biol. Chem. v.52 Purification and some properties of cholesterol oxidase produced by an inducible and a constitutive mutant Liu, W.;M. H. Meng;K. S. Chem.
  23. J. Agric. Food Chem. v.37 Properties of the purified extracellular cholesterol oxidase from Rhodococcus equi No.13 Watanabe, K.;H. Aihara;Y. Nakagawa;R. Nakamura;K. Susaki
  24. J. Biochem. v.85 Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin Fukuyama, M.;Y. Miyake
  25. Kor. J. Appl. Microbiol. Biotechnol. v.22 Cholesterol oxidase를 생산하는 방선균 분리주 HSL-613의 동정 Lee, H. S.;I. A. Lee;Y. K. Choe;H. G. Lee;K. C. Lee;Y. H. Park;T. K. Oh;I. S. Choe;T. W. Chung
  26. Kor. J. Appl. Microbiol. Biotechnol. v.20 Cholesterol oxidase를 생성하는 토양미생물의 분리 및 효소생산에 관한 연구 Lee, I. A.;Y. K. Choe;H. S. Lee;I. S. Choe;T. W. Chung