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해양미생물 Sphingomonas paucimobilis AS-1이 생산하는 새로운 extracelluar agarase의 정제 및 특성

Purification and Properties of a Novel Extracellular Agarase from Marine Bacterium, Sphingomonas paucimobilis AS-1

  • 정일선 (신라대학교 의생명과학대학 식품영양학과) ;
  • 김유정 (신라대학교 의생명과학대학 식품영양학과) ;
  • 송효주 (신라대학교 의생명과학대학 식품영양학과) ;
  • 갈상완 (진주산업대학교 미생물공학과) ;
  • 최영주 (신라대학교 의생명과학대학 식품영양학과)
  • Jung, Il-Sun (Department of Food and Nutrition, College of Medical Life Science Silla University) ;
  • Kim, Yu-Jung (Department of Food and Nutrition, College of Medical Life Science Silla University) ;
  • Song, Hyo-Ju (Department of Food and Nutrition, College of Medical Life Science Silla University) ;
  • Gal, Sang-Wan (Department of Microbiological Engineering, Jinju National University) ;
  • Choi, Young-Ju (Department of Food and Nutrition, College of Medical Life Science Silla University)
  • 발행 : 2008.01.31

초록

Agar로부터 oligosaccharides를 제조하기 위하여 효소(agarase)를 이용한 분해법이 시도되면서 agarase의 균주의 분리 및 유전자에 대한 많은 연구가 수행되고 있다. 본 연구에서는 해양으로부터 agarase를 생성하는 균주를 분리 동정하고 균주의 특성을 조사하였다. 분리된 균주는 VITEK 2 compact version 분석 결과 Sphingomonas paucimobilis AS-1로 동정되었다. 정제된 효소의 최적 활성 조건 및 agar 분해산물의 항산화활성을 조사하였다. 분리된 균주의 최적배양조건은 marine broth 2216에서 온도 $27^{\circ}C$, pH 7일 때 가장 균주의 생육이 높았다. Agarase 효소는 salt침전, ion exchange와 gel filtration chromatography에 의해 114.4 units/mg으로 104배 정제되었다. 정제된 agarase의 SDS-PACE 결과 분자량이 약 80 kDa의 band를 얻었다. 최적 효소활성은 온도 $40^{\circ}C$, pH 7일 때 나타났다. Agar 첨가한 배지에 agar 분해균을 접종한 후 분해산물의 시간에 따른 항산화활성은 12시간 배양 후 72%의 가장 높은 전자소거능(EDA)을 나타내었다. Agar 분해산물은 식중독균의 생육은 저해하는 것으로 나타났으며 젖산균의 생육은 약간 촉진하는 경향을 나타냈다.

An agar-degrading marine bacterium, strain AS-1 was isolated from the seawater. The strain AS-1 was identified as Sphingomonas paucimobilis (90% probability) by VITEK. The optimum medium for agarase activity of the isolated strain was determined to be marine medium, marine broth 2216 containing 0.1% agar as carbon source. An extracellular agarase was purified 104-fold from the culture supernatant by ammonium sulfate precipitation, ion exchange chromatography and gel filtration methods. The molecular weight of the purified enzyme was estimated to be 80 kDa by SDS-PAGE. The optimum pH and temperature for activity were 7.0 and $40^{\circ}C$, respectively. Antioxidative activity of the strain AS- was 72% in the supernatant cultured for 12 h. The culture supernatant of the strain AS-1 showed antibacterial activity against bacteria causing putrefaction and food poisoning such as Escherichia coli, Staphylococcus aureus and Proteus vulgaris. However, the cell growth of the lactic aicd forming strain, Lactobacillus plantarium was promoted by the treatment of 10% culture supernatant of an agar-degrading strain.

키워드

참고문헌

  1. Andrykovitch, G. and I. Maex. 1988. Isolation of a new polysaccharide - degrading bacterium from a salt marsh. Appl. Environ. Microbiol. 54, 1061-1062.
  2. Blois, M. S. 1958. Antioxidant determination by the use of a stable free radical. Nature 26, 1199-1200.
  3. Buttner, M. J., I. M. Feamley and M. J. Bibb. 1987. The agarase gene (dagA) of Streptomyces coelicolor A3(2) nucleotide sequence and transcriptional analysis. Mol. Gen. Genet. 209, 101-109. https://doi.org/10.1007/BF00329843
  4. Chiura, H. X. and K. Tsukamoto. 2000. Purification and characterization of novel agarase secreted by marine bacterium, Pseudoalteromonas sp. strain CKTl. Microb. Environ. 15, 11-22. https://doi.org/10.1264/jsme2.2000.11
  5. Duckworth, M. and J. R. Turvey. 1969 The action of bacterial agarase of agarose, porphyran and alkali treated porphyran. Biochem. J. 113, 687-692. https://doi.org/10.1042/bj1130687
  6. Fu, X. T., H. Lin and S. M. Kim. 2007. Purification and characterization of novel $\beta$-agarase, agaA34, from Agarivorans albus YKW-34. Appl. Microbiol. Biotechnol. in press.
  7. Giordano, A., G. Andreotti, A. Tramice and A. Trincone. 2006. Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides. Biotechnol. J. 1, 511-530. https://doi.org/10.1002/biot.200500036
  8. Jimenez-Escrig A and I. Goni Cambrodon. 1999. Nutritional evaluation and physiological effects of edible seaweeds. Arch Latinoam Nutr. 49, 114-120.
  9. Joo, D. S., S. Y. Cho and E. H. Lee. 1998. Preparation of agar hydrolysates by agarase and functionality of the hydrolysates. Korean, J. Biotechnol. Bioeng. 13, 378-382.
  10. Kang, N. Y., Y. L. Choi, Y. S. Cho, B. K. Kim, B. S. Jeon, J. Y. Cha, C. H. Kim and Y. C. Lee. 2003. Cloning, expression and characterization of a $\beta$-agarase gene from a marine bacterium, Pseudomonas sp. SK38. Biotechnology Letters 25, 1165-1170. https://doi.org/10.1023/A:1024586207392
  11. Kirimura, K., N. Masuda, Y. Iwasaki, H. Nkagawa, R. Kobayashi and S. Usami. 1999. Purification and characterization of a novel $\beta$-agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J. Biosci. Bioeng. 87, 436-441. https://doi.org/10.1016/S1389-1723(99)80091-7
  12. Kobayashi, R., M. Takisada, T. Suzuki, K. Kirimura and S. Usami. 1997. Neoagarobiose as a novel moisturizer with whitening effect. Biosci. Biotechnol. Biochem. 61, 162-163. https://doi.org/10.1271/bbb.61.162
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  14. Leon, O., L. Quintana, G. Peruzzo and J. C. Slebe. 1992. Purification and properties of an extracellular agarase from Alteromonas sp. strain C-1. Appl. Environ, Microbiol. 58, 4060-4063.
  15. Macmillan, J. D., H. J. Phaff and R. H. Vaughn. 1964. The Pattern of action of an exopolygalacturonic acid-transeliminase from Clostridium multifermentans. Biochemistry 3, 572-578. https://doi.org/10.1021/bi00892a017
  16. Miller, G. L. 1959. Use of dinitrosalicylic acid regent for determination of reducing sugar. Anal. Chem. 31, 426-428. https://doi.org/10.1021/ac60147a030
  17. Ohta Y., Y. Hatada, Y. Nogi, Z. Li, S. Ito and K. Horikoshi. 2004.Cloning, expression, and characterization of a glycoside hydrolase family 86 $\beta$-agarase from a deep-sea Microbulbifer-like isolate. Appl. Microbiol. Biotechnol. 66, 266-275. https://doi.org/10.1007/s00253-004-1757-5
  18. Ohta, Y., Y. Nogi, M. Myazaki, Z. u. Y. Hatada, S. Ito and K. Horikoshi. 2004. Enzymatic properties and nucleotide and amino acid sequences of a thermostable $\beta$-agarase from the novel marine isolate, JAMB-A94. Biosci. Biotechnol. Biochem. 68, 1073-1081. https://doi.org/10.1271/bbb.68.1073
  19. Sugano, Y., H. Kodama, I. Terada, Y. Yamazaki and M. Noma. 1994. Purification and characterization of a novel enzyme, $\alpha$-neoagarooligosaccharide hydrolase, from a marine bacterium, Vibrio sp. strain JT0107. J. Bacteriol. 176, 6812-6818. https://doi.org/10.1128/jb.176.22.6812-6818.1994
  20. Sugano, Y., I. Terada, M. Arita, M. Noma and Matsumoto. 1993. Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107. Appl. Environ. Microbiol. 59, 1649-1554.
  21. Suzuki, H., Y. Sawai, T. Suzuki and K. Kawai. 2003. Purification and characterization of an extracellular $\beta$-agarase from Bacillus sp. MK03. J. Biosci. Bioeng. 95, 328-334. https://doi.org/10.1016/S1389-1723(03)80063-4
  22. van der Meulen, H. and W. Harder. 1975. Production and characterization of the agarase of Cytophaga flevensis. Antonic Leeuwenhoek, 41, 431-447. https://doi.org/10.1007/BF02565087
  23. Vera, J., R. Alvarez, E. Murano, J. C. SIebe and O. Leon. 1998. Identification of a marine agarolytic Pseudoalteromonas isolate and characterization of its extracellular agarase. Appl. Environ. Microbiol. 64, 4378-4383.
  24. Wang, J. X., H. J. Mou, X. L Jiang and H. S. Guan. 2006. Characterization of a novel $\beta$-agarase from marine Alteromonas sp. SY37-12 and its degrading products. Appl. Microbiol Biotechnol. 71, 833-839. https://doi.org/10.1007/s00253-005-0207-3
  25. Wang, J. X., X. L. Jiang, H. J. Mou, and H. S. Guan. 2004. Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium. J. Appl. Phycol. 16, 333-340. https://doi.org/10.1023/B:JAPH.0000047944.40463.e6
  26. Yamaura, I., T. Matsumoto, M. Funatsu, H. Shigeiri and T. Shibata. 1991. Purification and some properties of agarase from Pseudomonas sp. PT-5. Agric. Biol. Chem. 55, 2531-2536. https://doi.org/10.1271/bbb1961.55.2531
  27. Young, K. S., and S. S. Bhattacharjee and W. Yaphe. 1978. Enzymic cleavage of the $\alpha$-linkages in agarose, to yield agaro-oligosaccharides. Carbohydr. Res. 66, 207-211. https://doi.org/10.1016/S0008-6215(00)83253-X

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