Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

The Optimal Production and Characteristics of an Alginate-degrading Enzyme from Vibrio sp. PKA 1003

Vibrio sp. PKA 1003의 알긴산 분해 조효소 생산 최적 조건과 조효소의 특성

  • Kim, Hyun-Jee (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Kim, Koth-Bong-Woo-Ri (Institute of Fisheries Sciences, Pukyong National University) ;
  • Kim, Dong-Hyun (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • SunWoo, Chan (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Jung, Seul-A (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Jeong, Da-Hyun (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Jung, Hee-Ye (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Lim, Sung-Mee (Dept. of Food Nutrition and Science, Tongmyong University) ;
  • Ahn, Dong-Hyun (Dept. of Food Science & Technology/Institute of Food Science, Pukyong National University)
  • 김현지 (부경대학교 식품공학과/식품연구소) ;
  • 김꽃봉우리 (부경대학교 수산과학연구소) ;
  • 김동현 (부경대학교 식품공학과/식품연구소) ;
  • 선우찬 (부경대학교 식품공학과/식품연구소) ;
  • 정슬아 (부경대학교 식품공학과/식품연구소) ;
  • 정다현 (부경대학교 식품공학과/식품연구소) ;
  • 정희예 (부경대학교 식품공학과/식품연구소) ;
  • 임성미 (동명대학교 식품영양과학과) ;
  • 안동현 (부경대학교 식품공학과/식품연구소)
  • Received : 2012.08.27
  • Accepted : 2013.02.27
  • Published : 2013.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

This study was conducted to screen the characteristics and alginate-degrading activity of marine bacterium isolated from brown seaweed (Sargassum thunbergii). The results of 16S ribosomal RNA sequence analysis the strain the genus Vibrio and the strain was subsequently named Vibrio sp. PKA 1003. The optimum culture conditions for the growth of Vibrio sp. PKA 1003 were at pH 7, 3% NaCl, $25^{\circ}C$, and 6% alginic acid, with a 48-hour incubation time. A crude enzyme preparation from Vibrio sp. PKA 1003, showed its highest levels of alginate-degradation activity when cultured at pH 9, $30^{\circ}C$, and 6% alginic acid, with a 63-hour incubation time. Thin layer chromatography analyses confirmed that the crude enzyme released monomers or oligomers from sodium alginate, and results from trypsin treatment showed that the alginate degrading activity depends on this enzyme produced by Vibrio sp. PKA 1003. These results suggest that Vibrio sp. PKA 1003 and its alginate-degrading crude enzyme is useful for the production of alginate oligosaccharides.

본 연구에서는 산업적으로 응용가능한 알긴산 올리고당을 효소적 분해를 이용하여 제조하기 위한 기초 연구로서 해조류로부터 알긴산 분해 활성이 우수한 미생물을 탐색하고, 분리하여 알긴산 분해 효소를 생산하는 미생물의 최적 생육 조건 및 그 조효소액의 알긴산 분해 특성을 알아보고자 하였다. 갈조류인 지충이로부터 균주를 분리하였고 16S rRNA 염기서열을 분석한 결과, Vibrio sp. V140으로 동정되었으며 PKA 1003으로 명명하였다. Vibrio sp. PKA 1003균주의 최적 생육 특성을 알아본 결과, pH 7, 3% NaCl, $25^{\circ}C$ 및 48시간이었으며, 6% 알긴산과 1:1 배양 시 분해 효율이 가장 좋은 것으로 나타났다. Vibrio sp. PKA 1003 균주가 생산하는 조효소의 알긴산 분해 최적 조건은 pH 9, $30^{\circ}C$, 6% 알긴산 및 63시간으로 나타났으며, 배양 시간에 따라 TLC를 실시한 결과, 배양 12시간부터 서서히 분해되어 2개의 spot을 형성하는 것을 확인하였으며, 조효소액에 의해 알긴산이 여러 가지 단당 또는 올리고당으로 저분자화되는 것을 확인하였다. 또한 조효소액에 trypsin을 처리하여 실험한 결과, trypsin 처리구 및 조효소액 처리구의 환원당 생성량이 각각 70.91 및 $538.62{\mu}g/mL$로써 알긴산 분해 활성이 효소에 의한 것임을 확인하였다. 이상의 결과들을 종합해 볼 때, Vibrio sp. PKA 1003의 최적 생육 조건 및 알긴산 분해 최적 조건에서 알긴산 분해 효소 및 알긴산 올리고당을 효율적으로 획득할 수 있을 것으로 사료된다.

Keywords

References

  1. Joo DS, Lee JS, Park JJ, Cho SY, Kim HK, Lee EH. 1996. Preparation of oligosaccharides from alginic acid by enzymic hydrolysis. Korean J Food Sci Technol 28: 146-151.
  2. Joo DS, Lee EH. 1993. Purification of extracellular enzyme produced by Vibrio sp. AL-145. J Korean Soc Food Nutr 22: 234-239.
  3. Shin JW. 2012. Cloning and characterization of oligomeric alginate lyase from Stenotrophomonas sp. KJ-2. MS Thesis. Kyung Hee University, Gyeonggi-do, Korea.
  4. Lee JH, Lee EY. 2003. Isolation of alginate-degrading marine bacteria and characterization of alginase. J Life Sci 13: 718-722. https://doi.org/10.5352/JLS.2003.13.5.718
  5. Jung DY, Son CW, Kim SK, Kim YJ, Chung CH, Lee JW. 2009. Biosorption of metal ions by seaweed alginate, polyguluronate and polymannuronate. J Life Sci 19: 553-560. https://doi.org/10.5352/JLS.2009.19.5.553
  6. Lange III LG, Spilburg CA. 1989. Use of sulfated polysaccharides to decrease cholesterol and fatty acid absorption. US Patent 5,063,210.
  7. Choi JH, Kim DW. 1997. Effect of alginic acid-added functional drink (HAEJOMIIN) in brown algae (Undaria pinnatifida) on obesity and biological activity of SD rats. Korean J Life Sci 7: 361-370.
  8. Lee BH, Lee SB, Kim WK. 2009. Alginate fiber. Fiber Tech Ind 13: 21-24.
  9. Davidson IW, Sutherland IW, Lawson CJ. 1976. Purification and properties of an alginate lyase from a marine bacterium. Biochem J 159: 707-713. https://doi.org/10.1042/bj1590707
  10. Güven KC, Özsoy Y, Ulutin ON. 1991. Anticoagulant, fibrinolytic and antiaggregant activity of carrageenans and alginic acid. Botanica Marina 34: 429-432.
  11. Joo DS, Lee JS, Park JJ, Cho SY, Ahn CB, Lee EH. 1995. Purification and characterization of the intracellular alginase from Vibrio sp. AL-145. Kor J Appl Microbiol Biotechnol 23: 432-438.
  12. Kaneko Y, Yonemoto Y, Okayama K, Kimura A, Murata K. 1990. Symbiotic formation of alginate lyase in mixed culture of bacteria isolated from soil. J Ferment Bioeng 69: 192-194. https://doi.org/10.1016/0922-338X(90)90047-Z
  13. Takeuchi T, Murata K, Kusakabe I. 1994. A method for depolymerization of alginate using the enzyme system of Flavobacterium multivolum. Nippon Shokuhin Kogyo Gakkaishi 41: 505-511. https://doi.org/10.3136/nskkk1962.41.505
  14. Joo DS, Lee JS, Cho SY, Shin SJ, Lee EH. 1995. Changes in functional properties of alginic acid by enzymatic degradation. Korean J Food Sci Technol 27: 86-91.
  15. Kim OJ, Lee DG, Lee SM, Lee SJ, Do HJ, Park HJ, Kim A, Lee JH, Ha JM. 2010. Isolation and characteristics of alginate-degrading Methylobacterium sp. HJM27. Kor J Microbiol Biotechnol 38: 144-150.
  16. Wasikiewicz JM, Yoshii F, Nagasawa N, Wach RA, Mitomo H. 2005. Degradation of chitosan and sodium alginate by gamma radiation, sonochemical and ultraviolet methods. Radiat Phys Chem 73: 287-295. https://doi.org/10.1016/j.radphyschem.2004.09.021
  17. Lee DS, Kim HR, Pyeun JH. 1998. Effect of low-molecularization on rheological properties of alginate. J Korean Fish Soc 31: 82-89.
  18. Lee JH, Bae MJ, Kim YC, Nam SW. 2009. Identification and characterization of alginate lyase producing Pseudomonas sp. N7151-6. Kor J Microbiol Biotechnol 37: 350-354.
  19. Kim OJ. 2011. Isolation and characterization of alginate degrading bacteria separated from brown seaweed. MS Thesis. Silla University, Busan, Korea.
  20. Uo MH, Joo DS, Cho SY, Min TS. 2006. Purification and characterization of the extracellular alginase produced by Bacillus licheniformis AL-577. J Korean Soc Food Sci Nutr 35: 231-237. https://doi.org/10.3746/jkfn.2006.35.2.231
  21. Gacesa P. 1987. Alginate modifying enzymes: a proposed unified mechanism of action for the lyases and epimerases. FEBS Lett 212: 199-202. https://doi.org/10.1016/0014-5793(87)81344-3
  22. Michaud P, Da Costa A, Courtois B, Courtois J. 2003. Polysaccharide lyases: recent developments as biotechnological tools. Crit Rev Biotechnol 23: 233-266. https://doi.org/10.1080/07388550390447043
  23. Wong TY, Preston LA, Schiller NL. 2000. Alginate lyase: review of major sources and enzyme characteristics, structure- function analysis, biological roles, and application. Annu Rev Microbiol 54: 289-340. https://doi.org/10.1146/annurev.micro.54.1.289
  24. Hu X, Jiang X, Hwang HM. 2006. Purification and characterization of an alginate lyase from marine bacterium Vibrio sp. mutant strain 510-64. Curr Microbiol 53: 135-140. https://doi.org/10.1007/s00284-005-0347-9
  25. Kim DE, Lee EY, Kim HS. 2009. Cloning and characterization of alginate lyase from a marine bacterium Streptomyces sp. ALG-5. Mar Biotechnol (NY) 11: 10-16. https://doi.org/10.1007/s10126-008-9114-9
  26. Iwamoto Y, Araki R, Iriyama K, Oda T, Fukuda H, Hayashida S, Muramatsu T. 2001. Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates. Biosci Biotechnol Biochem 65: 133-142. https://doi.org/10.1271/bbb.65.133
  27. Nakada HI, Sweeny PC. 1967. Alginic acid degradation by eliminases from abalone hepatopancreas. J Biol Chem 242: 845-851.
  28. Nisizawa K, Fujibayashi S, Kashiwabara Y. 1968. Alginate lyases in the hepatopancreas of a marine mollusc, Dolabella auricula Solander. J Biochem 64: 25-37. https://doi.org/10.1093/oxfordjournals.jbchem.a128859
  29. Eppley RW, Lasker R. 1959. Alginase in the sea urchin Strongylocentrotus purpuratus. Science 129: 214-215. https://doi.org/10.1126/science.129.3343.214
  30. Elyakova LA, Favorov VV. 1974. Isolation and certain properties of alginate lyase VI from the mollusk Littorina sp. Biochimica et Biophysica Acta (BBA)-Enzymology 358: 341-354. https://doi.org/10.1016/0005-2744(74)90464-1
  31. Muramatsu T, Hirose S, Kayose M. 1977. Isolation and properties of alginate lyase from the mid-gut gland of wreath shell Turbo cornutus. Agric Biol Chem 41: 1939-1946. https://doi.org/10.1271/bbb1961.41.1939
  32. Joo DS, Cho SY, Lee EH. 1993. Isolation of alginate-degrading bacteria and production of alginate-degrading activities by the bacteria. Kor J Appl Microbiol Biotechnol 21: 207-213.
  33. Eller J, Payne WJ. 1960. Studies on bacterial utilization of uronic acids IV. J Bacteriol 80: 193-199.
  34. Nakagawa A, Ozaki T, Chubachi K, Hosoyama T, Okubo T, Iyobe S, Suzuki T. 1998. An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase. J Appl Microbiol 84: 328-335. https://doi.org/10.1046/j.1365-2672.1998.00319.x
  35. Kim HS, Bae TJ. 2002. Studies on the hydrolysis of seaweed using microorganisms and its application. J Korean Fish Soc 35: 438-444. https://doi.org/10.5657/kfas.2002.35.4.438
  36. Stevens RA, Levin RE. 1976. Viscometric assay of bacterial alginase. Appl Environ Microbiol 31: 896-899.
  37. Nelson N. 1994. A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153: 375-380.
  38. Li JW, Dong S, Song J, Li CB, Chen XL, Xie BB, Zhang YZ. 2011. Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524. Mar Drugs 9: 109-123. https://doi.org/10.3390/md9010109
  39. Baxter RM. 1959. An interpretation of the effects of salts on the lactic dehydrogenase of Halobacterium salinarium. Can J Microbiol 5: 47-57. https://doi.org/10.1139/m59-006
  40. Uo MH, Joo DS, Cho SY. 2006. Screening and cultivation characteristics of alginate degrading bacteria. J Korean Soc Food Sci Nutr 35: 109-114. https://doi.org/10.3746/jkfn.2006.35.1.109
  41. Yonemoto Y, Mutata K, Kimura A, Yamaguchi H, Okayama K. 1991. Bacterial alginate lyase: characterization of alginate lyase-producing bacteria and purification of the enzyme. J Ferment Bioeng 72: 152-157. https://doi.org/10.1016/0922-338X(91)90208-X
  42. Takeshita S, Sato N, Igarashi M, Muramatsu T. 1993. A highly denaturant-durable alginate lyase from a marine bacterium: purification and properties. Biosci Biotech Biochem 57: 1125-1128. https://doi.org/10.1271/bbb.57.1125