생명과학회지 (Journal of Life Science)

  • 제22권1호
  • /
  • Pages.7-15
  • /
  • 2012
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

Streptomyces sp. M3 알긴산분해효소의 돌연변이에 의한 활성증대

Enhancing the Alginate Degrading Activity of Streptomyces sp. Strain M3 Alginate Lyase by Mutation

  • 김희숙 (경성대학교 식품생명공학과)
  • Kim, Hee-Sook (Department of Food Science and Biotechnology, Kyungsung University)
  • 투고 : 2011.10.14
  • 심사 : 2011.12.26
  • 발행 : 2012.01.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

이전 연구에서 Streptomyces sp. M3 균주로부터 polyguluronate에 기질특이성을 가지는 알긴산분해효소를 cloning하고 활성을 연구하였다. 이번 연구에서는 pColdI vector에 들어있는 M3 알긴산분해효소 유전자를 돌연변이시켜 알긴산분해효소의 활성을 증진시키고자 하였으며, 점-돌연변이 또는 무작위-돌연변이 방법을 사용하여 돌연변이를 실시하였다. Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu 및 Gly194Cys 등 6 종류의 돌연변이 단백질을 얻을 수 있었다. Phe99Leu 및 Lys191Glu 돌연변이 단백질은 알긴산을 분해하는 능력을 완전히 잃었으나 Gly194Cys 돌연변이 단백질의 활성은 원래 단백질에 비하여 10배 증가하였다. 또한 돌연변이된 M3 알긴산분해효소 단백질의 3차 구조는 Swiss-Model 자동모델러를 이용하여 생성하였으며 다른 알긴산분해효소의 결정구조와 비교하였다. 194 번째 아미노산인 글리신은 알긴산의 C-말단 보존서열인 YFKAGXYXQ의 Gly193과 Tyr195 사이에 위치한다. 이 연구에서 돌연변이된 글리신과 페닐알라닌 잔기들은 활성자리로부터 많이 떨어져있음에도 불구하고 돌연변이에 의하여 알긴산 분해활성이 강하게 영향을 받는 것으로 나타났다.

A polyguluronate-specific lyase from Streptomyces sp. strain M3 has been previously cloned and characterized. In this study, the M3 alginate lyase gene in the pColdI vector was mutated by site-directed mutagenesis and random mutagenesis to enhance the alginate degrading activity. Six mutants were obtained: Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu, and Gly194Cys. Phe99Leu and Lys191Glu mutants completely lost their alginate lyase activity, whereas the alginate degrading activity of Gly194Cys mutant increased by nearly 10 fold. The 3-D protein structure of M3 alginate lyase, which was constructed using the Swiss-Model automodeler, was also compared to the crystal structure of another alginate lyase. A mutated glycine residue was positioned between Gly193 and Tyr195 of the C-terminal conserved sequence, YFKAGXYXQ. A phenylalanine residue (at position 99) and a glycine residue (at position 194) mutated in this study were distant from the active site, but the degrading activity was strongly affected by their mutation.

키워드

참고문헌

  1. Arnold, K., L. Bordoli, J. Kopp, and T. Schwede. 2006. The SWISS-MODEL workspace, A web-based environment for protein structure homology modelling. Bioinformatics 22, 195-201. https://doi.org/10.1093/bioinformatics/bti770
  2. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  3. Choi, S. H., H. S. Kim, and E. Y. Lee. 2009. Comparative homology modeling-inspired protein engineering for improvement of catalytic activity of Mugil cephalus epoxide hydrolase. Biotechnol. Letters 31, 1617-1624. https://doi.org/10.1007/s10529-009-0055-9
  4. Gacesa, P. 1998. Bacterial alginate biosynthesis-recent progress and future prospects. Microbiol. 144, 1133-1143. https://doi.org/10.1099/00221287-144-5-1133
  5. Gimmestad, M., H. Ertesvag, T. M. B. Heggeset, O. Aarstad, B. I. G. Svanem, and S. Valla. 2009. Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination. J. Bacteriol. 191, 4845-4853. https://doi.org/10.1128/JB.00455-09
  6. Haug, A., B. Larsen, and O. Smidsrot. 1966. A study of the constitution of alginic acid by partial acid hydrolysis. Acta. Chem. Scand. 20, 183-190. https://doi.org/10.3891/acta.chem.scand.20-0183
  7. Huang, W., A. Matte, Y. Li, Y. S. Kim, R. J. Linhardt, H. Su, and M. Cygler. 1999. Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 $\AA$ resolution. J. Mol. Biol. 294, 1257-1269. https://doi.org/10.1006/jmbi.1999.3292
  8. Heyraud, A., C. Gey, C. Leonard, C. Rochas, S. Girond, and B. Kloareg. 1996. NMR spectroscopy analysis of oligoguluronates and oligomannuronates prepared by acid or enzymatic hydrolysis of homopolymeric blocks of alginic acid. application to the determination of the substrate specificity of Haliotis tuberculata alginate lyase. Carbohydr. Res. 289, 11-23. https://doi.org/10.1016/0008-6215(96)00060-2
  9. Kam, N., Y. J. Park, E. Y. Lee, and H. S. Kim. 2011. Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp. strain KS-408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate. Can. J. Microbiol. 57, 1032-1041. https://doi.org/10.1139/w11-106
  10. Kim, H. S. 2010. Characterization of recombinant polyG-specific lyase from a marine bacterium, Streptomyces sp. M3. J. Life Sci. 20, 1582-1588. https://doi.org/10.5352/JLS.2010.20.11.1582
  11. Kim, H. S. 2009. Cloning and expression of alginate lyase from a marine bacterium, Streptomyces sp. M3. J. Life Sci. 19, 1522-1528. https://doi.org/10.5352/JLS.2009.19.11.1522
  12. Kim, H. S., C. G. Lee, and E. Y. Lee. 2011. Alginate lyase; structure, property and application. Biotechnol. Bioproc. Eng. 16, 843-851. https://doi.org/10.1007/s12257-011-0352-8
  13. Kim, D. E., E. Y. Lee, and H. S. Kim. 2009. Cloning and characterization of alginate lyase from a marine bacterium Streptomyces sp. ALG-5. Marine Biotechnol. 11, 10-16. https://doi.org/10.1007/s10126-008-9114-9
  14. Miyake, O., W. Hashimoto, and K. Murata. 2003. An exotype alginate lyase in Sphingomonas sp. A1, Overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV). Protein Expres. Purif. 29, 33-41. https://doi.org/10.1016/S1046-5928(03)00018-4
  15. Ochiai, A., W. Hashimoto, and K. Murata. 2006. A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58, Molecular identification of Atu3025 as an exotype family PL-15 alginate lyase. Res. Microbiol. 157, 642-649. https://doi.org/10.1016/j.resmic.2006.02.006
  16. Ochiai, A., M. Yamasaki, B. Mikami, W. Hashimoto, and K. Murata. 2010. Crystal structure of exotype alginate lyase Atu3025 from Agrobacterium tumefaciens. J. Biol. Chem. 285, 24519-24528. https://doi.org/10.1074/jbc.M110.125450
  17. Ogura, K., M. Yamasaki, T. Yamada, B. Mikami, W. Hashimoto, and K. Murata. 2009. Crystal structure of family 14 polysaccharide lyase with pH-dependent modes of action. J. Biol. Chem. 284, 35572-35579. https://doi.org/10.1074/jbc.M109.068056
  18. Osawa, T., Y. Matsubara, T. Muramatsu, M. Kimura, and Y. Kakuta. 2005. Crystal structure of the alginate (poly $\alpha$-L-guluronate) lyase from Corynebacterium sp. at 1.2 $\AA$resolution. J. Mol. Biol. 345, 1111-1118. https://doi.org/10.1016/j.jmb.2004.10.081
  19. Park, H. H., N. Kam, E. Y. Lee, and H. S. Kim. 2011. Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar. Biotechnol. Online (DOI 10.1007/s10126-011-9402-7)
  20. Sawabe, T., H. Takahashi, Y. Ezura, and P. Gacesa. 2001. Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydr. Res. 335, 11-21. https://doi.org/10.1016/S0008-6215(01)00198-7
  21. Tondervik, A., G. Klinkenberg, O. A. Aarstad, F. Drablos, H. Ertesvag, T. E. Ellingsen, G. Skjak-Bræk, S. Valla, and H. Sletta. 2010. Isolation of mutant alginate lyases with cleavage specificity for di-guluronic acid linkages. J. Biol. Chem. 285, 35284-35292. https://doi.org/10.1074/jbc.M110.162800
  22. Weissbach, A. and J. Hurwitz. 1959. The formation of 2-keto- 3-deoxyheptonic acid in extracts of Escherichia. J. Biol.Chem. 234, 705-709.
  23. Wong, T. Y., L. A. Preston, and N. L. Schiller. 2000. Alginate lyase; Review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu. Rev. Microbiol. 54, 289-340. https://doi.org/10.1146/annurev.micro.54.1.289
  24. Xiao, L., F. Han, Z. Yang, X. Z. Lu, and W. G. Yu. 2006. A novel alginate lyase with high activity on acetylated alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03. World J. Microbiol. Biotechnol. 22, 81-88. https://doi.org/10.1007/s11274-005-7713-4
  25. Yamamoto, S., T. Sahara, D. Sato, K. Kawasaki, S. Ohgiya, A. Inoue, and T. Ojima. 2008. Catalytically important amino- acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis. Enz. Microb. Technol. 43, 396-402. https://doi.org/10.1016/j.enzmictec.2008.06.006
  26. Yamasaki, M., S. Moriwaki, O. Miyake, W. Hashimoto, K. Murata, and B. Mikami. 2004. Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7, a novel alginate lyase with a β -sandwich fold. J. Biol. Chem. 279, 31863-31872. https://doi.org/10.1074/jbc.M402466200
  27. Yoon, H. J., W. Hashimoto, O. Miyake, K. Murata, and B. Mikami. 2001. Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 $\AA$ resolution. J. Mol. Biol. 307, 9-16. https://doi.org/10.1006/jmbi.2000.4509