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

Streptomyces sp. M3 알긴산분해효소의 돌연변이에 의한 활성증대

  • Kim, Hee-Sook (Department of Food Science and Biotechnology, Kyungsung University)
  • 김희숙 (경성대학교 식품생명공학과)
  • Received : 2011.10.14
  • Accepted : 2011.12.26
  • Published : 2012.01.30


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.


  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.
  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.
  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.
  4. Gacesa, P. 1998. Bacterial alginate biosynthesis-recent progress and future prospects. Microbiol. 144, 1133-1143.
  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.
  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.
  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.
  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.
  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.
  10. Kim, H. S. 2010. Characterization of recombinant polyG-specific lyase from a marine bacterium, Streptomyces sp. M3. J. Life Sci. 20, 1582-1588.
  11. Kim, H. S. 2009. Cloning and expression of alginate lyase from a marine bacterium, Streptomyces sp. M3. J. Life Sci. 19, 1522-1528.
  12. Kim, H. S., C. G. Lee, and E. Y. Lee. 2011. Alginate lyase; structure, property and application. Biotechnol. Bioproc. Eng. 16, 843-851.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.
  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.