Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Enzymatic Characterization of a Thermostable 4-α-Glucanotransferase from Thermotoga neapolitana

Thermotoga neapolitana 유래 내열성 4-알파-글루칸전이효소의 효소적 특성

  • Choi, Kyoung-Hwa (Department of Microbiology, Pusan National University) ;
  • Seo, Ja-Yeong (Department of Microbiology, Pusan National University) ;
  • Kim, Ji-Eun (Department of Microbiology, Pusan National University) ;
  • Cha, Jae-Ho (Department of Microbiology, Pusan National University)
  • Received : 2011.01.10
  • Accepted : 2011.01.31
  • Published : 2011.02.28
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Abstract

The gene encoding 4-$\alpha$-glucanotransferase (mgtA) from Thermotoga neapolitana was cloned and expressed in Escherichia coli in order to investigate whether this enzyme was capable of producing cycloamylose for industrial applications. MgtA was purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatographies. The size of the enzyme as determined by SDS-PAGE was about 52 kDa, which was in good agreement with its deduced molecular mass of 51.9 kDa. The optimal temperature and pH for the activity of the 4-$\alpha$-glucanotransferase was found to be $85^{\circ}C$ and 6.5, respectively. The enzyme hydrolyzed the 1,4-$\alpha$-glucosidic bonds in oligomeric 1,4-$\alpha$-glucans and transferred oligosaccharides (maltotriose being the shortest one) to acceptor maltodextrins. However, the enzymes had no activity against pullulan, glycogen, and other di- or trioligosaccharides with rare types of $\alpha$-bond. MgtA is distinguished from 4-$\alpha$-glucanotransferase from Thermotoga maritima in that it can convert maltotriose into maltooligosaccharides. The treatment of glucoamylase after the reaction of MgtA with maltotriose, maltotetraose, maltopentaose, or maltohexaose as sole substrate revealed that MgtA yielded linear maltooligosaccharides instead of cycloamylose.

Thermotoga neapolitana 유래 내열성 4-알파-글루칸전이효소(MgtA)가 산업적 응용성을 지닌 싸이클로아밀로스를 생산할 수 있는지를 검사하기 위하여 그 유전자를 클로닝하고 대장균에서 발현시켰다. MgtA는 HiTrap Q와 Sephacryl S-200 분배 크로마토그래피를 이용하여 순수한 형태로 정제되었으며, SDS-PAGE를 통하여 분자량이 약 52 kDa으로 아미노산서열로부터 계산된 분자량과 일치하였다. 효소활성의 최적 pH와 온도는 6.5와 $85^{\circ}C$였으며 알파1,4결합을 갖는 글루칸의 1,4결합을 효율적으로 가수분해함과 동시에 작은 크기의 올리고당을 말토덱스트린에 전이하는 전이활성을 가지고 있었다. 그러나 플루란, 글리코겐 및 1,4결합 이외의 다른 알파결합을 갖는 글루칸에는 활성을 나타내지 않았다. MgtA는 말토트리오스를 말토올리고당으로 전환할 수 있는 능력에서 그렇지 못한 Thermotoga maritima 의 효소와 구별할 수 있었으며, 반응 후 glucoamylase의 처리결과로부터 그 전이산물이 싸이클로아밀로스 대신에 긴 연쇄상의 말토올리고당을 생산하는 효소로 확인할 수 있었다.

Keywords

References

  1. Berezina, O. V., V. V. Zverlov, N. A. Lunina, L. A. Chekanovskaya, E. N. Dubinina, W. Liebl, and G. A. Velikodvorskaya. 1999. Genes and properties of thermostable 4-${\alpha}$-glucanotransferase of Thermotoga neapolitana. Mol. Biol. 33, 801-806.
  2. Bhuiyan, S. H., M. Kitaoka, and K. Hayashi. 2003. A cycloamylose-forming hyperthermostable 4-${\alpha}$-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli. J. Mol. Catal. B: Enzym. 22, 45-53. https://doi.org/10.1016/S1381-1177(03)00005-5
  3. Bibel, M., C. Brettl, U. Gosslar, G. Kriegshauser, and W. Liebl. 1998. Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima. FEMS Microbiol. Lett. 158, 9-15. https://doi.org/10.1111/j.1574-6968.1998.tb12793.x
  4. Boos, W. and H. Shuman. 1998. Maltose/maltodextrin system of Escherichia coli: Transport, metabolism, and regulation. Microbiol. Mol. Biol. Rev. 62, 204-229.
  5. Coutinho, P. M. and B. Henrissat. 1999. Carbohydrate-active enzymes: An integrated database approach, in Recent Advances in Carbohydrate Bioengineering. In Gilbert H. J., G. Davies, B. Henrissat, and B. Svensson (eds.), pp. 3-12, The Royal Society of Chemistry, Cambridge, England.
  6. Goda, S. K., O. Eissa, M. Akhtar, and N. P. Minton. 1997. Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-${\alpha}$-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli. Microbiology 143, 3287-3294. https://doi.org/10.1099/00221287-143-10-3287
  7. Heinrich, P., W. Huber, and W. Liebl. 1994. Expression in Escherichia coli and structure of the gene encoding 4-${\alpha}$-glucanotransferase from Thermotoga maritima. Classification of maltodextrin glycosyltransferases into two distantly related enzyme subfamilies. Syst. Appl. Microbiol. 17, 297-305. https://doi.org/10.1016/S0723-2020(11)80044-5
  8. Jeon, B. S., H. Taguchi, H. Sakai, T. Ohshima, T. Wakagi, and H. Matsuzawa. 1997. 4-${\alpha}$-glucanotransferase from a hyperthermophilic archaeon, Thermococcus litoralis: enzyme purification and characterization, and gene cloning, sequencing and expression in Escherichia coli. Eur. J. Biochem. 248, 171-178. https://doi.org/10.1111/j.1432-1033.1997.00171.x
  9. Kaper, T., B. Talik, T. J. Ettema, H. Bos, M. J. E. C. Van der Maarel, and L. Dijkhuizen. 2005. The amylomaltase of Pyrobaculum aerophilum IM2 produces thermo-reversible starch gels. Appl. Environ. Microbiol. 71, 5098-5106. https://doi.org/10.1128/AEM.71.9.5098-5106.2005
  10. Liebl, W., R. Feil, J. Gabelsberger, J. Kellermann, and K. H. Schleifer. 1992. Purification and characterization of a novel thermostable 4-${\alpha}$-glucanotransferase of Thermotoga maritima cloned in Escherichia coli. Eur. J. Bochem. 207, 81-88. https://doi.org/10.1111/j.1432-1033.1992.tb17023.x
  11. MacGregor, E. A., S. Janecek, and B. Svensson. 2001. Relationship of sequence and structure to specificity in the ${\alpha}$-amylase family of enzymes. Biochim. Biophys. Acta 1546, 1-20. https://doi.org/10.1016/S0167-4838(00)00302-2
  12. Machida, S., S. Ogawa, S. Xiaohua, T. Takaha, K. Fujii, and K. Hayashi. 2000. Cycloamylose as an efficient artificial chaperone for protein refolding. FEBS Lett. 486, 131-135. https://doi.org/10.1016/S0014-5793(00)02258-4
  13. Monod, J. and A. M. Torriani. 1948. Synthese d’un polysaccharide de type amidon aux depens du maltose, en presence d’un extrait enzymatique d’origine bacterienne. C.R. Acad. Sci. 227, 240-242.
  14. Palmer, T. N., B. E. Ryman, and W. J. Whelan. 1976. The action pattern of amylolmaltase from Escherichia coli. Eur. J. Biochem. 69, 105-115. https://doi.org/10.1111/j.1432-1033.1976.tb10863.x
  15. Pazur, J. H. and S. Okada. 1968. The isolation and mode of action of a bacterial glucanotransferase. J. Biol. Chem. 243, 4732-4738.
  16. Schinzel, R. and B. Nidetzky. 1999. Bacetrial ${\alpha}$-glucan phosphorylases. FEMS Microbiol. Lett. 171, 73-79.
  17. Schmidt, J. and M. John. 1979. Starch metabolism in Pseudomonas stutzeri. II. Purification and properties of a dextrin glycosyl-transferase (D-enzyme) and amylomaltase. Biochim. Biophys. Acta 566, 100-114. https://doi.org/10.1016/0005-2744(79)90253-5
  18. Tachibana, Y., T. Takaha, S. Hujiwara, M. Takagi, and T. Imanaka. 2000. Acceptor specificity of 4-${\alpha}$-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose. J. Biosci. Bioeng. 90, 406-409. https://doi.org/10.1016/S1389-1723(01)80009-8
  19. Takaha, T., M. Yanase, S. Okada, and S. M. Smith. 1993. Disproportionating enzyme (4-${\alpha}$-glucanotransferase; EC 2.4.1.25) of potato. Purification, molecular cloning, and potential role in starch metabolism. J. Biol. Chem. 268, 1391-1396.
  20. Takaha, T., M. Yanase, H. Takata, S. Okada, and S. M. Smith. 1996. Potato D-enzyme catalyzes the cyclization of amylose to produce cycloamylose, a novel cyclic glucan. J. Biol. Chem. 271, 2902-2908. https://doi.org/10.1074/jbc.271.6.2902
  21. Takaha, T. and S. M. Smith. 1999. The functions of 4-${\alpha}$-glucanotransferases and their use for the production of cyclic glucans. Biotechnol. Genet. Eng. Rev. 16, 257-280. https://doi.org/10.1080/02648725.1999.10647978
  22. Terada, Y., K. Fujii, T. Takaha, and S. Okada. 1999. Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose. Appl. Environ. Microbiol. 65, 910-915.
  23. Park, T. H., K. W. Choi, C. S. Park, S. B. Lee, H. Y. Kang, K. J. Shon, J. S. Park, and J. Cha. 2005. Substrate specificity and transglycosylation catalyzed by a thermostable ${\beta}$-glucosidase form marine hyperthermophile Thermotoga neapolitana, Appl. Microbiol. Biotechnol. 69, 411-422. https://doi.org/10.1007/s00253-005-0055-1
  24. Van der Maarel, M. J. E. C., I. Caprona, G. J. W. Euverink, H. T. P. Bos, T. Kaper, D. J. Binnema, and P. Steeneken. 2005. A novel thermoreversible gelling product made by enzymatic modification of starch. Starch 57, 465-472. https://doi.org/10.1002/star.200500409
  25. Zona, R., F. Chang-Pi-Hin, M. J. O’Donohue, and S. Janecek. 2004. Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis. Eur. J. Bochem. 271, 2863-2872. https://doi.org/10.1111/j.1432-1033.2004.04144.x