Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Thermostable ${\alpha}$-Amyalse of Bacillus licheniformis YB-1234 Isolated from the Fermented Soybean of a Korean Buddhist Temple

사찰의 된장에서 분리된 Bacillus licheniformis YB-1234의 내열성 ${\alpha}$-Amyalse

  • Lee, Eun Ji (Department of Food Science & Biotechnology, Woosong University) ;
  • Yoon, Ki-Hong (Department of Food Science & Biotechnology, Woosong University)
  • 이은지 (우송대학교 식품생물과학과) ;
  • 윤기홍 (우송대학교 식품생물과학과)
  • Received : 2012.10.04
  • Accepted : 2012.11.21
  • Published : 2012.12.28
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Abstract

A bacterial strain was isolated from soybean paste fermented in a Korean Buddhist temple as a producer of the extracellular thermostable ${\alpha}$-amylase. The isolate YB-1234 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. A gene encoding the thermostable ${\alpha}$-amylase of B. licheniformis YB-1234 was cloned into Escherichia coli and its nucleotide sequence was determined. The deduced amino acid sequence of ${\alpha}$-amylase was very highly homologous to those of the thermostable ${\alpha}$-amylases of B. licheniformis belonging to the glycosyl hydrolase family 13. The ${\alpha}$-amylase produced by recombinant E. coli carrying the ${\alpha}$-amylase gene exhibited maximal activity at pH 6.0, identical to ${\alpha}$-amylase in the culture filtrate of B. licheniformis, while the temperature profile was somewhat different between the two. Particularly, ${\alpha}$-amylase produced from B. lcheniformis is much more thermostable than that from recombinant E. coli. The predominant products resulting from the ${\alpha}$-amylase hydrolysis were glucose, maltose and maltotriose for maltotetraose and maltohexaose.

국내 사찰에서 제조된 된장으로부터 내열성 ${\alpha}$-amylase 생산균으로 분리된 YB-1234는 형태적 특성, 생화학적 성질 및 16S rRNA 유전자 염기서열에 근거하여 Bacillus licheniformis로 동정되었다. B. licheniformis YB-1234의 ${\alpha}$-amylase 유전자를 클로닝하여 그 염기서열을 결정하였으며 그로부터 유추된 ${\alpha}$-amylase의 아미노산 서열은 glycosyl hydrolase family 13에 속하는 B. licheniformis의 내열성 ${\alpha}$-amylases와 매우 높은 상동성을 보였다. ${\alpha}$-Aamylase 유전자를 함유한 재조합 대장균과 B. licheniformis에 의해 각각 생산된 ${\alpha}$-amylase는 pH 6.0에서 최대활성을 보였으나, 최적 반응온도는 약간의 차이가 있었다. 또한 B. licheniformis로부터 ${\alpha}$-amylase는 재조합 대장균에서 생산된 효소보다 열안정성이 매우 높았다. 이들 효소에 의한 maltotetraose와 maltohexaose의 주된 가수분해산물로는 glucose, maltose 및 maltotriose가 관찰되었다.

Keywords

References

  1. Asoodeh, A., J. Chamani, and M. Lagzian. 2010. A novel thermostable, acidophilic $\alpha$-amylase from a new thermophilic "Bacillus sp. Ferdowsicous" isolated from Ferdows hot mineral spring in Iran: purification and biochemical characterization. Int. J. Biol. Macromol. 46: 289-297. https://doi.org/10.1016/j.ijbiomac.2010.01.013
  2. Bajpai, P. and P. K. Bajpai. 1989. High-temperature alkaline $\alpha$-amylase from Bacillus licheniformis TCRDC-B13. Biotechnol. Bioeng. 33: 72-78. https://doi.org/10.1002/bit.260330110
  3. Blakeney, A. B. and B. A. Stone. 1985. Activity and action pattern of Bacillus licheniformis $\alpha$-amylase in aqueous ethanol. FEBS Lett. 186: 229-232. https://doi.org/10.1016/0014-5793(85)80714-6
  4. Bose, K. and D. Das. 1996. Thermostable $\alpha$-amylase production using Bacillus licheniformis NRRL B14368. Indian J. Exp. Biol. 34: 1279-1282.
  5. Chakraborty, K., B. K. Bhattacharyya, and S. K. Sen. 2000. Purification and characterization of a thermostable $\alpha$- amylase from Bacillus stearothermophilus. Folia Microbiol. (Praha) 45: 207-210. https://doi.org/10.1007/BF02908945
  6. Declerck, N., P. Joyet, J. Y. Trosset, J. Garnier, and C. Gaillardin. 1995. Hyperthermostable mutants of Bacillus licheniformis $\alpha$- amylase: multiple amino acid replacements and molecular modelling. Protein Eng. 8: 1029-1037. https://doi.org/10.1093/protein/8.10.1029
  7. Ghalanbor, Z., N. Ghaemi, S. A. Marashi, M. Amanlou, M. Habibi-Rezaei, K. Khajeh, and B. Ranjbar. 2008. Binding of Tris to Bacillus licheniformis $\alpha$-amylase can affect its starch hydrolysis activity. Protein Pret. Lett. 15: 212-214. https://doi.org/10.2174/092986608783489616
  8. Hmidet, N., A. Bayoudh, J. G. Berrin, S. Kanoun, N. Juge, and M. Nasri. 2008. Purification and biochemical characterization of a novel $\alpha$-amylase from Bacillus licheniformis NH1: cloning, nucleotide sequence and expression of amyN gene in Escherichia coli. Proc. Biochem. 43: 499-510. https://doi.org/10.1016/j.procbio.2008.01.017
  9. Hmidet, N., H. Maalej, A. Haddar, and M.Nasri. 2010. A novel $\alpha$-amylase from Bacillus mojavensis A21: purification and biochemical characterization. Appl. Biochem. Biotechnol. 162: 1018-1030. https://doi.org/10.1007/s12010-009-8902-7
  10. Hwang, J. S., H. J. Yoo, S. J. Kim, and H. B. Kim. 2008. Characterization of $\beta$-1,4-glucanase activity of Bacillus licheniformis B1 in chungkookjang. Kor. J. Microbiol. 44: 69-73.
  11. Hwang, K. Y., H. K. Song, C. Chang, J. Lee, S. Y. Lee, K. K. Kim, S. Choe, R. M. Sweet, and S. W. Suh. 1997. Crystal structure of thermostable $\alpha$-amylase from Bacillus licheniformis refined at 1.7 A resolution. Mol. Cells 7: 251-258.
  12. Khajeh, K., S. Khezre-Barati, and M. Nemat-Gorgani. 2001. Proteolysis of mesophilic and thermophilic $\alpha$-amylases: acomparative study. Appl. Biochem. Biotechnol. 94: 97-109. https://doi.org/10.1385/ABAB:94:2:097
  13. Kikani, B. A. and S. P. Singh. 2011. Single step purification and characterization of a thermostable and calcium independent $\alpha$-amylase from Bacillus amyloliquefaciens TSWK1- 1 isolated from Tulsi Shyam hot spring reservoir, Gujarat (India). Int. J. Biol. Macromol. 48: 676-681. https://doi.org/10.1016/j.ijbiomac.2011.02.010
  14. Kim, D. H., N. Morimoto, W. Saburi, A. Mukai, K. Imoto, T. Takehana, S. Koike, H. Mori, and H. Matsui. 2012. Purification and characterization of a liquefying $\alpha$-amylase from alkalophilic thermophilic Bacillus sp. AAH-31. Biosci. Biotechnol. Biochem. 76: 1378-1383. https://doi.org/10.1271/bbb.120164
  15. Kweun, M. A., H. S. Kim, M. S. Lee, J. H. Choi, and K.-H. Yoon. 2003. Mannanase production by a soybean isolate, Bacillus subtilis WL-7 Kor. J. Microbiol. Biotechnol. 31: 277-283.
  16. Maassen, A. 1991. Comparison between $\alpha$-amylase from B. amyloliquefaciens and B. licheniformis. Biomed. Biochim. Acta 50: 213-217.
  17. Malhotra, R., S. M. Noorwez, and T. Satyanarayana. 2000. Production and partial characterization of thermostable and calcium-independent $\alpha$-amylase of an extreme thermophile Bacillus thermooleovorans NP54. Lett. Appl. Microbiol. 31: 378-384. https://doi.org/10.1046/j.1472-765x.2000.00830.x
  18. Niu, D., Z. Zuo, G. Y. Shi, and Z. X. Wang. 2009. High yield recombinant thermostable $\alpha$-amylase production using an improved Bacillus licheniformis system. Microb. Cell Fact. 8: 58. https://doi.org/10.1186/1475-2859-8-58
  19. Oh, Y.-S., J.-E. Park, H.-J. Oh, J. H. Kim, and M.-C. Oh, 2010. Isolation and characteristics of microorganisms producing extracellular enzymes from Jeju traditional fermented soybean paste (Doenjang). J. Kor. Soc. Food Sci. Nutr. 39: 47-53. https://doi.org/10.3746/jkfn.2010.39.1.047
  20. Pancha, I., D. Jain, A. Shrivastav, S. K. Mishra, B. Shethia, S. Mishra, P. M. V., and B. Jha. 2010. A thermoactive $\alpha$- amylase from a Bacillus sp. isolated from CSMCRI salt farm. Int. J. Biol. Macromol. 47: 288-291. https://doi.org/10.1016/j.ijbiomac.2010.04.006
  21. Priyadharshini, R., D. Hemalatha, and P. Gunasekaran. 2010. Role of Val289 residue in the $\alpha$-amylase of Bacillus amyloliquefaciens MTCC 610: an analysis by site directed mutagenesis. J. Microbiol. Biotechnol. 20: 563-568.
  22. Qin, Y., Z. Fang, F. Pan, Y. Zhao, H. Li, H. Wu, and X. Meng. 2012. Significance of Tyr302, His235 and Asp194 in the $\alpha$- amylase from Bacillus licheniformis. Biotechnol. Lett. 34: 895-899. https://doi.org/10.1007/s10529-011-0843-x
  23. Roy, J. K., S. K. Rai, and A. K. Mukherjee. 2012. Characterization and application of a detergent-stable alkaline $\alpha$- amylase from Bacillus subtilis strain AS-S01a. Int. J. Biol. Macromol. 50: 219-229. https://doi.org/10.1016/j.ijbiomac.2011.10.026
  24. Stephens, M. A., S. A. Ortlepp, J. F. Ollington, and D. J. McConnell. 1984. Nucleotide sequence of the 5' region of the Bacillus licheniformis $\alpha$-amylase gene: comparison with the B. amyloliquefaciens gene. J. Bacteriol. 158: 369-372.
  25. Yoon, K.-H. and H. Y. Shin. 2010. Medium optimization for the protease production by Bacillus licheniformis isolated from cheongkookjang. Kor. J. Microbiol. Biotechnol. 38: 385-390.
  26. Yuuki, T., T. Nomura, H. Tezuka, A. Tsuboi, H. Yamagata, N. Tsukagoshi, and S. Udaka. 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable $\alpha$- amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying $\alpha$-amylases deduced from the DNA sequences. J. Biochem. 98: 1147- 1156.

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