Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Characterization of a Novel ${\alpha}$-Amylase from a Fecal Microbial Metagenome

  • Xu, Bo (School of Life Science, Yunnan Normal University) ;
  • Yang, Fuya (School of Life Science, Yunnan Normal University) ;
  • Xiong, Caiyun (School of Life Science, Yunnan Normal University) ;
  • Li, Junjun (School of Life Science, Yunnan Normal University) ;
  • Tang, Xianghua (School of Life Science, Yunnan Normal University) ;
  • Zhou, Junpei (School of Life Science, Yunnan Normal University) ;
  • Xie, Zhenrong (School of Life Science, Yunnan Normal University) ;
  • Ding, Junmei (School of Life Science, Yunnan Normal University) ;
  • Yang, Yunjuan (School of Life Science, Yunnan Normal University) ;
  • Huang, Zunxi (School of Life Science, Yunnan Normal University)
  • Received : 2013.11.05
  • Accepted : 2014.01.02
  • Published : 2014.04.28
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Abstract

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for ${\alpha}$-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel ${\alpha}$-amylase from a gastrointestinal metagenomic library.

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References

  1. Bao L, Huang Q, Chang L, Sun Q, Zhou J, Lu H. 2012. Cloning and characterization of two $\beta$-glucosidase/xylosidase enzymes from yak rumen metagenome. Appl. Biochem. Biotechnol. 166: 72-86. https://doi.org/10.1007/s12010-011-9405-x
  2. Da Lage JL, Feller G, Jane ek S. 2004. Horizontal gene transfer from Eukarya to bacteria and domain shuffling: the $\alpha$-amylase model. Cell Mol. Life Sci. 61: 97-109. https://doi.org/10.1007/s00018-003-3334-y
  3. Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, et al. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl. Microbiol. Biotechnol. 75: 319-328. https://doi.org/10.1007/s00253-006-0820-9
  4. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, et al. 2005. Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ. Microbiol. 7: 1996-2010. https://doi.org/10.1111/j.1462-2920.2005.00920.x
  5. Ferrer M, Ghazi A, Beloqui A, Vieites JM, Lopez-Cortes N, Marin-Navarro J, et al. 2012. Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen. PLoS One 7: e38134. https://doi.org/10.1371/journal.pone.0038134
  6. Gong X, Gruninger RJ, Qi M, Paterson L, Forster RJ, Teather RM, McAllister TA. 2012. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene. BMC Res. Notes 5: 566. https://doi.org/10.1186/1756-0500-5-566
  7. Guo H, Feng Y, Mo X, Duan C, Tang J, Feng J. 2008. Cloning and expression of a beta-glucosidase gene umcel3G from metagenome of buffalo rumen and characterization of the translated product. Sheng Wu G ong C heng X ue B ao (Chinese) 24: 232-238.
  8. Hostinova E, Jane ek S, Gasper k J. 2010. Gene sequence, bioinformatics and enzymatic characterization of $\alpha$-amylase from Saccharomycopsis fibuligera KZ. Protein J. 29: 355-364. https://doi.org/10.1007/s10930-010-9260-6
  9. Janecek S. 2002. How many conserved sequence regions are there in the $\alpha$-amylase family? Biologia 57(Suppl. 11): 29-41.
  10. MacGregor EA, Janecek S, Svensson B. 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
  11. Machovi M, Jane ek S. 2003. The invariant residues in the $\alpha$-amylase family: just the catalytic triad. Biologia (Bratisl) 58: 1127-1132.
  12. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 32: 426-428.
  13. Morita H, Kuwahara T, Ohshima K, Sasamoto H, Itoh K, Hattori M, et al. 2007. An improved DNA isolation method for metagenomic analysis of the microbial flora of the human intestine. Microb. Environ. 22: 214-222. https://doi.org/10.1264/jsme2.22.214
  14. Nimchua T, Thongaram T, Uengwetwanit T, Pongpattanakitshote S, Eurwilaichitr L. 2012. Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes. J. Microbiol. Biotechnol. 22: 462-469. https://doi.org/10.4014/jmb.1108.08037
  15. Rashamuse KJ, Visser DF, Hennessy F, Kemp J, Roux-van der Merwe MP, Badenhorst J, et al. 2013. Characterisation of two bifunctional cellulase-xylanase enzymes isolated from a bovine rumen metagenome library. Curr. Microbiol. 66: 145-151. https://doi.org/10.1007/s00284-012-0251-z
  16. Stam MR, Danchin EGJ, Rancurel C, Coutinho PM, Henrissat B. 2006. Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of $\alpha$-amylase-related proteins. Protein Eng. Des. Sel. 19: 555-562. https://doi.org/10.1093/protein/gzl044
  17. Streit WR, Schmitz RA. 2004. Metagenomics: the key to the uncultured microbes. Curr. Opin. Biotechnol. 7: 492-498.
  18. Tasse L, Bercovici J , Pizzut-Serin S , Robe P, Tap J , Klopp C, et al. 2010. Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes. Genome Res. 20: 1605-1612. https://doi.org/10.1101/gr.108332.110
  19. Van der Kaaij RM, Jane ek S, van der Maarel MJEC, Dijkhuizen L. 2007. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal $\alpha$-amylase enzymes. Microbiology 153: 4003-4015. https://doi.org/10.1099/mic.0.2007/008607-0
  20. Walter J, Mangold M, Tannock GW. 2005. Construction, analysis, and $\beta$-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice. Appl. Environ. Microbiol. 71: 2347-2354. https://doi.org/10.1128/AEM.71.5.2347-2354.2005
  21. Warnecke F, Luginbuhl P, Ivanova N, Ghassemian M, Richardson TH, Stege JT, et al. 2007. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450: 560-565. https://doi.org/10.1038/nature06269
  22. Xu B, Xu W, Yang F, Li J, Yang Y, Tang X, et al. 2013. Metagenomic analysis of the pygmy loris fecal microbiome reveals unique functional capacity related to metabolism of aromatic compounds. PLoS One 8: e56565. https://doi.org/10.1371/journal.pone.0056565
  23. Zhao S, Wang J, Liu K, Zhu Y, Bu D, Li D, Yu P. 2009. Screening and characterization of lipase from a metagenome library of dairy rumen microflora. Sheng Wu Gong Cheng Xue Bao (Chinese) 25: 869-874.
  24. Zhao S, Wang J, Bu D, Liu K, Zhu Y, Dong Z, Yu Z. 2010. Novel glycoside hydrolases identified by screening a Chinese Holstein dairy cow rumen-derived metagenome library. Appl. Environ. Microbiol. 76: 6701-6705. https://doi.org/10.1128/AEM.00361-10
  25. Zhou J, Dong Y, Li J, Zhang R, Tang X, Mu Y, et al. 2012. Cloning, heterologous expression, and characterization of novel protease-resistant $\alpha$-galactosidase from new Sphingomonas strain. J. Microbiol. Biotechnol. 22: 1532-1539. https://doi.org/10.4014/jmb.1112.12036

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