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Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases

Abstract

A metagenomic cosmid library was prepared in Escherichia coli from DNA extracted from the contents of rabbit cecum and screened for cellulase activities. Eleven independent clones expressing cellulase activities (four endo-β-1,4-glucanases and seven β-glucosidases) were isolated. Subcloning and sequencing analysis of these clones identified 11 cellulase genes; the encoded products of which shared less than 50% identities and 70% similarities to cellulases in the databases. All four endo-β-1,4-glucanases and all seven β-glucosidases, respectively, belonged to glycosyl hydrolase family 5 (GHF 5) and family 3 (GHF 3) and formed two separate branches in the phylogenetic tree. Ten of the 11 cloned cellulases exhibited highest activities at pH 5.5 ∼ 7.0 and 40 ∼ 55°C, a condition similar to that in the rabbit cecum. All the four endo-β-1,4-glucanases could hydrolyze a wide range of β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides. One endo-β-1, 4-glucanase gene, umcel5G, was overexpressed in E. coli, and the purified recombinant enzyme was characterized in detail. The enzymes cloned in this work represented at least some of the cellulases operating efficiently in the rabbit cecum. This work provides the first snapshot on the cellulases produced by bacteria in rabbit cecum.

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References

  1. Abecia L, Fondevila M, Balcells J, Edwards JE, Newbold CJ, McEwan NR (2005) Molecular profiling of bacterial species in the rabbit cecum. FEMS Microbiol Lett 244:111–115

  2. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

  3. Bhatia Y, Mishra S, Bisaria V (2005) Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II with high hydrolytic activity on sophorose. Appl Microbiol Biotechnol 66:527–535

  4. Blanco A, Diaz P, Martinez J, Vidal T, Torres AL, Pastor FIJ (1998) Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw. Appl Microbiol Biotechnol 50:48–54

  5. Bradford MM (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

  6. Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23:321–329

  7. Eberhart B, Cross DF, Chase LR (1964) β-Glucosidase system of Neurospora crassa I. β-Glucosidase and cellulase activities of mutant and wild-type strains. J Bacteriol 87:761–770

  8. Eckert K, Schneider E (2003) A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases. Eur J Biochem 270:3593–3602

  9. Feng JX, Karita S, Fujino E, Fujino T, Kimura T, Sakka K, Ohmiya K (2000) Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product. Biosci Biotechnol Biochem 64:2614–2624

  10. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Martins Dos Santos VAP, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov MM, Timmis KN, Golyshin PN (2005) Novel hydrolase diversity retrieved from a metagenomic library of bovine rumen microflora. Environ Microbiol 7:1996–2010

  11. Gidenne T (1997) Caeco-colic digestion in the growing rabbit: impact of nutritional factors and related disturbances. Livest Prod Sci 51:73–88

  12. Healy FG, Ray RM, Aldrich HC, Wilkie AC, Ingram LO, Shanmugam KT (1995) Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose. Appl Microbiol Biotechnol 43:667–674

  13. Inoue T, Moriya S, Ohkuma M, Kudo T (2005) Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus. Gene 349:67–75

  14. Kamra DN (2005) Rumen microbial ecosystem. Curr Sci 89:124–135

  15. Krause DO, Denman SE, Mackie RI, Morrison M, Rae AL, Attwood GT, McSweeney CS (2003) Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics. FEMS Microbiol Rev 27:663–693

  16. Kuske CR, Banton KL, Adorada DL, Stark PC (1998) Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl Environ Microbiol 64:2463–2472

  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  18. Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, Moller K (2002) Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol 68:673–690

  19. Lynd LR, Weimer PJ, Willem HZ, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577

  20. Mackie RI (1997) Gut environment and evolution of mutualistic fermentative digestion. In: Mackie RI, White BA (eds) Gastrointestinal Microbiology, vol 1. Chapman and Hall, New York, pp 13–35

  21. McLaughlin CA, Chiasson RB (1990) Laboratory Anatomy of the Rabbit, 3rd edn. William C. Brown, Dubuque, IA, pp 59–64

  22. Michalet-Doreau B, Fernandez I, Peyron C, Millet L, Fonty G (2001) Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents. Reprod Nutr Dev 41:187–194

  23. Miller GL (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Biotechnol Bioeng Symp 5:193–219

  24. Odoux E, Escoute J, Verdeil L, Brillouet M (2003) Localization of β-glucosidase activity and glucovanillin in vanilla bean (Vanilla planifolia Andrews). Ann Bot 92:437–444

  25. Rees HC, Grant S, Jones B, Grant WD, Heaphy S (2003) Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries. Extremophiles 7:415–421

  26. Reinhold-Hurek B, Hurek T, Claeyssens M, Montagu M (1993) Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph. J Bacteriol 175:7056–7065

  27. Seed B, Parker RC, Davidson N (1982) Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion. Gene 19:201–209

  28. Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128

  29. Sugimura M, Watanabe H, Lo N, Saito H (2003) Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris. Eur J Biochem 270:3455–3460

  30. Taylor LE II, Henrissat B, Coutinho PM, Ekborg NA, Hutcheson SW, Weiner RM (2006) Complete cellulase system in the marine bacterium Saccharophagus degradans strain 2-40T. J Bacteriol 188:3849–3861

  31. Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–780

  32. Voget S, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit WR (2003) Prospecting for novel biocatalysts in a soil metagenome. Appl Environ Microbiol 69:6235–6242

  33. Voget S, Steele HL, Streit WR (2006) Characterization of a metagenome-derived halotolerant cellulase. J Biotechnol 126:26–36

  34. 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

  35. Zhou JZ, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322

  36. Zverlov VV, Kellermann J, Schwarz WH (2005) Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes. Proteomics 5:3646–3653

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Acknowledgment

This work was supported by the National Natural Science Foundation of China (30560003), International Science and Technology Collaborating Key Project Program of China (2002AA217121) and Program for New Century Excellent Talents in University of China (NCET-05-0752). The authors are grateful to Kunio Ohmiya and Shuichi Karita for their helpful discussions.

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Correspondence to Jia-Xun Feng.

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Feng, Y., Duan, C., Pang, H. et al. 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 (2007). https://doi.org/10.1007/s00253-006-0820-9

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Keywords

  • Cellulase
  • Cloning
  • Metagenome
  • Rabbit cecum
  • Expression
  • Enzyme
  • Properties