Applied Microbiology and Biotechnology

, Volume 43, Issue 4, pp 667–674 | Cite as

Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose

  • F. G. Healy
  • R. M. Ray
  • H. C. Aldrich
  • A. C. Wilkie
  • L. O. Ingram
  • K. T. Shanmugam
Original Paper


Gene libraries (“zoolibraries”) were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years. Clones expressing cellulase and xylosidase were readily recovered from these libraries. Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-β-d-cellobiopyranoside were characterized. All four cellulases exhibited temperature optima (60–65° C) and pH optima (pH 6–7) in accordance with conditions of the enrichment. The DNA sequence of the insert in one clone (plasmid pFGH1) was determined. This plasmid encoded an endoglucanase (celA) and part of a putative β-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to β-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications.


Cellulase Clostridium Anaerobic Digester Carboxymethylcellulose Enrichment Culture 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
  2. Amann R, Springer N, Ludwig W, Gortz H-D, Schleifer K-H (1991) Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351:161–164Google Scholar
  3. Angert ER, Clements KD, Pace NR (1993) The largest bacterium. Nature 362:239–241Google Scholar
  4. Béguin P (1990) Molecular biology of cellulose degradation. Annu Rev Microbiol 44:219–248Google Scholar
  5. Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  6. Castle LA, Smith KD, Morris RO (1992) Cloning and sequencing of an Agrobacterium tumefaciens β-glucosidase gene involved in modifying a vir-inducing plant signal molecule. J Bacteriol 174:1478–1486Google Scholar
  7. Chauvaux S, Béguin P, Aubert J-P (1992) Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD. J Biol Chem 267:4472–4478Google Scholar
  8. Collmer A, Wilson DB (1983) Cloning and expression of a Thermomonospora YX endocellulase gene in E. coli Biotechnology 1:594–601Google Scholar
  9. Coughlan MP (1985) The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol Genet Eng Rev 3:39–109Google Scholar
  10. Deveraux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  11. Erb RW, Wagner-Dobler I (1993) Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol 59:4065–4073Google Scholar
  12. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, Warren RAJ (1991) Domains in microbial β-1,4-glycanases: sequence conservation, function and enzyme families. Microbiol Rev 55:303–315Google Scholar
  13. Gräbnitz F, Rücknagel KP, Seiss M, Staudenbauer WL (1989) Nucleotide sequence of the Clostridium thermocellum bglB gene encoding thermostable β-glucosidase B: homology to fungal β-glucosidases. Mol Gen Genet 217:70–76Google Scholar
  14. Hattori M, Sakaki Y (1986) Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152:232–238Google Scholar
  15. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316Google Scholar
  16. Henson JM, Bordeaux FM, Rivard CJ, Smith PH (1986) Quantitative influences of butyrate and propionate on thermophilic production of methane from biomass. Appl Environ Microbiol 51:288–292Google Scholar
  17. Juy M, Amit AG, Alzari PM, Poljak RJ, Claeyssens M, Béguin P. Aubert J-P (1992) Three dimensional structure of a thermostable bacterial cellulase. Nature 357:89–91Google Scholar
  18. Lejeune A, Colson C, Eveleigh DE (1991) Molecular cloning of cellulase genes into noncelluloytic microorganisms. In: Haigler CH, Weimer PJ (eds) Biosynthesis and biodegradation of cellulose. Dekker, New York, pp 623–671Google Scholar
  19. Ljungdahl, LG, Erikkson K-E (1985) Ecology of microbial cellulose degradation. In: Marshall KC (ed), Advances in microbial ecology. Plenum, New York, pp 237–299Google Scholar
  20. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  21. Marmur J (1961) A procedure for isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218Google Scholar
  22. McGavin MJ, Forsberg CW, Crosby B, Bell AW, Dignard D, Thomas DY (1989) Structure of cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product endoglucanase 3. J Bacteriol 171:5587–5595Google Scholar
  23. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  24. Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375–380Google Scholar
  25. Presutti DG, Hughes TA, Stutzenberger FJ (1993) Characterization of a Thermomonospora curvata endoglucanase expressed in Escherichia coli. J Biotechnol 29:307–320Google Scholar
  26. Poole DM, Hazlewood GP, Laurie JI, Barker PJ, Gilbert HJ (1990) Nucleotide sequence of the Ruminococcus albus SY3 endoglucanase genes celA and celB. Mol Gen Genet 223:217–223Google Scholar
  27. Rixon RE, Ferreira LM, Durrant AJ, Laurie JI, Hazlewood GP, Gilbert HJ (1992) Characterization of the gene celD and its encoded product, 1,4-β-d-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa. Biochem J 285:947–955Google Scholar
  28. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  29. Sayler GS, Layton AC (1990) Environmental application of nucleic acid hybridization. Annu Rev Microbiol 44:625–648Google Scholar
  30. Schwarz WH, Schimming S, Rücknagel KP, Burgschwaiger S, Kreil G, Staudenbauer WL (1988) Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum. Gene 63:23–30Google Scholar
  31. Shima S, Igarashi Y, Kodama T (1991) Nucleotide sequence analysis of the endoglucanase encoding gene, celCCD, of Clostridium cellulolyticum. Gene 104:33–38Google Scholar
  32. Smith PH, Bordeaux FM, Goto M, Shiralipour A, Wilkie A, Andrews JF, Ide S, Barnett MW (1988a) Biological production of methane from biomass. In: Smith WH, Frank JR (eds) Methane from biomass: a systems approach. Elseiver, New York, pp 291–334Google Scholar
  33. Smith PH, Bordeaux FM, Wilkie A. Yang J, Boone D, Mah RA, Chynoweth D, Jerger D (1988b) Microbial aspects of biogas production. In: Smith WH, Frank JR (eds) Methane from biomass: a systems approach. Elsevier, New York, pp 335–353Google Scholar
  34. Steffan RJ, Atlas RM (1988) DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl Environ Microbiol 54:2185–2191Google Scholar
  35. Stewart CS, Bryant MP (1988) The rumen bacteria. In: Hobson PN (ed) The rumen microbial ecosystem, Elsevier, New York, pp 21–75Google Scholar
  36. Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from bovine rumem. Appl Environ Microbiol 43:777–780Google Scholar
  37. Tomme P, Chauvaux S, Béguin P, Millet J, Aubert J-P, Claeyssens M (1991) Identification of a histidyl residue in the active center of endoglucanase from Clostridium thermocellum. J Biol Chem 266:10313–10318Google Scholar
  38. Voordouw G, Shen Y, Harrington CS, Telang AJ, Jack TR, Westlake DWS (1993) Quantitative reverse sample genome probing of microbiol communities and its application to oil field production waters. Appl Environ Microbiol 59:4101–4114Google Scholar
  39. Wilbur WJ, Lipman DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci USA 80:726–730Google Scholar
  40. Wilkie A, Goto M, Bordeaux FM, Smith PH (1986) Enhancement of anerobic methanogenesis from Napiergrass by addition of micronutrients. Biomass 11:135–146Google Scholar
  41. Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Methods Enzymol 160:87–112.Google Scholar
  42. Yagüe E, Béguin P, Aubert J-P (1990) Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. Gene 89:61–67Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • F. G. Healy
    • 1
  • R. M. Ray
    • 1
  • H. C. Aldrich
    • 1
  • A. C. Wilkie
    • 2
  • L. O. Ingram
    • 1
  • K. T. Shanmugam
    • 1
  1. 1.Department of Microbiology and Cell ScienceUniversity of FloridaGainesvilleUSA
  2. 2.Soil and Water Science DepartmentUniversity of FloridaGainesvilleUSA

Personalised recommendations