Original Paper

Applied Microbiology and Biotechnology

, Volume 43, Issue 4, pp 667-674

First online:

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

  • F. G. HealyAffiliated withDepartment of Microbiology and Cell Science, University of Florida
  • , R. M. RayAffiliated withDepartment of Microbiology and Cell Science, University of Florida
  • , H. C. AldrichAffiliated withDepartment of Microbiology and Cell Science, University of Florida
  • , A. C. WilkieAffiliated withSoil and Water Science Department, University of Florida
  • , L. O. IngramAffiliated withDepartment of Microbiology and Cell Science, University of Florida
  • , K. T. ShanmugamAffiliated withDepartment of Microbiology and Cell Science, University of Florida

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Abstract

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.