DIRECT ISOLATION OF FUNCTIONAL GENES ENCODING CELLULASES FROM THE MICROBIAL CONSORTIA IN A THERMOPHILIC, ANAEROBIC DIGESTER MAINTAINED ON LIGNOCELLULOSE

Gene libraries (''zoolibraries'') were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic d igesters, which were in continuous operation with lignocellulosic feed stocks for over 10 years. Clones expressing cellulase and xylosidase w ere readily recovered from these libraries. Four clones that hydrolyze d carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranos ide were characterized. All four cellulases exhibited temperature opti ma (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditi ons of the enrichment. The DNA sequence of the insert in one clone (pl asmid pFGH1) was determined. This plasmid encoded an endoglucanase (ce lA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues. CelA pr otein shared limited homology with members of the A3 subfamily of cell ulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity). The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp. cellulosa (28% homology). The use of zoolibraries constructed from natural or labora tory enrichment cultures offers the potential to discover many new enz ymes for biotechnological applications.