Clostridium thermocellum Cel5L – Cloning and Characterization of a New, Thermostable GH5 Cellulase

Aims: Clostridium thermocellum is a thermophilic, anaerobic bacterium that ferments cellulose and produces ethanol. While many of the predicted cellulases have been characterized from C. thermocellum, characterization of additional members is still needed to unravel the function of these enzymes.

Study Design: This report describes the first cloning, expression, characterization, and evaluation in cellulose degradation of Clostridium thermocellum Cel5L as well as its relationship to other cellulases.

Results: Cel5L, a 526 amino acid protein that shows low homology to other cellulases of C. thermocellum, possesses both endo and exo activity on b-glucan, carboxymethylcellulose, and cellooligosaccharides; the enzyme does not hydrolyze xylan, xyloglucan or glucomannan. When combined with exo-cellulases and b-glucosidase, Cel5L generates more glucose from cellulose than other tested C. thermocellum GH5 cellulases, which is surprising due to the lack of an annotated carbohydrate binding module. The Cel5L phylogenetic tree shows orthologs in both mesophilic and thermophilic cellulose degraders.

Conclusion: The presence of these orthologs in a variety of molecular constructs demonstrates the importance of Cel5L and its orthologs in cellulose degradation, both in C. thermocellum as well as other gram-positive cellulose degraders.