Cel9D, an Atypical 1,4--D-Glucan glucohydrolase: Characteristics, Catalytic Residues and Synergistic Interactions with Other Cellulases

Department of Molecular and Cellular Biology, University of Guelph Guelph, ON, Canada

^* To whom correspondence should be addressed. Email: cforsber{at}uoguelph.ca.

	Abstract

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes a cell extract of cellulose-grown cells was separated by ion-exchange chromatography, cellulases located by zymogram analysis, and identified by peptide-mass fingerprinting. An atypical family 9 glycoside hydrolase (GH), Cel9D, with less than 20% identity to typical GH9 cellulases was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5 to 4-fold when separately mixed with each of 4 other glucanases although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity and its activity was restricted to -14 linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the non-reducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased k_cat/K_m values on cello-oligosaccharides with higher degree of polymerization. The k_cat/K_m value on cellohexaose was 2300 times higher than on cellobiose. This indicates that Cel9D is a 1,4--D-glucan glucohydrolase (EC 3.2.1.74) in GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues while Ser168, which is not present in typical GH9 cellulases has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cellooligosaccharides.