S55: Novel glycoside hydrolases from the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus for biomass deconstruction

The complete genome sequence of Caldicellulosiruptor saccharolyticus (Topt 70-75oC), a bacterium that has the ability to utilize a range of hemicelluloses and crystalline cellulose as carbon and energy sources, has been examined for novel glycoside hydrolases (GHs). To this end, whole genome oligonucleotide microarrays were used for the functional genomics-based enzyme discovery and to understand the function of specific GHs with respect to biomass deconstruction. We are focusing on several carbohydrate-active enzymes that potentially play major roles in degrading recalcitrant lignocellulosic substrates, such as switch grass and poplar. One endoglucanase, which is composed of a signal peptide, a glycoside hydrolase family (GH) 5 catalytic domain, a carbohydrate binding module, and three S-layer homology domains, is a particularly intriguing candidate cellulase. The gene encoding this endoglucanase has been expressed in Escherichia coli and shown to be active on microcrystalline cellulose, filter paper, carboxymethyl cellulose (CMC), xylan, lichenan, and arabinose. Western blot analysis indicated that this endoglucanase is localized to the cell wall and seems to play a key role in recruiting substrates for C. saccharolyticus growth. Benchmarking studies showed that the activity of the endoglucanase is comparable to commercial cellulases and xylanases. Synergism studies of this enzyme with other C. saccharolyticus GHs to improve the activity on biomass substrates are also being pursued. Along these lines, CelB from C. saccharolyticus, which contains one GH10 catalytic domain, one carbohydrate binding module, and one GH5 catalytic domain, is among the enzymes being examined.