The degradation of cellulose and hemicellulose, the main components of the plant cell wall, is a pivotal step in the carbon cycle on earth. The hydrolysis of plant biomass to soluble sugar requires the synergistic actions of several glycoside hydrolases (GHs) including endoglucanases, exoglucanases or cellobiohydrolases, and β-glucosidases. This process is of major importance for the realization of a renewable energy source (biomass) for liquid fuel that does not contribute net CO2 to the atmosphere.
Geobacillus stearothermophilus is a thermophilic Gram positive soil bacterium that possesses an extensive system for utilization of the plant cell-wall polysaccharides. Genome analysis revealed two genes encoding for family 1 glycoside hydrolases. The first gene, cel1A, encodes for a 6-phospho-β-glucosidase and is part of an operon which encodes also for celB, celC, and celD comprising a phosphotransferase system (PTS). This operon is induced by cellobiose and biochemical characterization showed that Cel1A hydrolyzes cellobiose-6-phosphate and 2-nitrophenyl-β-D-glucopyranoside-6-phosphate. These results suggest the involvement of this operon in the utilization of cellobiose.
The second GH1 gene, gan1D, encodes for a putative 6-phospho-β-galactosidase. The gene is induced by galactan and galactose, suggesting its involvement in galactan utilization. Gan1D seems to have a bi-functional activity and hydrolases 2-nitrophenyl-β-D-galactopyranoside-6-phosphate, 2-nitrophenyl-β-D-glucopyranoside-6-phosphate, lactose-6-phosphate and cellobiose-6-phosphate. Recently, we determined the crystal structure of Gan1D, as native and in complex with its reaction products, glucose-6-phosphate and galactose-6-phosphate.
In this study we demonstrate the substrate specificity of these enzymes using biochemical characterization and structure analysis.
