Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Enzymes are traditionally viewed as having exquisite substrate specificity; however, recent evidence supports the notion that many enzymes have evolved activities against a range of substrates. The diversity of activities across glycoside hydrolase family 5 (GH5) suggests that this family of enzymes may contain members with activities on multiple substrates. In this study, we combined structure- and sequence-based phylogenetic analysis with biochemical characterization to survey the prevalence of multiple-specificity for glucan- and mannan-based substrates in the GH5 family. Examination of amino acid profile differences between the subfamilies led to the identification and subsequent experimental confirmation of an active site motif indicative of dual-specificity. The motif enabled us to successfully discover several new dually-specific members of GH5 and this pattern is present in over seventy other enzymes, strongly suggesting that dual endoglucanase-mannanase activity is widespread in this family. In addition, reinstatement of the conserved motif in a wild type member of GH5 enhanced its catalytic efficiency on glucan and mannan substrates by 175% and 1,600%, respectively. Furthermore, a novel enzyme in this GH5 subfamily shows activities not only on cellulose and mannan as expected but also on xylan. Together with a novel beta-glucosidase active on the corresponding disaccharides (cellobiose, mannobiose and xylobiose), this tri-specific GH5 was used for developing a “minimal” enzyme cocktail for lignocellulosic biomass deconstruction. This novel cocktail would significantly reduce the enzyme loading for saccharification, and simplify the efforts for enzyme production and engineering.