5-104: Biochemical Comparison of a Gycosyl Hydrolase family 7 Library

A library of Glycosyl Hydrolase family seven (GH7) cellobiohydrolases was assembled using sequences gleaned from the Carbohydrate-Active Enzyme (CAZy) database and NCBI Entrez. The protein sequences identified on the CAZy webserver as GH7 cellobiohydrolases were analyzed by CLUSTAL and PHYLIP to generate an unrooted phylogenetic tree.  Representative sequences were selected for expression in Aspergillus and purified to homogeneity using column chromatography.  The proteins were characterized and compared using a variety of biochemical tests including activity on cellulose and pretreated corn stover. Assays were run in parallel with Cel7A from Hypocrea jecorina (anamorph: Trichoderma reeseii) to permit unbiased comparisons of activity with the best-studied cellobiohydrolase. Representatives were also homology modeled using the software package Modeler^TM and structural variability between enzymes compared.  These efforts are expected to further the understanding of sequence-structure-function relationships in cellobiohydrolases and to correlate differences in active site topology with differences in activity on model and realistic cellulose substrates.