Poster Presentation 1B-30

Exploring the Natural Diversity of Glycosyl Hydrolase Cel7 Cellobiohydrolases

Frits Goedegebuur,^1* Paulien Neefe,¹ Peter Gualfetti² and Colin Mitchinson²

¹Genencor International BV

Archimedesweg 30

2333CN Leiden, the Netherlands

Phone: +31 71 5686 143

Fax: +31 71 5686 320

E-mail: goedegebuur@genencor.com

²Genencor International Inc.

925 Page Mill Road

Palo Alto, CA 94304

Genencor International has been working under a subcontract from the Office of Biomass Program, within the DOE Office of Energy Efficiency and Renewable Energy, for cellulase cost reduction for biomass conversion to fermentable sugars. The goal of this three-year program was to reduce the cost of cellulase by ten-fold. Reaching this aggressive target required improvements in both the production, and in the specific performance, of the cellulases. Cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. Within the cellulase mixture produced by Trichoderma reesei, CBHI (Hypocrea jecorina Cel7A) is ~50% of the secreted protein and is an essential enzyme for cellulose degradation. A program was started to improve the activity and thermal stability of this molecule. As part of this, many CBHI homologs have been cloned, sequenced and expressed. Genes were obtained by either specific or homologous cloning approach, and expressed using Aspergillus niger var. nidulans as a heterologous host. Successfully expressed enzymes were purified and the thermal stability evaluated by circular dichroism. The stability and sequence comparisons between homologs informed a site-saturation mutagenesis approach to produce improved T. reesei CBHI mutants.