Sunday, April 29, 2007

Enhanced biomass-bioenergy conversion through enzyme engineering of thermoacidophiles

Blake A. Simmons, Rajat Sapra, Diana Roe, George Buffleben, Joanne Volponi, and Jean-Loup Faulon. Energy Systems, Sandia National Laboratories, 7011 East Avenue, Livermore, CA 94551

Evolutionary processes have optimized the structure of enzymes to deliver a desired catalytic function.  Proteins, however, are fragile and easily succumb to extreme environmental conditions that are typically associated with the pretreatment of biomass. We are attempting to modify the sequence, structure, and optimal operating characteristics of these enzymes so that a consolidated bioprocessing approach can be implemented to lower costs dramatically. However, instead of engineering pH and temperature tolerance into the enzyme and the making it highly efficient catalyst, we chose to work with enzymes from extremophilic archaea.  The crenarchaeote Sulfolobus solfataricus, which thrives in acidic volcanic hot springs, is a thermoacidophile growing optimally at approx. 80 °C and pH 2– 4. The genome of S. solfataricus has been sequenced, and three genes encoding potentially secreted endo-β-glucanases of glycoside hydrolases (GH) family 12 are found in the genome. We focused our attention on the sso1949 gene which has been shown to exhibit activity at extremely low pH and is thermostable. As the sequence alignment of S. sofataricus to the other enzymes in this family was limited (15-27%), we  tested 9 different homology modeling programs and compared the homology structure and sequence to the most active enzyme of the set, T. reesei, and identified mutations that are hypothesized to increase performance.  The sso1949 was cloned into pET-28c-1949Nhis and expressed. Mutations were inserted using site-specific mutagenesis kits.  We will present the results obtained comparing the predicted changes from those determined by assays of the actual mutants.