The goal of this project is to demonstrate that enzyme engineering of cellulases can enable the development of consolidated lignocellulosic ethanol biorefinery unit operations; specifically a consolidation of biomass pretreatment and enzymatic hydrolysis. Our year two efforts are focused on the development, manipulation, and optimization of novel classes of cellulase enzymes that have been isolated from extremophiles. Extremophiles are organisms that exist in unique environments, such as sulfurous calderons and thermal hot springs, and possess enzymes that perform optimally in those environments. The majority of industrial cellulase enzymes to date have been derived from bacteria and fungi. Archaea are the third domain of known life and are finding more applications in biotechnology. This is in no small part because most of the archaeal species identified to date have been identified from extreme environments. Environments such as geothermal and deep-sea volcanic sites (80–121 °C), polar regions (-20 °C), acidic solfatara fields (pH < 4) and alkaline springs (pH > 8) and hypersaline lakes (2–5 M NaCl) which were thought to be inhospitable for life to exist have been shown to be teeming with life forms that are unique sources of enzymes with biotechnological importance.

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 established pretreatment technologies for biomass processing, such as dilute acid. We are attempting to modify the sequence, structure, and optimal operating characteristics of these enzymes so that a consolidated bioprocessing approach, collapsing the pretreatment and enzymatic hydrolysis steps into one unit operation, can be implemented to lower costs dramatically. The current estimates of the state-of-the-art pretreatment and enzymatic hydrolysis steps are 25-32% of the estimated total operating costs of a lignocellulosic biorefinery. Three-dimensional structures for each member of the enzyme family are either directly acquired from the protein data base (PDB) or if structural information is not available, homology modeling techniques will be used to build three-dimensional models (if a template structure exists). The active sites of the enzymes are then aligned and compared, and a geometric and chemical "fingerprint" is generated for the site. The active site fingerprint is compared with a set of synthetically accessible scaffolds and/or fragments to identify matches with significant geometric and chemical similarity. The set of scaffolds thus identified are tested and optimized using an iterative process involving detailed atomistic simulations and in-house techniques to suggest modifications followed by the production of new engineered and tailored enzymes for the hydrolysis of cellulosic and lignocellulosic materials.

The hyperthermophilic archaea that thrive under extremes of temperature, pH, salinity etc., have received considerable attention because of the high thermostability of their associated enzymes. The crenarchaeote Sulfolobus solfataricus is a thermoacidophile that grows optimally at approximately 80 °C and pH 2– 4. As such, the proteins isolated from this organism are functional at high temperature and low pH, making them ideal candidates for use in a consolidated dilute acid biomass pretreatment scenario. The genome of S. solfataricus has been sequenced, and three genes (designated sso1354, sso1949 and sso2534) encoding potentially secreted endo-â-glucanases of glycoside hydrolases (GH) family 12 are found in the genome. We selected the SSO1949 gene that generates an endo-â-glucanase (EC 3.2.1.4), exhibits an exceptional activity at extremely low pH, and is thermostable. Our objective is to engineer this enzyme to be a highly efficient catalyst as defined by high turnover and low affinity for binding of the cellulose substrate. This enzyme has a molecular weight of 37 kDa and catalyzes the hydrolysis of carboxymethylcellulose and other oligomers of cellulose to cellobiose and cellotriose. These products can be easily screened for using established commercial assays. We obtained a clone of this gene from the lab of Dr. G. Lipps at the Institute of Biochemistry, University of Bayreuth, Bayreuth, Germany and our current experimental objective is to establish conditions for expression of the proteins, high throughput purification and enzymatic analysis of the protein.

The SSO1949 was cloned into pET-28c-1949Nhis, where the SSO1949 protein carries a fusion of six histidine residues at its N-terminus to enable affinity chromatographic separation of the SSO1949. This plasmid was then transformed into E. coli for expression and protein expression induced via IPTG addition. The active and soluble portion of the purified enzyme demonstrates that the SSO1949 is significantly more active than other commercial endo-â-glucanase at optimal temperature and pH. We will present the results obtained from the computationally predicted sequence changes in terms of improved performance and efficiency.