Engineering thermostable fungal cellobiohydrolases

Meeting the world's growing energy demands while protecting our fragile environment is a challenging issue. Second generation biofuels are liquid fuels like long-chain alcohols produced from lignocellulosic biomass. To reduce the cost of biofuel production, we engineered fungal family 6 cellobiohydrolases (Cel6A) for enhanced thermostability using random mutagenesis and recombination of beneficial mutations. During long-time hydrolysis, engineered thermostable cellulases hydrolyze more sugars than wild-type Cel6A as single enzymes and binary mixtures at their respective optimum temperatures. Engineered thermostable cellulases exhibit synergy in binary mixtures similar to wild-type cellulases, demonstrating the utility of engineering individual cellulases to produce novel thermostable mixtures. Crystal structures of the engineered thermostable cellulases indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by proline substitutions. At high temperature, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A and wild-type Cel6A. The mechanism of thermal inactivation in this cellulase family is consistent with disulfide bond degradation and thiol-disulfide exchange. Enhancing the thermostability of Cel6A also increases tolerance to pretreatment chemicals, demonstrated by the strong correlation between thermostability and tolerance to 1-ethyl-3-methylimidazolium acetate. Several semi-rational protein engineering approaches – on the basis of consensus sequence analysis, proline stabilization, FoldX energy calculation, and high B-factors – were evaluated to further enhance the thermostability of Cel6A.

Long Bay hypoxia study: A collaborative and multidisciplinary approach

The nearshore waters along the Myrtle Beach area are oceanographically referred to as Long Bay. Long Bay is the last in a series of semi-circular indentations located along the South Atlantic seaboard. The Bay extends for approximately 150 km from the Cape Fear River in North Carolina to Winyah Bay in South Carolina and has a number of small inlets (Figure 1). This region of the S.C. coast, commonly referred to as the “Grand Strand,” has a significant tourism base that accounts for a substantial portion of the South Carolina economy (i.e., 40% of the state’s total in 2002) (TIAA 2003). In 2004, the Grand Strand had an estimated 13.2 million visitors of which 90% went to the beach (MBCC 2006). In addition, Long Bay supports a shore-based hook and line fishery comprised of anglers fishing from recreational fishing piers, the beach, and small recreational boats just offshore. (PDF contains 4 pages)