Enzymatic liquefaction of untreated corn stover

There is an ever increasing need for renewable alternatives to fossil fuels derived from petroleum. This makes feedstocks in the form of lignocellulosic biomass attractive substrates for the production of ethanol and value added chemicals. However, the economics of converting lignocellulosic materials involve high processing costs attributed with pretreatment of the biomass and the use of enzymes for saccharification. Corn stover was obtained for the examination of an upstream processing step to transform the material into a pumpable slurry for subsequent pretreatment and saccharification. Biomass liquefaction was carried out using the enzyme Depol 692L (Biocatalysts, Wales, UK) at 50°C with a mixing speed of 290 RPM using a dual-impeller design. Solids were added in a fed-batch manner over a course of 8 hours to reach a final solids concentration of 15% (w/v). Slurry viscosity was analyzed in real time to determine the flowability of the mixture. Minor changes to the particle size distribution were observed as liquefaction proceeded. A decrease in insoluble solids concentration was seen in liquefied corn stover versus the control, which could help explain the approximately 200 cP drop in viscosity over the control. When pretreatment (190°C for 15 min. at 10% and 15% solids) of liquefied material was carried out, an increase in glucose formation was seen over raw, pretreated corn stover. Total glucose formation after pretreatment andenzyme hydrolysis (50°C and 2.5 FPU per gram glucan of Cellic ® CTec3) was higher for liquefied material than raw material

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance