Hemicellulases from anaerobic thermophiles. Progress report

The longterm goal of this research effort is to obtain an anaerobic thermophilic bacterium that efficiently converts various hemicellulose-containing biomass to ethanol over a broad pH range. The strategy is to modify the outfit and regulation of the rate-limiting xylanases, glycosidases and xylan esterases in the ethanologenic, anaerobic thermophile Thermoanaerobacter ethanolicus, which grows between pH 4.5 and 9.5. Although it utilizes xylans, the xylanase, acetyl(xylan) esterase and O-methylglucuronidase activities in T. ethanolicus are barely measurable and regarded as the rate limiting steps in its xylan utilization. Thus, and also due to the presently limited knowledge of hemicellulases in anaerobic thermophiles, we characterize the hemicellulolytic enzymes from this and other anaerobic thermophiles as enzyme donors. Beside the active xylosidase/arabinosidase from T. ethanolicus, exhibiting the two different activities, we characterized 2 xylosidases, two acetyl(xylan) esterases, and an O-methylglucuronidase from Thermoanaerobacterium spec. We will continue with the characterization of xylanases from novel isolated slightly acidophilic, neutrophilic and slightly alkalophilic thermophiles. We have cloned, subcloned and partially sequenced the 165,000 Da (2 x 85,000) xylosidase/arabinosidase from T. ethanolicus and started with the cloning of the esterases from Thermoanaerobacterium spec. Consequently, we will develop a shuttle vector and continue to apply electroporation of autoplasts as a method for cloning into T. ethanolicus