Sunday, April 29, 2007

The characterization of a beta-xylosidase (xlnD) from A. niger with respect to the enzymatic saccharification of lignocellulosic biomass

Michael J. Selig, Eric P. Knoshaug, William S. Adney, and Stephen R. Decker. National Renewable Energy Laboratory, 1617 Cole Blvd MS 3323, Golden, CO 80401

The complete conversion and utilization of xylan in biomass is one significant barrier to the economic viability of the lignocellulose-to-ethanol industry. One avenue to overcome this involves the complete enzymatic saccharification of post-pretreatment xylan as well as xylo-oligomers present in hydrolyzates. A stronger understanding of how xylan degrading enzymes and their accessory enzymes work to depolymerize the plant cell wall structure should allow for higher recoveries of monomeric xylose to be achieved and potentially reduce the pretreatment severity required for optimal utilization of lignocellulosic feedstocks. A key protein in most xylanolytic enzyme systems, beta-xylosidases cleave beta-1-4 linkages in smaller xylo-oligomers, primarily xylobiose, produced by the interactions of xylanases and other hemicellulolytic enzymes. The presence of this enzyme type is key to achieving high enzymatic conversion to monomeric xylose and acts synergistically to improve the efficacy of the entire xylanolytic system. For this study we have successfully expressed and purified a beta-xylosidase (xlnD) from Aspergillus niger in A. awamori. We have characterized this protein on standard substrates such as ρNP-beta-D-xylopyranoside and xylobiose, and investigated end-product limitations of this enzyme. We have probed the ability of xlnD to reduce small xylo-oligomers and characterized the ability of the enzyme to work in concert with an endoxylanase, xynA from Thermomyces lanuginosus, to hydrolyze native xylan and xylan present in a range of pretreated lignocellulosic substrates. We have also investigated the ability of the xynA/xlnD complex to work in synergy with a simplified cellulase mix to more efficiently convert glucan present in pretreated substrates.