Heterologous expression of Aspergillus niger beta-D-xylosidase (XlnD): characterization on lignocellulosic substrates.
The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T (max) was determined using differential scanning microcalorimetry (DSC) to be 78.2 degrees C; the K (m) and k (cat) were found to be 255 microM and 13.7 s(-1), respectively, using pNP-beta-D-xylopyranoside as substrate. End-product inhibition by D-xylose was also verified and shown to be competitive; the K (i) for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.
Selig
Michael J
MJ
National Renewable Energy Laboratory, Chemical and Biosciences Center, Golden, CO 80401, USA. michael_selig@nrel.gov
Knoshaug
Eric P
EP
Decker
Stephen R
SR
Baker
John O
JO
Himmel
Michael E
ME
Adney
William S
WS
D000327:Adsorption; D001234:Aspergillus niger / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D008031:Lignin / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C036909:lignocellulose; D008031:Lignin; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase