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United States Department of Agriculture

Agricultural Research Service

Research Project: EVOLUTIONARY ENZYMES AND SEPARATION PROCESSES FOR IMPROVED BIOREFINING OF CROPS AND RESIDUES Title: Purification and characterization of a glycoside hydrolase family 43 Beta-xylosidase from Geobacillus thermoleovorans IT-08

Authors
item Wagschal, Kurt
item Heng, Chamroeun
item Lee, Charles
item Robertson, George
item Orts, William
item Wong, Dominic

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 4, 2008
Publication Date: December 9, 2008
Citation: Wagschal, K.C., Heng, C., Lee, C.C., Robertson, G.H., Orts, W.J., Wong, D. 2008. Purification and characterization of a glycoside hydrolase family 43 Beta-xylosidase from Geobacillus thermoleovorans IT-08. Applied Biochemistry and Biotechnology. 155(1-3):1-10.

Interpretive Summary: The United States produces nearly 3 billion gallons containing 2.28X1014 BTU of ethanol annually from grain. This work is part of a larger effort to expand the biomass sources used to make ethanol to include crop residues. Hemicellulose is a major chemical constituent of crop residues, and this work will help define new optins for biorefining processes to reduce the chemical and energy costs associated with ethanol production, such that the use of crop residues is economically feasible. The project strategy starts with the identification of enzymes that are able to degrade hemicellulose to fermentable sugars, which can then be readily converted to ethanol or other chemical feedstocks. To do so enzymatically requires a suite of different snzymes, a critical one of which is fò-xylosidase that may find use in the breakdown of hemicellulose.

Technical Abstract: The gene encoding a glycoside hydrolase family 43 enzyme termed deAX was isolated and subcloned from a culture seeded with a compost starter mixed bacterium population, expressed with a C-terminal His6-tag, and purified to apparent homogeneity. deAX was monomeric in solution, and had a broad pH maximum between pH 5.5 and pH 7. A two-fold greater kcat/Km for the p-nitrophenyl derivative of a-L-arabinofuranose (4NPA) versus that for the isomeric substrate b-D-xylopyranose (4NPX) was due to an appreciably lower Km for the arabinofuranosyl substrate. Substrate inhibition was observed for both 4-methylumbelliferryl arabinofuranoside (muA) and the xylopyranoside cogener (muX). While no loss of activity was observed over 4 h at 40 °C, the observed t1/2 value rapidly decreased from 630 min at 49°C to 47 min at 53 °C. The enzyme exhibited end-product inhibition, with a Ki for xylose of 145 mM, 18.5 mM for arabinose, and 740 mM for glucose. Regarding natural substrate specificity, deAX had arabinofuranosidase activity on sugarbeet arabinan, 1,5-a-L-arabinobiose, and 1,5-a-L-arabinotriose, and wheat and rye arabinoxylan, while xylosidase activity was detected for the substrates xylobiose, xylotriose, and arabinoxylan from beech and birch. Thus, deAX can be classified as a mixed-function xylosidase/arabinofuranosidase with respect to both artificial and natural substrate specificity.

Last Modified: 10/21/2014
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