Title: Biochemical characterization of a novel dual-function a-L-arabinofuranosidase/b-xylosidase isolated from a compost starter mixture Authors
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 6, 2008
Publication Date: January 15, 2009
Citation: Wagschal, K.C., Heng, C., Lee, C.C., Wong, D. 2009. Biochemical characterization of a novel dual-function a-L-arabinofuranosidase/b-xylosidase isolated from a compost starter mixture. Applied Microbiology and Biotechnology. 81:855-863. Interpretive Summary: The gene encoding a glycoside hydrolase family 43 xylosidase (deAX) was isolated from a compost starter mixture, cloned and expressed in E. coli, and the recombinant protein purified to apparent homogeneity. The usefulness of the enzyme under potential biomass utilization process conditions was explored by determining that the pH maximum was 6.5, maximal activity was at 53 °C, and thermal instability occurred above 45 °C. A series of natural substrates was used to determine the substrate specificity of the enzyme. It was found that deAX hydrolyzed both arabionosyl and xylosyl glycosidic linkages, and thus is a dual-function enzyme. This work is important since this shows that the enzyme has potential utility for the enzymatic for break down of biomass for use as a feedstock for chemicals and fuel.
Technical Abstract: The gene encoding a glycoside hydrolase family 43 enzyme was isolated from a culture seeded with a compost starter mixed bacterium population. The gene was cloned into E. coli with a C-terminal His-tag and its recombinant product termed deAX was expressed and purified to apparent homogeneity. The enzyme was monomeric under the gel-permeation chromatography conditions employed. deAX showed 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). deAX had a broad pH maximum between pH 5.5 and pH 7. 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 and a rather low KI of 19 mM for arabinose. The natural substrate specificity of the enzyme was remarkably broad. The enzyme hydrolyzed xylobiose and xylotriose, albeit with kcats that were 30-50 fold lower than those for the artificial substrates 4NPA and 4NPX. deAX was inactive on debranched sugar-beet arabinan, but was active on sugarbeet arabinan containing 1,3- and 1,2-a-linked arabinose branches. The enzyme released arabinose from both 1,5-a-L-arabinobiose and 1,5-a-L-arabinotriose, arabinose only from wheat and rye arabinoxylan, xylose only from beech and birch arabinoxylan, and both arabinose and xylose from oat-spelt arabinoxylan. Thus, deAX can be classified as a mixed-function xylosidase/arabinofuranosidase with respect to both artificial and natural substrate specificity.