5-59: Discovery and characterization of a novel GH43 xylosidase enzyme isolated from a rice straw culture inoculated with municipal solid waste sludge

Our group has been pursuing the discovery and characterization of hemicellulolytic enzymes, through database mining and by DNA isolation from microorganism consortia degrading various biomass feedstocks. One class of enzymes necessary for the enzymatic breakdown of arabinoxylan are the β-D-xylosidases (EC 3.2.1.37), which catalyze hydrolysis of non-reducing end xylose from xylooligosaccharides. An activated sludge sample from a municipal solid waste treatment facility was used to inoculate a culture  grown on rice straw as a carbon source. Genomic DNA was isolated that encoded hemicellulolytic enzymes including a novel xylosidase termed RS223bx (975 bp). Sequence analysis of the putative catalytic domain indicated that the enzyme is a glycoside hydrolase (GH) family 43 member, and interestingly RS223bx is significantly smaller than the majority of GH43 enzymes examined to date. The gene was cloned into E. coli with a C-terminal His-tag, and the recombinant enzyme was expressed, purified and characterized. The enzyme was dimeric under the size exclusion chromatography conditions employed, with a pH optimum near pH 6.5, and is active on both chromophore labeled xylopyranoside and arabinofuranosidic substrates, as well as being able to hydrolyze xylobiose.