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Title: Highly thermostable GH39 ß-xylosidase from a Geobacillus sp. strain WSUCF1

item BHALLA, ADITYA - South Dakota School Of Mines And Technology
item Bischoff, Kenneth
item SANI, RAJESH - South Dakota School Of Mines And Technology

Submitted to: BMC Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2014
Publication Date: 12/23/2014
Publication URL:
Citation: Bhalla, A., Bischoff, K.M., Sani, R.K. 2014. Highly thermostable GH39 ß-xylosidase from a Geobacillus sp. strain WSUCF1. BMC Biotechnology. 14:963.

Interpretive Summary: New enzymes that function under harsh industrial conditions of extreme temperature and pH are needed to help overcome some of the technical barriers to using agricultural residues as feedstocks for fuel ethanol production. Thermophilic bacteria are microorganisms that grow at high temperatures and may possess robust enzymes useful to the fermentation industry. In the present study, a gene for a xylosidase enzyme, which helps break down hemicellulose, was isolated from a thermophilic bacterium, expressed in a laboratory strain of bacteria, and the recombinant enzyme purified. The recombinant enzyme was active over a broad pH range, and was very stable at high temperatures. Results will be valuable to researchers developing new enzymes to serve as biocatalysts in the conversion of agricultural residues to fermentable sugars.

Technical Abstract: Background Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and ß-xylosidase. ß-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable ß-xylosidases have been a focus of attention as industrially important enzymes due to their long shelf life and role in the relief of end-product inhibition of xylanases caused by xylo-oligosaccharides. Therefore, a highly thermostable ß-xylosidase with high specific activity has significant potential in lignocellulose bioconversion. Results A gene encoding a highly thermostable GH39 ß-xylosidase was cloned from Geobacillus sp. strain WSUCF1 and expressed in Escherichia coli. Recombinant ß-xylosidase was active over a wide range of temperatures and pH with optimum temperature of 70°C and pH 6.5. It exhibited very high thermostability, retaining 50% activity at 70°C after 9 days. WSUCF1 ß-xylosidase is more thermostable than ß-xylosidases reported from other thermophiles (growth temperature'='70°C). Specific activity was 133 U/mg when incubated with p-nitrophenyl xylopyranoside, with Km and Vmax values of 2.38 mM and 147 U/mg, respectively. SDS-PAGE analysis indicated that the recombinant enzyme had a mass of 58 kDa, but omitting heating prior to electrophoresis increased the apparent mass to 230 kDa, suggesting the enzyme exists as a tetramer. Enzyme exhibited high tolerance to xylose, retained approximately 70% of relative activity at 210 mM xylose concentration. Thin layer chromatography showed that the enzyme had potential to convert xylo-oligomers (xylobiose, triose, tetraose, and pentaose) into fermentable xylose. WSUCF1 ß-xylosidase along with WSUCF1 endo-xylanase synergistically converted the xylan into fermentable xylose with more than 90% conversion. Conclusions Properties of the WSUCF1 ß-xylosidase i.e. high tolerance to elevated temperatures, high specific activity, conversion of xylo-oligomers to xylose, and resistance to inhibition from xylose, make this enzyme potentially suitable for various biotechnological applications.