Bacterial Expression and Characterization of Recombinant beta-Xylosidase from the Thermophilic Xylanolytic Bacterium Bacillus sp

摘要

With the passage of time, energy sources are decreasing day by day. In order to meet the world's demand, much attention is being paid to the study of enzymes with xylanolytic activity as a potential means of generating energy. A thermophilic xylanolytic bacterium, Bacillus sp., was isolated from naturally decaying material by enrichment culture and serial dilution methods. The bacterium was grown in MH medium at 50 degrees C and pH 7 for 10 h. The xylanolytic Bacillus sp. produced clear yellow haloes around the colonies in the presence of p-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate. After condition optimization, it was found that the organism produced the higher level of xylosidase activity after 14 h in the presence of arabinose as a carbon source and ammonium sulfate as a nitrogen source in the pH 7 medium of at 55 degrees C. The maximum beta-xylosidase activity after optimizing the culture condition was 5.0 U/mL. Later this thennophilic Bacillus sp. was used as a donor in cloning of the beta-xylosidase gene. A genomic library of Bacillus sp. was prepared by digesting the genomic DNA of the Bacillus with the restriction endonuclease BamHI, ligating the fragments in the pIJC18 cloning vector and then transforming the competent E. coli DH5 alpha cells with the resultant chimeric plasmid. The beta-xylosidase gene was identified by screening the transformanIs in duplicates on LB agar plates overlaid with pNPX as a substrate. Commercial production of beta-xylosidase to be used as a methanol-producing enzyme can help to overcome fuel shortages.