Cloning, Expression, Characterization Of Beta-glucosidases From Different Thermophilic Microorganisms

β-glucosidase, known as P-D-glucoside hydrolase (EC3.2.1.21), can specifically catalyze the oligosaccharides (typically2-6monosaccharide residues), and the β-D-glucoside bond of alkyl and aromatic hydrocarbon. P-glucosidase enzyme is the key rate-limiting enzyme of cellulase enzymes. To obtain some kinds of β-glucosidase with high specific activity and thermal stability has been the hot study of enzymatic hydrolysis of cellulose.Four kinds of P-glucosidase encoding gene named tabgl、gebgll、gebgl2and gebgl3was cloned from Thermoascus aurantiacus and Geobacillus thermodenitrificans, they were inserted in expression vector pGEX-2LT and pRX2and transformed into Escherichia coli BL21(DE3), finally seven recombinant strains were obtained, and their expression in E. coli. were achieved.The study of enzymatic properties of the purified P-glucosidase expressed by strain BL21(pGEX-gebgl1) was found that, the optimum temperature and optimum pH of the recombinant expression β-glucosidase were60℃-70℃and7.0, respectively, the purity can reach to90%. the enzyme is stable for4h under the conditions of pH5-10,60℃, and it maintains its high enzymatic activity at the high salt concentration (up to880mmol/L K+).In the potassium phosphate buffer, the specific activity of this recombinant expression P-glucosidase enzyme reached to0.0058IU/mg, when adding citrate buffer and10mM Al3+, the enzyme specific activity can be achieved to0.043IU/mg. The P-glucosidase GST fusion protein exists in different oligomers by a Superdex G-200gel filtration analysis, and the different oligomers of enzymes all have hydrolase activity.