Cloning,Expression And Characterization Of ?-glucosidase Gene From Enterococcus Faecalis CTB

Cellulose is the most abundant carbohydrate in nature.It is an important issue how to hydrolyze cellulose.The hydrolysis of cellulose depends on the synergism of endoglucanase?EC 3.2.1.4?,exoglucanase?EC 3.2.1.74?,cellobiohydrolase?EC3.2.1.176?and beta-glucosidase?EC 3.2.1.21?.Among them,?-glucosidase hydrolyzes cellobiose or oligosaccharide into non-reducing terminal glucose,which can effectively alleviate the inhibition of cellulase by cellobiose during cellulase hydrolysis,and is an important factor limiting the complete hydrolysis of cellulose.A cellulolytic Enterococcus faecalis,named as Enterococcus faecalis CTB,was isolated from the rumen of ruminant sheep in previous study of our team and detected to have?-glucosidase activity.In order to investigate why there is?-glucosidase activity in Enterococcus faecalis,what are the characteristics or specificities of the enzyme,sequencing the whole genome of Enterococcus faecalis.The genome sequencing results showed that the strain had multiple?-glucosidase genes.Two representative genes were selected,and the two gene fragments were amplified by PCR.The proteins were expressed by E.coli BL21?DE3?as host.The expressed proteins were named bgl1213 and bgl2557,respectively.bgl1213 showed?-glucosidase activity,and bgl2557 exhibited?-galactosidase activity.The optimal conditions for CTB-2557 expression as follows:optimal temperature was 30°C,optimal incubation time was 12 h,and IPTG concentration was 1.2 mmol/L.The optimal conditions for CTB-1213 expression as follows:optimal temperature was20?,optimal culture time was 16 h,and IPTG concentration was 1.2 mmol/L.The enzymatic properties of the purified protein were studied.The optimum reaction temperature of bgl1213 is 40?,and the optimum pH is 8.5;100?mol/L Er³⁺can increase enzyme activity by 10%,Tris and Urea increased enzyme activity by 24%and 12%,respectively.,SDS can completely inactivate the enzyme;Glucose has a greater inhibitory effect on bgl1213;The characteristic constants K_m and V_max of this enzyme for pNPG are 1.38 mmol/L and 19.33 U/mg,respectively,and K_cat is 1.53.The optimum reaction temperature of bgl2557 is 50?,and the optimum pH is 7.0.Mg²⁺?1 mmol/L?can increase the enzyme activity by about 40%;Ethanol?1%?can increase the enzyme activity by about 25%,and it is also highly resistant to high concentration of organic solvent.0.5%of SDS can completely inactivate the enzyme;When the glucose concentration is 1 mol/L,the relative enzyme activity can reach more than 45%of the maximum enzyme activity;Using pNPG as a substrate,K_m was0.69 mM and V_max was 35.42 U/mg.