Gene Cloning,Expression And Characterization Of Three Glycoside Hydrolase From Cellulomonas Bogoriensis

Glycoside hydrolases?GHs?,a widely distributed group of enzymes,cleave glycosidic bonds in glycosides,glycans and glycoconjugates,and they can play key roles in the development of biofuels and in disease research.With the development of molecular biology techniques,more and more microbial genome sequence were determined,and the glycoside hydrolase gene can be cloned and expressed directly by using molecular biology method which is effective to obtain a large number of glycosidase.Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of Cellulomonas bogoriensis shows a large number of uncharacterized genes for glycoside hydrolase potentially.To investigate the enzymatic activity of potential glycoside hydrolase in Cellulomonas bogoriensis,genes encoding two GH1 enzymes and one GH3 enzyme were cloned and recombinant proteins were expressed in E.coli.We analysis biochemical of these proteins.CbBgl1A displayed an optimum temperature of 40_?C and optimum pH was 7.0.Its protein concentration was 0.29 mg/mL and specific activity was 4.28 U/mg.Significant inactivation was observed with Co²⁺,Cu²⁺,Fe³⁺and Zn²⁺.EDTA,methanol and Tween-80 did not affect enzyme activity significantiy,whereas chemical reagents such as EDTA and Tween-80 have a certain inhibitory effect on enzyme activity.CbBgl1A has good tolerance for salinity.CbBgl1A exhibited different substrate specificities with hydrolysis of pNP?-Glu/Cel/Fuc/Lac/Gal,cello-oligosaccharide,sophorose and laminaribiose,and with transglycosylation activities on cellobiose,sophorose and laminaribiose.The calculated K_m and K_cat/K_m of the enzyme was 5.96±1.53 mM and 4.45 s^-1mM^-1 for pNP?Glu,respectively.In terms of substrate inhibition,1.35 M of galactose did not affect the enzyme activity,and 2.5M of fucose still made the residual activity of the enzyme higher than 60%,and the Ki of glucose for CbBgl1A was 0.52 M.CbBgl1A has a synergistic effect with other cellulases.CbBgl1B displayed an optimum temperature of 40_?C and optimum pH was 7.0.Its protein concentration was 0.32 mg/mL and specific activity was 2.19 U/mg.Enzyme activity was stimulated by 1 mM Mg²⁺,Ca²⁺,Mn²⁺and Co²⁺.The addition of methanol,ethanol and glycerin resulted in a slight activity enhancement,whereas chemical reagents such as EDTA and Tween-80 have a certain inhibitory effect on enzyme activity.CbBgl1B has good tolerance for salinity.CbBgl1B was found to hydrolyze pNP?-Glu/Fuc,as well as transglycosylate cellobiose and laminaribiose.The calculated K_m and K_cat/K_m of the enzyme was 1.17±0.35 mM and 2.23 s^-1mM^-1 for pNP?Glu.The appropriate concentration of glucose and fucose has promoted activity,so CbBgl1B has good tolerance for hydrolysis product.CbBgl1B has a synergistic effect with other cellulases.CbXyl3A displayed an optimum temperature of 50_?C and optimum pH was 7.0.Its protein concentration was 0.62 mg/mL and specific activity was 3.37 U/mg.Significant inactivation of CbXyl3A was observed with Mn²⁺,Co²⁺,Cu²⁺,Fe³⁺and Zn²⁺.Light concentration of methanol,ethanol,glycerin and Tween-80 did not affect enzyme activity significantiy.CbXyl3A has good tolerance for salinity.CbXyl3A is a bifunctional enzyme that is mainly xylosidase activity,based on pNP?-Glu/Araf/Xyl and xylo-oligosaccharide.The calculated K_m and K_cat/K_m of the enzyme was1.33±0.26 mM and 5.34 s^-1mM^-1 for pNP?Xyl.And the Ki of xylose for CbXyl3A was 6.25 M,which is higher than most xylosidase.CbXyl3A has a synergistic effect with other xylanase.The three glycoside hydrolases were stable in high salinity and monosaccharide and have synergistic effect with corresponding enzyme,which would be advantageous for biotechnological applications in the development of biofuels and in disease research and so on.