Properties Of β-glucosidase CHU₂273from Cytophaga Hutchinsonii

In the case of the gradual depletion of the world’s resources and energy shortages, countries are committed to the development of biomass energy.Including the development,more and more attention was paid to the utilization of lignocellulose. However, cellulose is difficult for enzymatic degradation due to its compact structure and the presence of crystalline region.Therefore,the screening and research of high efficient cellulolytic microorganisms is significant.Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that can rapidly digest crystalline cellulose, and it does not secrete soluble extracellular cellulolytic enzymes and has no cellulosome-like structure. Presumably,Cytophaga hutchinsonii has a novel mechanism of cellulose degration.P-glucosidase is an enzyme which can hydrolyze cellobiose and short chain cellooligosaccharide to glucose.β-glucosidase is a key component of the cellulase system.It further hydrolyzes cellobiose into glucose. In addition,(3-glucosidase can regulate the velocity of the cellulose degradation.Therefore,in depth study of β-glucosidase have a profit of the high veloeity of the cellulose degradation.It has great significant for cellulose degration and exploitation the efficient cellulose degradation mechanisms.In this paper,I report the study of an β-glucosidase which identified from Cytophaga hutchinsonii by Native PAGE,including it’s identification,cloning, heterologous expression,isolation, purification and characterization.The main contents and results are as follows:1.Previous studies have found that most of the β-glucosidase activity was located on the cell surface of C. hutchinsonii from the enzyme assay,so I extract outer membrane proteins of C. hutchinsonii to Native PAGE.Found only a single band have β-glucosidase activity. Identified with Mass Spectrometry,it’s a β-glucosidase,with a length of2643bp,encoding820amino acids,molecular weight89613.Homologous comparing shows that the enzyme belongs to glycosyl hydrolase falmily3,has three disulfide bridges and transmembrane domain does not exist.Specifically,it’s amino acid sequence shows characteristics of cold-active enzymes.2.Cloning the enzyme CHU₂273into pMAL C2X vector and transformed into the competent expression host, E. coli JM109.Optimizing the expressing condition,the enzyme showed efficient expression in E.coli JM109and the enzyme is purified using MBP affinity purification system.3.The enayme exhibits the best activity at PH6.8,20℃and a high specific activity with PNPGlu and an apparent Km value of0.00543mM.It shows poor thermal stability. Ca2+can activate the enzyme activity whereas Ni+, Co2+, Cu2+, K+, Mg2+showing different levels of inhibition to the enzyme activity. Meanwhile,glucono-delta-lactone,SDS can strongly inhibited the enzyme activity,and methanol, ethanol also have strong inhibition. Mercaptoethanol and EDTA had no effect on the activity. CHU₂273can hydrolyze β-1,4and β-1,6glycosidic bond and can completely hydrolyze cellotriose,cellotetraose and cellopentaose.But it does’t have the ability of hydrolyze a-glycosidic bond.It is worth mentioning that CHU₂273remain high activity at low temperature,which shows that it’s cold active enzyme.