Functional Identification Of Three GH1 Family β-glucosidases In Novosphingobium Sp. GX9

β-glucosidase is a kind of enzymes that catalyze the transfer of glycosyl groups between oxygen nucleophiles.It is widely distributed in nature,and has important application value in food industry,biomass conversion,medicine and biosynthesis.In this study,threeβ-glucosidase genes encoding GH1,sbgl2,sbgl3,and sbgl5,were cloned from the genome of Novosphingobium sp.GX9,and recombinant plasmids were constructed and expressed in E.coli.After verifying the successful construction,the target proteins SBGL2,SBGL3,and SBGL5were purified,their enzymatic properties,transglycosidic functions and hydrolysis functions were studied respectively.Using pNPG as the substrate,the optimal pH and temperature of SBGL2are 5.5 and 40℃,and the stability was good in the range of pH 3.5-10 and temperature 25-50℃.The K_m and V_max values were 1.776±0.08 m M and43.69±0.55μmol*min^-1*mg^-1,respectively.Ki value of glucose inhibition constant is 330.2±29.62 m M.It can hydrolyze o NPG,pNPGal,gentiobiose,salicin,cellobiose,amygdalin,rebaudioside A.1%Triton 100-X can activate150%of the enzyme activity.Using pNPG as the substrate,the optimal pH and temperature of SBGL3are 5.5 and 50℃,and the stability was good in the range of pH 4.5-10.5 and temperature 25-50℃.The K_m and V_max values were 6.158±0.26 m M and104.2±1.27μmol*min^-1*mg^-1,respectively.Ki value of glucose inhibition constant is 143.2±7.65 m M.It can hydrolyze o NPG,pNPGal,cellobiose,gentiobiose,sophorose,lactose,rubusoside,rebaudioside C.1%Triton 100-X can activate 240%of the enzyme activity.Using pNPG as the substrate,the optimal pH and temperature of SBGL5are 6.0 and 60℃,and the stability was good in the range of pH 4.5-10.0 and temperature 25-45℃.The K_m and V_max values were 0.2891±0.03 m M and30.94±1.11μmol*min^-1*mg^-1,respectively.It can hydrolyze o NPG,pNPGal,amygdalin,cellobiose（235.76 U/mg）,gentiobiose（17.98 U/mg）,sophorose（147.90 U/mg）,lactose（44.90 U/mg）.10%ethylene glycol can activate 220%of the enzyme activity.In studying the ability to hydrolyze terpenoid compounds,it was found that SBGL2,SBGL3,and SBGL5 can completely hydrolyze soy isoflavones,epimedium flavonoids,polydatin.They are all capable of hydrolyzing stevioside and steviolbioside in steviol glycosides,and their hydrolysis ability is SBGL3﹥SBGL2﹥SBGL5.In the study of transglycoside function,it was found that when pNPG was used as a glycosyl donor,SBGL2,SBGL3,and SBGL5 all showed a transglycosidic activity of methanol,ethanol,n-propanol,1,2-propanediol,ethylene glycol,（+）-atechins and caffeic acid.SBGL5 can use cellobiose or glucose as glycosyl donors for a variety of transglycoside reactions.SBGL3 can hydrolyze rebaudioside C withβ-glucosidase SBGL that has been isolated in this laboratory.According to mass spectrometry analysis,the product may be a substance dulcoside A/dulcoside B and a substance with a CAS number of 64432-08-2.