Identification,Characterization Of Inulosucrase And ?-galactosidase

Fructans is a functional polysaccharide widely used in food,health products and pharmaceutical industry.Currently,there are two ways to obtain fructans,one is plant source,the other is directly synthesized by microbe enzymes.There are two enzymes which from bacteria for synthesizing fructans:inulosucrase?EC 2.4.1.9?and levansucrase?EC 2.4.1.10?.Among them,inulosucrase has been rarely studied,and enzymatic properties and crystal structure information are not very clear.In order to obtain the inulosucrase with better catalytic efficiency,a section of glycoside hydrolase sequence from Brevibacillus formosus was selected from the CAZy database.Through the analysis of the glycoside hydrolase sequence from the B.formosus,the similarity with the sequence of levansucrase from Bacillus korlensis?wp₀66052162.1?is 78.55%,and the similarity with the sequence of inulosucrase from Bacillus agaradh.aerens WDG185?ATN45518.1?is 72.01%.It was named Inu-Bfor.High expression of soluble protein was obtained through the Escherichia coli expression system?This protein content is about 1.5 mg/mL?.The enzymatic properties of the purified protein were studied.Inu-Bfor optimum temperature was 35?,with excellent thermal stability,the half-life is up to 34 h at 45 ?.The optimal pH value was 6.0.At pH5.0,the enzyme activity was still retained 80%after 148 h,and the half-life is up to 100 h at pH7.0,it indicating that Inu-Bfor had excellent pH stability under moderate and acidic conditions.Inu-Bfor only hydrolyzing substrates containing fructose molecules and glucose molecules that are linked by(3-?2,1?glycosidic bonds.Except that SDS can completely inhibit the enzyme activity of Inu-Bfor,metal ions and other chemical reagents showed different promoting and inhibiting effects on Inu-Bfor.High concentration of sucrose was beneficial to Inu-Bfor fructans synthesis.The maximum amount of fructans synthesis reached 7 mg/mL within 8 h.It was confirmed by NMR,HPLC and FTIR that the fructans synthesized by Inu-Bfor was inulin.Therefore,Inu-Bfor is inulosucrase which has a wide range of potential applications.P-galactosidase?EC.3.2.2.23?is an important industrial enzyme that hydrolyzes lactose into glucose and galactose.There are two main types of commercial?-galactosidase,one is from Aspergillus Niger and the other is from Yeast.The main application of commerciall P-galactosidase is the hydrolysis of lactose in milk,which has a neutral pH.There is also a lack of ?-galactosidase at both neutral pH and high temperatures.However,high temperature P-galactosidase from bacteria can catalyze the lactose in milk at high temperature and prevent the milk from being contaminated by bacteria.In order to obtain high catalytic efficiency of P-galactosidase at high temperature,a section of glycoside hydrolase sequence from Truepera radiovictrix was selected from the CAZy database.Through the analysis of the glycoside hydrolase sequence from T.radiovictrix,the similarity with the sequence of glycoside hydrolase from GHl family?API85502.1?is 61.22%,and the similarity with the sequence of P-galactosidase from Thermus thermophiles?AAS82372.1?is 57%.It had a typical P-galactosidase domain,and it was named BgalT.The soluble protein was obtained by Escherichia coli expression system.The enzymatic properties of purified protein were studied.BgalT optimum temperature was 65 ?,it has a good thermal stability,the half-life is up to 2 h at 65?.The optimal pH value was 6.5.Among pH 4.0-9.0,with the increase of pH value,its stability becomes better and better.BaglT has broad substrate specificity.Except that 5 mM Cd2+and 10 mM SDS could completely inhibit the enzyme activity,metal ions showed different promoting and inhibiting effects on BgalT,it showed certain tolerance to other chemical reagents.High concentrations of organic reagents,glucose and galactose can inhibit the enzyme activity of BgalT.The enzyme activity of BgalT is 878.6+14.6 U/mg.When oNPG is used as the substrate,Km=6.24 mM,Kcat=1657.2 S-1,Kcat/Km=65.6 S-1·mM-1,when lactose was used as substrate,Km=288.5 mM,Kcat=22.7 S-1,Kcat/Km =0.08 S-1mM-1.Compared with the reported?-galactosidase from bacteria,BgalT can completely hydrolysis lactose in skim milk with only 5 h in 55 ? and enzyme amount of 1.8 U/mL,fully shows that BgalT has high catalytic efficiency.