Cloning And Functional Analysis Of Two Glycoside Hydrolase Genes

The(?/?)8 barrel structure is a typical feature of the glycoside hydrolase 13(GH13)family.The enzymes of the glycoside hydrolase 13 family can catalyze a variety of covalent bond substrates and transglycosylation reactions^[1,2],where?-Amylase catalyzes?-1,4glycosidic bonds in starch and related?-glucans^[3]to generate monosaccharides or malto-oligosaccharides;Trehalose synthase Tre S reverses the?-1,4 glycosidic bonds in maltose molecules The synthesis of trehalose for the?-1,1 glycosidic bond^[4]has broad prospects in industrial applications.In this paper,a maltosyl trehalose hydrolase encoding gene and a trehalose synthase encoding gene were cloned from Myxococcus sp.V11 and Streptomyces lavendulae X33,respectively,and the expression vector was constructed and used in the E.coli expression system.Express,purify the recombinase by nickel column affinity chromatography,use DNS method to study the enzymatic properties,use HPLC to detect the reaction product,and use site-directed mutagenesis to modify the gene.The nucleic acid sequence of malto-oligosaccharyl trehalose hydrolase Mthase is 1869bp,and the encoded protein is 68KDa;the product produced after the recombinase Mthase hydrolyzes corn starch is detected by HPLC.Maltohexaose is the main product,and a small amount of glucose and Maltose;Malto-oligosaccharyl trehalose hydrolase Mthase has an optimal reaction temperature of 40°C,which is a mesothermal enzyme;most of the divalent metal ions have a strong inhibitory effect on the recombinase Mthase;Triton X-100 has a slight effect on the recombinase Mthase Promoting effect,relative enzyme activity is107.21%;K_m of recombinase Mthase is 14.28 mg/m L,V_max is 8.88?mol/min.The active center of the maltosyl seaweed oligosaccharide hydrolase Mthase was modified,and the active center site was verified by observing the enzymatic properties,and the triplet active center of the recombinase Mthase was verified by experiments to be composed of Asp256-Glu291-Asp386.Using site-directed mutagenesis,the Leu362 and Tyr436 of the recombinase Mthase were mutated to Glu362 and phe436,respectively.The optimal reaction temperature of the mutant recombinase Mthase(Y436F)and the wild-type recombinase Mthase were the same at 40°C;the mutant recombinase Mthase(L362E))The optimum reaction temperature is35?.The optimal reaction p H of mutant recombinase Mthase(Y436F)is 7.0,the optimal reaction p H of wild-type recombinase Mthase is 6.5;the optimal reaction p H of mutant recombinase Mthase(L362E)is 7.5.Under the optimal reaction conditions,the specific enzyme activities of recombinase Mthase(Y436F),mutant recombinase(L362E),and wild-type recombinase Mthase were 2.78 U/mg,0.38 U/mg,and 3.75 U/mg,respectively.The stability of mutant recombinase Mthase(Y436F)between p H3.0-5.0 is higher than that of wild-type recombinase Mthase.The sequence length of the trehalose synthase Slts gene is 1716 bp,and the encoded protein is 66KDa.The reaction product of the recombinase Slts and maltose was detected by HPLC,and the recombinase Slts was analyzed to synthesize trehalose from maltose.The enzyme activity of recombinase Slts is 67.6 U/mg under the optimum conditions,and the optimum reaction temperature is 20?,which is a low temperature enzyme;the enzyme activity of recombinase Slts is strongly inhibited by Ni²⁺,Zn²⁺,and Cu²⁺,and Ca²⁺has an effect on the activity of recombinase Slts.Activation,the enzyme activity reaches 115%;EDTA has no significant effect on the activity of the recombinase Slts.