Cloning And Site-directed Mutation Of Trehalose Synthase Gene From Myxococcus Sp.V11

Trehalose has been considered as a kind of non reducing multifunctional disaccharide with a wide range of uses.Trehalose synthase is a glycosidase that converts maltose(?,?-1,4-glucose)into trehalose(?,?-1,1-glucose)in one step by transglycosylation.It has attracted much attention in various fields.A trehalose synthase gene designated tres I was cloned from the Myxococcus sp.strain V11.The gene had a length of 1656 bp and encoded for a trehalose synthase(TreS ?)with 551amino acids.The molecular weight of TreS ? was calculated as 64.7 k Da,and the isoelectric point(p I)was predicted to be 5.6.The catalytic cleft consists of Asp202-Glu244-Asp310 and various conserved residues.The gene(tres I)was cloned by PCR amplification and expressed in E.coli BL21(DE3).The recombinant(His)₆-tag enzyme was purified by Ni²⁺-NTA resin,with a specific activity of up to 172.7 U/mg.The result of TLC and HPLC showed that the recombinant rTreS ? can convert maltose into trehalose,with a conversion rate of 60%.The Km and Vmax of recombinant r TreS ? for maltose were 0.62 m M and 25.5 m M·min^-1 mg^-1protein respectively.The recombinant r TreS ? was optimally active at 40? in sodium phosphate buffer(20 m M,p H 6.5)and stable at temperatures of<30?.The recombinant rTreS ? was stable within a narrow range of p H values(from p H 5.5 to 7.0).The recombinant r TreS ? was slightly stimulated by Mg²⁺and strongly inhibited by Fe³⁺,Co²⁺and Cu²⁺.The recombinant r TreS ? was inhibited strongly by SDS and weakly by EDTA and Triton X-100.However,tres I enzyme is extremely unstable to temperature.Through the comparison of tertiary structure prediction and free energy prediction,the temperature stable sites G139?D295 and S523were mutated to obtain trehalose synthase with stable.Finally,the temperature stability of D295P and S523L mutant strains was improved.Tres ? gene was cloned from the genome of Myxococcus sp.V11.The full length was1650 bp,encoding 549 amino acids(63.2 k Da).The crude enzyme of tres ? was collected by a large number of fermentation.The results of HPLC and TLC showed that maltose could be transformed into trehalose.The results showed that the conversion efficiency was 71%,the K_mwas 1.918 m M,the optimal reaction temperature was 40?,the optimal p H was 6.0(PBS),and the unit enzyme activity was 100.35 U/mg,and the predicted active sites D201,E243,D310 were site mutated.By comparing metal ion binding sites,structure comparison and free energy prediction,site directed mutagenesis of possible temperature stability sites Q3,A283,Y537,W374 and R449was carried out,and the mutants A283R and Y537H with improved temperature stability were obtained.Successfully achieved heterologous expression of TreS ? and TreS ?I,studied the enzymatic properties,and identified the amino acid residues of their active centers.Both TreS ? and TreS ?I have the characteristics of not producing by-products,and TreS ? has higher enzymatic activity and TreS ?I has stronger thermal stability.Through the research on the enzymatic properties and site-directed mutation of TreS ? and TreS ?I,a new enzyme resource is provided for the production of trehalose by enzymatic method.