Study On The Heterologous Coexpression Of MTSase And MTHase

Trehalose is called "sugar of life" because of its excellent resistance to biological macromolecules,It has wide application in food transportation and medicine preservation.At present,the production of trehalose by the double enzyme method has become the first choice for industrial production of domestic and foreign enterprises,this production method has higher product yield and lower production cost,which is suitable for industrial production.Because the double enzyme method is produced by the continuous action of Maltooligosaccharide trehalose synthetase(MTSase)and Maltooligosaccharide trehalose hydrolase(MTHase),trehalose is produced.The two enzymes are expressed separately by the two strains,which will cause manpower and waste of material resources.In this research,the following work has been done to solve this problem(1)In this study,the MTSase and MTHase encoding genes treY gene and treZ gene derived from Sulfolobus acidocaldarius ATCC 33909 were transformed into E.coli for recombinant expression,and the expression strain E.coli BL21(DE3)/pET-28a-treY/treZ The target protein expressed by enzymatic properties was found to find that the optimal temperature of MTSase is 65? and the optimal pH is 5.5,the optimal temperature of MTHase is 60? and the optimal pH is 6.0,the optimal temperature for the double enzyme co-conversion substrate is 65?,the optimal pH is 5.5,the optimal substrate concentration is 20%,and found that adding 5%CGTase can significantly improve the conversion rate of double enzyme.Through the analysis and detection of the purified enzyme,it was found that when the molar mass ratio of the purified enzyme MTSase:MTHase=3:1,the double enzyme has the highest conversion rate 70%,the enzyme activity of MTSase is 119.2 U/mg,and the enzyme activity of MTHase was 473.2 U/mg.(2)By connecting the SpyTag-SpyCatcher self-assembly system derived from the CnaB2 domain to the treY and treZ genes,construct a single plasmid expression strain that self-assembles in vitro and a single plasmid expression strain that self-assembles in vivo E.coli BL21/pETDuet-SpyCatcher-treY-SpyTag-treZ,and a dual plasmid expression strain capable of self-assembly in vivo.Verification of enzyme production by fermentation found that both in vivo self-assembly system and in vitro self-assembly system can successfully connect MTSase and MTHase together and have enzyme activity.The enzymatic properties of the self-assembled complex enzyme were studied and found to be consistent with the non-assembled double enzyme enzymatic properties.And a strain of in vivo self-assembled recombinant E.coli with a transformation rate of 75%was obtained.(3)For the constructed recombinant strain E.coli BL21(DE3)/pET28a-SpyCatcher-treY/pETDuet-SpyTag-treZ optimized the medium,and finally determined that the components of the optimized TB medium were pH 7.0,10 mM Mg2+,10 mM Cu2+,10 g/L glycerin,20 g/L yeast extract,10 g/L tryptone.The fermentation conditions of the fermentor were explored,and the optimal culture conditions of the strain were:the optimal induction time was OD600 reached 6,the optimal induction temperature was 25?,and the lactose flow rate was0.2 g/L/h.After adding 5%CGTase,the compound enzyme converted 20%maltodextrin under the conditions of 65? and pH5.5,and the highest conversion rate reached 85.3%.