Trehalose is a widely used and safe natural disaccharide.Maltooligosyl trehalose hydrolase(MTHase)is one of the key enzymes for trehalose preparation by double enzyme method using starch or dextrin as substrate.In ind...Trehalose is a widely used and safe natural disaccharide.Maltooligosyl trehalose hydrolase(MTHase)is one of the key enzymes for trehalose preparation by double enzyme method using starch or dextrin as substrate.In industrial production,the thermalstability of MTHase is of great significance.We first heterogeneously expressed MTHase from Arthrobacter in E.coli strains BL21(DE3).Based on the overall stability of the protein after virtual saturation mutation predicted by FoldX and the evolutionary information from PSSM,15 mutations were selected and combined.Finally,the combinatorial mutant G589P/A57P was obtained.At 60℃,the t_(1/2)of G589P/A57P and wild type are 37 min and 19 min,respectively,which is 1.9 times higher than that of wild type.The enzyme kinetic parameters of G589P/A57P were analyzed.The K_(M)and k_(cat)are 4.82 mM and 1136 s^(−1),respectively,and the results were close to the wild type,indicating that the mutation did not reduce the catalytic efficiency of the enzyme.The molecular dynamics simulation results show that the rigidity and thermal stability of G589P/A57P protein increase in the range of residues 50-100 and 400-500,which may be due to the proline effect caused by the introduction of proline.展开更多
Maltooligosyl trehalose synthase(MTSase)and maltooligosyl trehalose hydrolase(MTHase)are used to produce trehalose,a disaccharide of interest to many different industries,from starch.MTSase and MTHase from Arthrobacte...Maltooligosyl trehalose synthase(MTSase)and maltooligosyl trehalose hydrolase(MTHase)are used to produce trehalose,a disaccharide of interest to many different industries,from starch.MTSase and MTHase from Arthrobacter ramosus S34 were first produced using separate Escherichia coli BL21(DE3)strains.The activities obtained in a 3-L fermenter under optimized conditions were 1608.3 UmL^(-1) and 8766.2 UmL^(-1),respectively.Then,MTSase and MTHase were co-produced in E.coli BL21(DE3)using a co-expression construct.After optimizing induction conditions,the MTSase and MTHase activities produced by the superior strain reached 1827.4 UmL^(-1) and 2944.9 UmL^(-1),respectively.When the co-produced enzymes were used to synthesize trehalose from starch,a conversion rate identical to that achieved using separately produced enzymes(about 67%)was obtained.This is the first describing the co-production of the MTSase and MTHase in a 3-L fermentor.The results represented the highest MTSase production level reported to date,and the MTHase activity from co-production was sufficient for trehalose synthesis.Using co-produced enzymes during trehalose synthesis would lower costs without sacrific-ing yield.Therefore,this study provided a foundation for the industrial synthesis of trehalose using co-produced enzymes.展开更多
基金supported by the National Natural Science Foundation of China(No.32171471)Key Research and Development Project of Shandong Province,China(2019JZZY020605)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions,Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘Trehalose is a widely used and safe natural disaccharide.Maltooligosyl trehalose hydrolase(MTHase)is one of the key enzymes for trehalose preparation by double enzyme method using starch or dextrin as substrate.In industrial production,the thermalstability of MTHase is of great significance.We first heterogeneously expressed MTHase from Arthrobacter in E.coli strains BL21(DE3).Based on the overall stability of the protein after virtual saturation mutation predicted by FoldX and the evolutionary information from PSSM,15 mutations were selected and combined.Finally,the combinatorial mutant G589P/A57P was obtained.At 60℃,the t_(1/2)of G589P/A57P and wild type are 37 min and 19 min,respectively,which is 1.9 times higher than that of wild type.The enzyme kinetic parameters of G589P/A57P were analyzed.The K_(M)and k_(cat)are 4.82 mM and 1136 s^(−1),respectively,and the results were close to the wild type,indicating that the mutation did not reduce the catalytic efficiency of the enzyme.The molecular dynamics simulation results show that the rigidity and thermal stability of G589P/A57P protein increase in the range of residues 50-100 and 400-500,which may be due to the proline effect caused by the introduction of proline.
基金financed by the National Natural Science Foundation of China(31771916)the Fundamental Research Funds for the Central Universities(5812050205226750)the Research Program of State Key Laboratory of Food Science and Resources,Jiangnan University(No.SKLF-ZZA-202302).
文摘Maltooligosyl trehalose synthase(MTSase)and maltooligosyl trehalose hydrolase(MTHase)are used to produce trehalose,a disaccharide of interest to many different industries,from starch.MTSase and MTHase from Arthrobacter ramosus S34 were first produced using separate Escherichia coli BL21(DE3)strains.The activities obtained in a 3-L fermenter under optimized conditions were 1608.3 UmL^(-1) and 8766.2 UmL^(-1),respectively.Then,MTSase and MTHase were co-produced in E.coli BL21(DE3)using a co-expression construct.After optimizing induction conditions,the MTSase and MTHase activities produced by the superior strain reached 1827.4 UmL^(-1) and 2944.9 UmL^(-1),respectively.When the co-produced enzymes were used to synthesize trehalose from starch,a conversion rate identical to that achieved using separately produced enzymes(about 67%)was obtained.This is the first describing the co-production of the MTSase and MTHase in a 3-L fermentor.The results represented the highest MTSase production level reported to date,and the MTHase activity from co-production was sufficient for trehalose synthesis.Using co-produced enzymes during trehalose synthesis would lower costs without sacrific-ing yield.Therefore,this study provided a foundation for the industrial synthesis of trehalose using co-produced enzymes.
