Studies On Molecule Evolution And The Enzyme Properties Of The Trehalose Synthase From Corynebacterium Glutamicum

Trehalose synthase is the key enzyme in enzymatic process to produce trehalose.It can catalyze maltose bonded in anα,α-1,4-glycosidic linkage to produce trehalose withα,α-1,4-glycosidic linkage.Recently,Trehalose has been widely attended because of it's non-special protecting function on big molecule of biology.The scientists from many countries have been engaging in studying on the technology of production and application to reduce it's cost of production. The existing trehalose synthase have some disadvantages:the transform efficiency is not enough high;The thermostability is low;The reaction time is long and so on.Therefore,it is necessary to perform mutagenesis on trehalose synthase to improve the industrial capability.In this paper,trehalose synthase was evolved by half-rational design.The three-dimensional model of TreS from Corynebacterium glutamicum was constructed based on the crystal structure of trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45 on the SWISS-MODEL Internet.The energy of primal structure was optimized.Combined with analyzing the conserved region of trehalose synthase amio sequence from different origin.The conserved amio acid R245 and E289 were predicted likely as active site and were mutated.Further more,the position F244,D247and A288 near the two sites(R245 and E289)were performed site-mutagenesis.The results indicated that the activity was lost after R245 and E289 were converted to other 19 kinds of amio acids and that the mutants D247E and D247N were also shown no activities.The reason of activity lost probably was that these positions were important to maintain the structure stability of the protein,and they possibly were crucial residues or active sites of the enzyme.The specific activity of the mutants F244C,F244L,F244W,F244Y and A288G were decreased largely and reduced to 38%,24%,62%,64%and 35%of the wild-type respectively.After the position of A288 was changed to A288T,it was shown that the activity was disrupted.Compared with the wild-type,The Km of F244C,F244W,A288G were not significantly altered,but the affinity of F244L and F244Y enzyme for the maltose substrate were decreased;The optimum temperature of F244Y was 27℃which the same as the wild-type,and the mutants F244C,F244L,F244W and A288G were enhanced 5℃more than the wild-type;The optimum pH of all the mutants were decreased,The optimum pH of F244C,F244Y and A288G were 7.5 that dropped 0.5 pH units compared with those of the wild-type,and the F244L and F244W were reduced 1.5 units;The thermostability of all the mutants were increased in various degree,The mutant F244Y,244W and A288G increased about more one degree than that of the wild type;F244L increased two degree and F244C enhanced four degree approximately.It can not only provide useful datum and information in studying the relation of the configuration and function by analyzing and studying these changes about the properties of the mutants but also can afford the theoretic basis to perform molecule evolution on trehalose synthase for the future.