Site-Directed Mutagenesis Of Active Center Of Trehalose Synthase And Preparation Of Trehalulose

Trehalose synthase converts maltose into trehalose by catalyzing intramolecular transglycosylation from α,α-1,4-glycosidic linkage to α,α-1,1-glycosidic linkage, and could be applied in industrial production of trehalose. Improving the product yield and reducing the formation of byproduct for trehalose synthase conversing maltose, which could increase the utilization of substrate, thereby reducing the production costs of trehalose. So it is necessary to research into the reaction characteristics of trehalose synthase.Four open reading frames (ORFs) annotated as "trehalose synthase" or "putative trehalose synthase" from Streptomyces coelicolor, Thermomonospora curvata, Streptomyces avermitilis, and Streptomyces griseus subsp. griseus were cloned and characterized. The results showed that all the four recombinant proteins (SCTreS, TCTreS, SATreS, and SGTreS) could be identified as trehalose synthase since they could catalyze the conversion of maltose into trehalose. Molecular weight analysis by Native gradient PAGE gel electrophoresis indicated that trehalose synthases studied in this thesis were all homotrimers. Using maltose as substrate, the optimum pH values of SCTreS, TCTreS, SATreS, and SGTreS were between7.0and7.5, and the optimum reaction temperatures were20℃,35℃,20℃, and25℃, respectively. The maximum yield of trehalose was87%with very low level of byproduct (3.9%) on SGTreS under the optimal conditions. In contrast, the trehalose yields of SCTreS, SATreS, and TCTreS were76.95%,70.30%, and82.34%accompanying with6.21%,8.23%, and5.11%byproducts, respectively.Twelve sugars (sucrose, cellobiose, maltotriose, etc.) were used to test the enzymes’substrate specificity, all the SCTreS, TCTreS, SATreS, SGTreS, CGTreS (from Corynebacterium glutamicum), and GTase (from Thermus thermophilus HB-8) could convert sucrose into trehalulose, but not the other11kinds of sugars. The specific activities of SCTreS, TCTreS, SATreS, SGTreS, CGTreS, and Gtase on maltose were56,65,57,60,55, and62(U/mg protein) respectively, which were almost2times more than trehalose (25,35,22,32,26, and33) or sucrose (10,34,11,28,18, and35) as substrates.It was speculated that the five amino acids including H120, D217, E259, H328, and D329of TCTreS were critical amino acids forming the catalytic "bag" by homology modeling and alignment analysis. Thirty-five functional amino acid sites having significant influence on enzymatic characteristics close to the active center of TCTreS were determined from a total of120mutants by site-directed mutagenesis. The closer to the catalytic "bag", the higher distribution rate of functional amino acid was (the distribution rate FAA%= 62%at a distance≤3.5A, FAA%=33%between3.5and6A, and FAA%=15%between6and10A).Site-saturation mutagenesis showed that site L116significantly affected substrate specificity, and showed inconsistent trends on maltose and sucrose substrates. Among the19saturated mutants, L116Y, L116H, and L116A had an approximately2.5times higher specific activity on sucrose than the wild type, but lower activity on maltose. All19mutants of site E330only showed hydrolytic but no intermolecular transglycosylation activity on maltose and sucrose. Mutagenesis of site V160could affect the product yield and the content of byproduct, and mutant V160K had an obvious decline on yield of trehalose from70%(wild type) to45%.Correlations analysis between energy and product formation on the mutagenesis of sites E330and V160in TCTreS showed that almost all the mutants with increased local energy had negative effects on product formation. It was known that high level of energy was not conducive to the stability of protein. Therefore, we inferred that the product yield and byproduct formation on trehalose synthase were related with the stability of the enzyme. Through energy analysis and alignment of several trehalose synthases (CGTreS, SCTreS, SGTreS, etc.), two regions with a high-energy level were found in CGTreS with low yield of product and high content of byproduct. Then mutants were constructed in these two regions according to energy minimization. Among them, one mutant K279A which could improve product yield from25%(wild type) to 30%was obtained.Since there were no byproducts like isomaltose, isomaltulose, or isomelezitose formed in the reacting systems of trehalose synthase converting sucrose into trehalulose, a simple, fast, and efficient process for producing and purifying trehalulose was established based on assimilating miscellaneous sugars ability of Saccharomyces cerevisiae cells. Purified trehalulose showed a single peak detecting by HPLC.