Gene Cloning, Expression, Charactrization And Molecular Modification Of The Trehalose Synthase From Thermus Ruber

Trehalose synthase is an intramolecular transglycosylase, which could catalyze the conversion of maltose into trehalose or convert sucrose into trehalulose. There is a huge market potential of trehalose and trehalulose in industrial production because of their characteristics, such as moisturizing and preservation. It is significant to study enzymatic properties and catalytic properties of trehalose synthetase for industrial productions of trehalose and trehalulose. It lays foundation for understanding the space structure and function of the enzyme by analyzing substrate binding sites and catalytic mechanism of trehalose synthetase.Trehalose synthase gene from Thermus ruber was cloned and expressed in Escherichia coil XL-10Glod strain. The recombinant TR-TreS was purified by nickel affinity chromatography. The recombinant enzyme showed its optimal activity was at55℃and pH6.5when it catalyzed maltose, and its specific activity was6.76U/mg. The result of HPLC system analyzed that TR-TreS had captable of reciprocal reaction. It could convert maltose to about86%trehalose and14%glucose. When it catalyzed sucrose as a substrate, the recombinant enzyme showed its optimal activity was at55℃and pH6.5, and its specific activity was1.21U/mg. The result of HPLC analyzed that TR-TreS could convert sucrose to about66%trehalulose and34%glucose and fructose. Its enzymatic properties was relatively stable after60days at4℃.The results of rite-directed mutagenesis showed that the mutant Y205L+E206K couldn’t use sucrose after mutating the205th tyrosine to leucine and the206th glutamic to lysine from recombinant enzyme, and the ability of using maltose and trehalose was significantly lower. the mutant Y205L could not utilize sucrose, but it could catalyze maltose increasing by0.1times and decrease ability to utilize trehalose. the mutant E206K had a weaker ability of using trehalose, maltose and sucrose. These mutants revealed that the two amino acids residues were very important to the enzyme binding site. To futher study the role of other amino acids residues in trehalose synthetase, site-directed saturation mutagenesis of the206th glutamic has built. The results of mutants E206V and E206W suggested that they could not transform sucrose and maltose, and had a low utilization rate of trehalose, and the mutants E206C, E206S and E206R could not convert sucrose, and had a low utilization rate of maltose and trehalose. Studying on the enzymatic properties of Y205L, we have acknowledged that its optimal activity was at50℃and pH7.0, and its specific activity was7.6U/mg. HPLC analyzed that Y205L could convert maltose to about92%trehalose and8%glucose.