| Trehalose is a kind of non-redcing disaccharide and is widespread in nature.Because trehalose has special physical, chemical properties and biological function, it could be widely used in molecular biology, food, cosmetic, pharmaceutical industry and agriculture etc. Trehalose synthase can convert maltose into trehalose by intramolecular transglucosylation. It is one of the most important ways about the synthesis of trehalose.TreS gene encoding trehalose synthase were successfully amplified from Pseudomonas putida S1 by PCR.The amplified fragment were linked to pMD19-T vector by TA cloning method, and then transformed the competent cells E.coli JM109. The transformants were screened on plate which have amplicillin/Xgal-IPTG and get the recombinant plasmid pMD-TS2. After identifying the recombinant plasmid by restriction enzyme digest analysis and sequencing, using restriction endonucleases BamHI and HindⅢdigest both pMD-TS2 and PQE30T.Object gene fragment were recollected and linked to each other by T4 DNA ligase.Identified by enzyme digest, the recombinant plasmid pQE-TS2 were constructed. The competent cells E.coli M15 were transformed with plasmid pQE-TS2 and induced by IPTG. Detected by SDS-PAGE, the trehalose synthase gene was expressed in E.coli and its molecular weight were about 77 kDa, but them were existed in the form of inclusion body in the host cel1s.The conditions for E. coli M15(pQE-TS2) cell growth and expression in shake flask were optimized. The optimized condition is that when the cells grew into OD600≈0.6, IPTG was added to the final concentration of 0.01mmol/L and last for 20 hours at 20℃. The expression of trehalose synthase could be as high as 18.6% of the total soluble proteins. The activity of trehalose synthase in the fermentation broth was detected as 1.9U /mL which was about 50-fold higher than that of original strain.The optimized medium suitable for high density fermentation is composed of: glucose 10 g/L, (NH4)2SO4 3 g/L, peptone 1.5 g/L, citric acid 1.5g/L, KH2PO4 12.5g/L, MgSO4·7H2O 1 g/L, trace elements solution (TES) 5 mL/L.The effect of dissolved oxygen (DO) concentration and the feeding of carbon and nitrogen source for the growth of E. coli M15(pQE-TS2) were studied at a 5L fermentor. When the DO was controlled above 30%, carbon and nitrogen source was feeded at log phase, the dry cell weight reached 17.2 g/L at last. The expression level of trehalose synthase reached 10.2% of the total soluble protein, which was 6.9 times than that with flask fermentation in unit volume. Recombinant trehalose synthase could convert substrate maltose into trehalose in one step. The influences of buffer pH, temperature, reaction time, substrate concentration, the amount of enzyme on the conversion of the maltose were studied. Its optimum pH and optimum temperature were pH 8.5 and 20℃, respectively. The enzyme was stable under 30℃and between pH 7.5 to pH9.0. It should be stored at low temperatures. The reacting conditions of trehalose synthase were researched through the orthogonal test. The results showed that: the optimum conditions for conversion of maltose by trehalose synthase were: pH 8.5, temperature 10℃, the amount of enzyme 6U/g maltose, the lasting time of reaction 36 hours. Under these conditions, the degree of conversion of the maltose was 70.1%.
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