| Trehalose, a non-reducing disaccharide, is widely distributed in nature. It has been reported that this heat and acid-stable sugar protects tissues of some plants and animals, biological macromolecules, single-cell organisms, in vivo and in vitro. As an additive or stabilizer, trehalose can be used in foods, cosmetics and medicines.The industrial production methods of trehalose include chemical synthesis, extraction from microbiology, microbial fermentation, and enzymatic synthesis. In all, the enzymatic synthesis method costs leastly. There are five approaches to synthesize trehalose in vivo. Trehalose synthase can produce trehalose with maltose in one step. For the cheap substrate and simple process, it would provide a prospective biochemical method for trehalose production. At present, the study on trehalose synthase has only just begun, reports about it are limited. In this study, we cloned a noble trehalose synthase gene from Arthrobacter aurescens ATCC 13344, identified its biochemical function, and also characterized its main enzyme features. This was the first TreS report in Arthrobacter genus and it could afford some useful information for the industrial production of trehalose. The research includes the items as follows:1. All the published TreS in GenBank were classified into two groups by ClustalX1.83, and five conserved amino acid sequences were found in the first group.2. Primers were designed based on the DNA sequences of trehalose synthase (gi: 119951278) from Arthrobacter aurescens ATCC BAA-1386. A gene of 1797bp was amplified from Arthrobacter aurescens ATCC 13344. The DNA and protein sequences had been registered in GenBank with accession number 220683605, 220683606.3. The recombinant TreS was expressed in E. coli BL21(DE3)pLysS. In the optimum conditions, protein amount of soluble recombinant TreS were 1246 mg/L, and the enzyme activities in the shake flask medium were 182U/L.4. The characteristics of recombinant TreS: Its optimal activity pH was 6.5. Within the range of pH 5.0~8.0, its relative activity was beyond 60%. When the pH was beyond 7.5, its activity dropped dramatically. After being treated in pH 9.0 for 30 min, the residual relative activity was less than 4%. Its optimal activity temperature was 35℃. In the range from 20℃to 35℃, the residual relative activity was beyond 60%. After being kept in 60℃for 30 minutes, the residual relative activity was less than 5%. It indicated that the recombinant TreS was thermal un-stable. The influence of metallic ions and other chemical reagents on the recombinant TreS activity were different. When the concentration was 1 mmol/L, Mg2+,Mn2+,Ca2+ could activate its activity; DTT,EDTA,Zn2+,Co2+,Sr2+,Ba2+,Ni2+ had little influence on its activity; Pb2+,Fe2+,Cd2+,Tris inhibited its activity moderately; SDS,Al3+,Hg2+,Cu2+ inhibited its activity strongly. When the concentration raised up to 5 mmol/L, Ca2+,Mg2+,Mn2+,DTT,EDTA had little influence on its activity; the residual relative activity were more than 80%; other metallic ions and chemical reagents strongly inhabited its activity, the residual relative activity were less than 40%. The recombinant TreS also had hydrolytisis activity, it could produce glucose when translating the maltose into trehalose. Glucose was a competitive inhibitor, it could raise the Km value of maltose, but it did not change its initial velocity. And the inhibition function strengthened with the increasing concentration of glucose in the reaction mixture.
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