Cloning, Expression And Characterization Of A Trehalose Synthase Gene From Rhodococcus Opacus

Trehalose is a non-reducing disaccharide in which two glucose molecules are linked via α, α-（1,1）glycosidic bond. Trehalose and its derivatives can play a role as protectants against variousenvironmental stresses such as cold, heat, dehydration and so on. Due to its properties, it can be used asa useful supplement in industrial applications, like foods, cosmetics and pharmaceutical industries.Fivedistinct biosynthetic pathways can lead to the formation of trehalose:（a） TPS-TPP;（b） TreP;（c） TreT;（d） TreY-TreZ; and （e） TreS. TreS is an enzyme that allows one-step formation of trehalose frommaltose through intramolecular transglycosylation. In summary, this pathway is capable of being used inthe industrial manufacture of trehalose.Members of the Rhodococcus genus have a marked ability to metabolize a wide variety of xenobioticcompounds, and can survive in extreme environment. Rhodococcus opacus B4was isolated as anorganic solvent-tolerant bacterium, and can survive in nonaqueous environments with less than1%（w/v）water. The hydrophobic feature of Rhodococcus may be attributed to its cell envelope, which alsocontains many glycolipids, typically trehalose mycolates. The genome sequence of R. opacus B4waspublished3years ago.However, to our knowledge, very little work has been presented on thebiochemical and biophysical properties of its putative TreS. This is the first report on TreS fromRhodococcus genus and could provide some useful information for the further study both on TreS andon Rhodococcus.1、TreS gene was cloned by PCR-mediated cloning from genomic DNA of R. opacus. A gene of1857bp was amplified from R. opacus B4. The DNA sequence had been registered in GenBank withaccession number KC473564;2、Constructed expressing vector pRSET-B-treS with restriction enzyme cutting sites of BglII andHindIII and overexpressed it under the control of T7promoter in Escherichia coli BL21（DE3）pLysS.The purified recombinant protein had a molecular weight of79kDa. The amount of solublerecombinant TreS were321μg/mL;3、We identified its biochemical function and characterized its main enzyme features. The resultsfrom thin layer chromatography （TLC） and high-performance liquid chromatography （HPLC） indicatedthat this protein had the ability to catalyze the conversion between maltose and trehalose in one step.The purified recombinant enzyme showed an optimum temperature of25°C and pH optimum around7.0.The optimum OD600of the recombinant TreS was around0.6, and the optical induction time of therecombinant TreS was about6h. Kinetic analysis showed the TreS has a4-fold higher catalyticefficiency （kcat/Km） for maltose than trehalose. Enzyme activity was inhibited by Hg²⁺, Cu²⁺and Al³⁺,while it was increased by Ca²⁺and Mn²⁺. The existence of EDTA had little influence on its activity.Fiveamino acid residues, Asp²⁴⁴, Glu²⁸⁶, Asp³⁵⁴, His¹⁴⁷and His³⁵³, were shown to be conserved in R. opacusTreS, which were also important for α-amylase family enzyme catalysis. The recombinant TreS not onlyhad the ability to catalyze the conversion between maltose and trehalose, in which it palyed a role ofinvertase, but also had the ability to work as a hydrolase to hydrolyze maltose into glucose. Glucose would appear in the processes of producing trehalose. The extsitance of glucose would inhibit theinvertase activity of recombinant TreS, and as a result, the production of trehalose would be reduced.