Cloning,Expression And Characterization Of TPS/TPP Genes From The Microbacterium Sediminis YLB-01

Trehalose is linked by a-1,1 glycosidic bond, with excellent biological performance and food processing properties. The physicochemical properties of trehalose have made it with various applications in food industry. Herein we describe the characterization of recombinant trehalose 6-phosphate synthase (TPS) and trehalose 6-phosphate phosphatase (TPP) from the piezotolerant and psychrotolerant actinomycete Microbacterium sediminis YLB-01. Theoretical pI/Mw (average) for msTPS and msTPP were 5.89/55.15kDa and 4.59/28.59kDa, respectively.First, the genes encoding TPS and TPP of Microbacterium sediminis YLB-01 were cloned respectively. Then the recombinant plasmids were constructed and introduced into the host bacterial of E.coli BL21 to express of the corresponding enzyme proteins. The proteins were purified by Ni Sepharose FF resin,which is able to bind His Tags specifically, followed by dialysis and ultrafiltration. Finally,we describe the characterization of the recombinant msTPS and msTPP. The researches show that the optimum temperature of the recombinant TPS and TPP was 40℃ and 30℃, respectively. The optimum pH both was 7.5. Mg2+, Co2+ and Ba2+ can promote the activity of the recombinant TPS mostly, while Mn2+, Zn2+, Ca2+ and Hg2+ had no effect on the activity of the recombinant TPS. For the recombinant TPP, Mg2+ can promote the activity mostly, followed by Co2+ and Ba2+, and Mn2+, Zn2+, Ca2+ and Hg2+ had no effect on the activity. The Km value of the recombinant TPS for G6P and UDPG was 1.38 mM and 1.53mM, respectively. The Vmax for the recombinant TPS was 62.5 U/min·mg. However, the Km value of the recombinant TPP for T6P was 2.45 mM. The Vmax for the recombinant TPP was 82.64U/min·mg.On one hand, this study enriches the information on the properties of the trehalose biosynthetic enzymes in the deep-sea microorganisms, and provides new candidate genes for the biological engineering industry of trehalose synthetic. On the other hand, it provides theoretical guidance for the production of trehalose by enzymatic synthesis.