The Process Optimization Of Producing Trehalose Synthase Of The Recombinant Escherichia Coli (BL21)

Trehalose is a kind of special disaccharide, has good physiological function, better moisture resistance, drought resistance, osmotic pressure resistance and other properties, so it is widely used in food, medicine, cosmetics, agriculture and other fields, and it has good security. Currently the ways of generating the trehalose include TreT pathway, TreS pathway, TPS/TPP pathway and TreY/TreZ pathway. TreS pathway is considered to be the most advantageous way, because trehalose synthase can transform maltose into trehalose in one conversion step with low cost and simple process, which conform to the industrial production design.Studies showed that the original strain produced less trehalose synthase and the enzyme activity was relatively low, so they can not meet the design requirements of industrialization, therefore through genetic engineering techniques, the heterologous. expression of the trehalose synthase gene has become an important way to obtain trehalose synthase. In previous research reports, there have been some trehalose synthase gene were cloned and transformed into E. coli and were expressed, but the studies found that the recombinant E. coli were not applied to large-scale production.In this paper, through constructing the trehalose synthase gene plasmid pET22b-tres, and made it transformed into Escherichia coliBL21 (DE3) to be efficiently expressed. And the lactose of 4g/L was as the inducer, the expression of trehalose synthase accounted for larger proportion in total protein content. Ni-column was used to combine the recombinant trehalose synthase to obtain pure enzyme solution, and studied the enzymatic properties of pure enzyme solution. The results showed trehalose synthase had optimum activity at 25℃,pH8.0.In the fermentation process of using recombinant E. coli, the fermentatton broth was found to have high content of acetic acid, which had impact on the production and activity of trehalose synthase. We also confirmed inhibition of acetic acid (sodium) on recombinant bacteria by experiments and discussed the inhibition mechanism. On this basis, the fermentation conditions were optimized, including the nitrogen source, carbon source, dissolved oxygen control method for future optimization of fermentation to lay a good foundation.