High Efficiency Expression of Trehalose Synthase in Escherichia coli and Its Use in the Production of Trehalose

Abstract

The trehalose synthase (TreS) from Pseudomonas putida ATCC47054 was highly expressed in E.coli BL21(DE3) using pET15b and pET22b as expression vector. The expression of TreS protein in recombinant E.coli BL21(DE3)/pET15b-treS reached 34.3% of total protein, is better than E.coli BL21(DE3)/pET22b-treS reached 19.9%. The optimum pH and temperature of purified TreS by Ni²⁺ affinity chromatography is 8.0 and 25 ℃, and stable between pH 6.5 to 9.0, and exhibited the thermalstability from 15 to 40 ℃. When the conversion system temperature of TreS is more than 40 ℃, enzyme activity will accelerate loss. But a special phenomenon was discovered that high substrate maltose concentration is advantageous to the thermal stability of enzyme activity. When the substrate maltose concentration reached 300 g/L, the TreS still has a stable conversion efficiency at 50 ℃. In order to improve the intracellular expression of TreS, the fermentation and induction conditions of recombinant E.coli BL21(DE3)/pET15b-treS were optimized. Using final concentration 4 g/L lactose as inducing agent to induce 7 h at 27 ℃ in 5-L fermentor, the enzyme activity reached 39,866 ± 1420 U per gram dry cell weight. For avoid the cost of enzyme purification, the cell lysis solution was directly used to convert high maltose syrups for preparation of trehalose, and over 64% of maltose in conversion system can be converted into trehalose with substrate maltose concentration 300 g/L at 50 ℃ and pH 8.0.