Expression And Application Of Sucrose Isomerase From Pantoea Dispersa

Sucrose isomerase(EC 5.4.99.11) could convert sucrose to the isomer, isomaltulose and trehalulose. So far, most of sucrose isomerases primarily produce isomaltulose. Isomaltulose had higher sweetness, soluble in water and prevent the diabetes and irregular teeth. Thus, isomaltulose was suitable for diabetics and children, and widely applied in the candies and pastries.In the present study, the gene of sucrose isomerase from Pantoea dispersa UQ68 J was expressed in Escherichia coli and Bacillus brevis. The condition of fermentation was optimized for improving the yield of sucrose isomerase. The crude sucrose isomerase was purified and characterized. The main contents were listed as follow.(1) The gene of sucrose isomerase which containing the signal peptide and the mature protein, coming from P. dispersa UQ68 J, was optimized and connected with the expression vector pET-24a(+). The recombinant plasmid was transformed into E. coli BL21(DE3) and named as ORI strain. The ORI strain was cultured in TB media and the extracellular and total enzyme activity reached 65 U·m L-1 and 85 U·m L-1. To further enhance the yield of sucrose isomerase, the effects of PelB and OmpA signal peptide on sucrose isomerase were studied. The mature protein, which started from the 22 th amino acid, was connected with the PelB signal peptide to construct P22 strain. The total activity of P22 reached 143 U·mL-1, which was 1.5 times of ORI strain. And the sum of sucrose isomerase activity coming from extracellular and periplasmic space was 1.3 times of the corresponding activity of ORI strain. There are a lot of sucrose isomerase existed in the P22 strain’s periplasmic space and cytoplasm, the effects of glycine concentration and induction time were studied to improve the extracellular activity of sucrose isomerase in 3 L fermentor. Induction when the DCW reached 18 g·L-1(OD600 =30), with 0.5% glycine, the extracellular enzyme activity reached 1981 U·mL-1, and the total enzyme activity reached 2640 U·mL-1.(2) The gene of sucrose isomerase without the signal peptide, the start site of which was the 22 th or 34 th amino acid, was connected with the extracellular expression plasmid of Bacillus brevis, respectively. Then the recombinant expression vector, contained the gene of sucrose isomerase, was transformed into B. brevis and the B22 strain and the B34 strain were constructed. After cultured in TM media for 48 h, the B22 strain and B34 strain’s extracellular enzyme activity were 50 and 35 U·mL-1, respectively. When optimized the culture media of B22 strain in flask, the extracellular sucrose isomerase activity reached 138 U·m L-1. For improving the yield of sucrose isomerase, the effects of nitrogen contention and impeller speed were studied in 3 L fermentor. When the nitrogen contention is 30 g·L-1 and the speed is 400 r·min-1, the extracellular sucrose isomerase activity increased to 190 U·m L-1.(3) The crude extracellular sucrose isomerases expressed in different host strains were purified. Then the related enzymatic properties were investigated. The result showed that the sucrose isomerases expressed in E. coli and Bacillus brevis exhibited specific activities of 597.7 and 555.0 U·mg-1, respectively. The optimal temperature and pH were both 30℃ and 6.0. Kinetic analysis showed that the Vmax of the sucrose isomerase expressed in E. coli and Bacillus brevis were 660.9 and 657.2 U·mg-1, respectively; the Km of them were 21.3 and 20.6 mM, respectively; the Kcat of them were 730 and 724 s-1, respectively; and the Kcat/Km were 34.3 and 35.1 mM-1·s-1, respectively.(4) This study also studied the effects of different conditions on the yield of isomaltulose production using the recombinant sucrose isomerase. The optimal conditions for the conversion was 20 U·g-1, 35℃, pH 6.5 and 400 g·L-1 sucrose, the maximum yield was 92.0%. In addition, even the concentration of substrate increased up to 700 g·L-1, the yield was still maintained at about 90%.