Résumé
@#Objective To express the sucrose isomerase(SI) fused with the tetrameric coiled-coil domain of the cell surface protein tetrabrachion(TdoT),and study the enzymatic properties of the recombinant enzymes.Methods The gene of SI fused with TdoT at the N/C terminus was cloned into the expression vectors respectively to construct the recombinant expression vectors pET-24a-TdoT-SI and pET-24b-SI-TdoT,which were transformed into E.coli BL21(DE3) and induced to express recombinant enzymes.The enzymatic properties and product specificity of the purified recombinant enzymes were studied.Results TdoT-SI and SI-TdoT were expressed as inclusion bodies with catalytic activity,while SI inclusion bodies without TdoT showed no catalytic activity.The results of enzymatic property analysis showed that the optimum reaction temperature for TdoT-SI and SI-TdoT active inclusion bodies was 40 ℃,and the optimum reaction pH was 5.5 and 5.0,respectively.The K_m of TdoT-SI active inclusion bodies was(103.9±9.5) mmol/L and the k_(cat)/K_m was(0.06±0.002) L/(mmol·s),while the K_m of SI-TdoT active inclusion bodies was(54.4±6.6) mmol/L and the k_(cat)/K_m was(0.03±0.002) L/(mmol·s).The results of product specificity analysis exhibited that the proportion of isomaltulose in the product did not change significantly,while the proportion of trehalose decreased,and the proportion of monosaccharides increased with increasing reaction temperature.Conclusion The active inclusion bodies of SI fused with coiled-coil domain were successfully prepared by fusion expression technology.As a novel self-immobilized enzyme,it has the advantage of simultaneous expression and immobilization,which provides a new strategy for large-scale preparation and efficient utilization of recombinant SI.
Résumé
To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.