Aim To study the mechanism of Escherichia coli DNA photolyase(EC 4.1.99.3) in repairing the ultraviolet induced pyrimidine dimmer lesions in DNA.Methods UV-Vis Specturum was used to measure the relationships between the enzyme activity and different state flavin ademine denucleotide(FAD).Results It was shown that the enzyme activity was the highest when FAD chromophore was in reduced state and lowest when it was in oxidated state,while the activity ranked between when the enzyme was in radical state.Partial denature was observed when the enzyme was in oxidated state and under over 30℃,yet the enzyme activity remained unchanged.The presence of MTHF chromophore enhanced the enzyme activity.Conclusion These findings provide clues for clinical application of DNA photolyase in the future.

As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni²⁺ affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by β-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.
Escherichia coli/genetics* ; Hexosyltransferases/genetics* ; Inulin ; Oligosaccharides ; Sucrose