The synergistic effect of metal ions and amino acids on the fermentation of ß-CGTase-producing statin DF257.

ABSTRACT

To meet the growing demand of ß-cyclodextrin (CD), innovative approaches are being developed to improve the production of ß-CD by ß-cyclodextrin glucose-transferase (CGTase). Considering the low production and efficacy of wild-type ß-CGTase-producing strains, to obtain the strains suitable for industrial production of ß-CGTase, the recombinant engineered bacteria strain DF257 is constructed by transfecting with the plasmid expressing His tagged ß-CGTase. The fermentation of ß-CGTase-expressing DF257 was optimized in the presence of different metal ions, amino acids, and incubated at a certain temperature and pH condition. The results showed that when Mg2+ and isoleucine were added into the culture medium at 0.5 mM and 0.5 g/L, respectively, the enzyme activity of ß-CGTase increased significantly after incubation at 37 °C with the initial pH of 7.5. In addition, the optimal temperature for ß-CGTase with the addition of Mg2+ and isoleucine was also determined. The T half of ß-CGTase under 50, 55, 60 and 65 °C was 9.5, 8.8, 6.2 and 1.2 h, respectively. Further investigation showed that ß-CGTase kept stable under the pH 6.0-10.0, and pH 7.5 was identified as the optimal pH condition of ß-CGTase. With the addition of Mg2+ and isoleucine, the kinetic properties of ß-CGTase in the cyclization reaction had a similar form with Michaelis equation under 50 °C and pH 7.5, and Vmax, Km, and Kcat was 3.74 mg/mL/min, 3.28 mg/mL, and 31.17/s, respectively. The possible underlying mechanism by which Mg2+ and isoleucine synergistically improved the thermostability of ß-CGTase was investigated by the surface hydrophobicity index analysis, Fourier transform infrared spectroscopy and differential scanning calorimetry (DSC) analysis. The results indicated that addition of Mg2+ and isoleucine maintained the spatial structure and enhanced the thermostability of ß-CGTase. These findings provided a theoretical basis for realizing the industrialization application of ß-CGTase in promoting the generation of ß-CD.