Abstract
To meet the growing demand of β-cyclodextrin (CD), innovative approaches are being developed to improve the production of β-CD by β-cyclodextrin glucose-transferase (CGTase). Considering the low production and efficacy of wild-type β-CGTase-producing strains, to obtain the strains suitable for industrial production of β-CGTase, the recombinant engineered bacteria strain DF257 is constructed by transfecting with the plasmid expressing His tagged β-CGTase. The fermentation of β-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 Mg(2+) and isoleucine were added into the culture medium at 0.5 mM and 0.5 g/L, respectively, the enzyme activity of β-CGTase increased significantly after incubation at 37 °C with the initial pH of 7.5. In addition, the optimal temperature for β-CGTase with the addition of Mg(2+) and isoleucine was also determined. The T half of β-CGTase under 50, 55, 60 and 65 °C was 9.5, 8.8, 6.2 and 1.2 h, respectively. Further investigation showed that β-CGTase kept stable under the pH 6.0-10.0, and pH 7.5 was identified as the optimal pH condition of β-CGTase. With the addition of Mg(2+) and isoleucine, the kinetic properties of β-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 Mg(2+) and isoleucine synergistically improved the thermostability of β-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 Mg(2+) and isoleucine maintained the spatial structure and enhanced the thermostability of β-CGTase. These findings provided a theoretical basis for realizing the industrialization application of β-CGTase in promoting the generation of β-CD.