Enzyme Characterization，Fermentation and Purification for FAD-Conjugated Glucose Dehydrogenase

Abstract:

Bacterial-derived FAD-conjugated glucose dehydrogenase is rare. Using lactose as an inducer，fed-batch fermentation of E. coli BL21（DE3） was carried out to produce FAD-GDH in 7.5 L fermenter under stepped temperature control strategy. Enzyme activity and dry cell weight reached 1 341 U/L and 12.4 g/L respectively. By using Histrap HP，HiPre TM 26/10 Desalting and DEAE anion exchange chromatography，a purified enzyme was obtained with a specific enzyme activity of 109 U/mg.The molecular weight conformed to the theory about approximately 60 000 as determined by SDS-PAGE. The isoelectric point of the enzyme was 5.3. Absorption spectroscopy of full wavelength scanning by microplate reader showed that the enzyme was FAD conjugated. Compared with other substances，the Km of the enzyme was lowest of 2.56 mmol/L when glucose was used as substrate，and k_cat/K_m was 3 487.7 L/（mmol·s）. The enzyme kept relatively stable when the pH ranged from 5.5 to 8.0，with optimal pH of 7.0. When the pH is higher than 8.0， the enzyme activity decreases rapidly. The Tm of the enzyme measured by differential scanning calorimetry（DSC） was 77 ℃ showing high thermal stability. This experiment provides reference for the further industrial production and application of the enzyme.