Study on purification and characterization of 5'-phosphodiesterase from Aspergillus fumigatus

The 5'-phosphodiesterase was purified from the crude enzyme solution of Aspergillus fumigatus XD9 by microfiltration through 0.22 μ m membrane, ultrafiltration through 10ku and 4ku membrane followed by ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and DEAE-Sepharose CL6B ion exchange chromatography. This protocol improved 82-fold purification with the specific activity of 1036.76U/mg protein and 7.82% recovery of enzyme activity. The purified enzyme showed a single protein band on SDS-PAGE and the molecular mass was estimated to be 9.5ku. The enzyme showed an optimum pH of 5.0 and an optimum temperature of 60℃. The enzyme was stable in the pH range from 5.0 to 9.0 and up to 55℃. Km and Vmax for the purified enzyme were 13.60mg/mL and 0.71mmol/ (mg·min) , respectively, with RNA as substrate. Its activity was activated by K+and strongly inhibited by Fe2+, Fe3+and Zn2+.