Purification and Properties of Extracellular Endoinulinase from Aspergillus niger 20 OSM

Extracellular inulinase (E.C. 3.2.1.7) produced by Aspergillus niger 20 OSM culture in a 2-litre fermentor was isolated and purified by ion exchange chromatography on DEAE Sepharose, hydrophobic interaction on phenyl Sepharose, chromatofocusing on PBE-94, and size exclusion chromatography (SEC) on Sephadex G-200. The enzyme was homogeneous, as measured by SDS-PAGE with an apparent molecular mass of 69 or 64 kDa, as determined by SEC. Carbohydrate content of the enzyme was estimated at approx. 44.5 %. The optimum temperature and pH for enzyme activity were 55 C and 5.0, respectively. Some physicochemical properties of the purified inulinase were also determined (isoelectric point, Km, Vmax and Ea). Enzyme activity was inhibited by EDTA, pCMB, Hg , Mn, and some other metal ions. Calcium cations showed a positive effect on the enzyme activity. Thin layer chromatography indicated that the purified enzyme is a typical endoinulinase.