Aspergillus niger exo-inulinase purification by three phase partitioning

Inulinase (2, 1-beta-D-fructan fructanohydrolase, EC 3.2.1.7) hydrolyses inulin into nearly pure fructose, which is an excellent alternative for the production of fructose syrup. Growing inulinase utilization in different industries encourages the search for high benefit/cost ratio purification techniques for such enzymes. Here, we adapted the three-phase partitioning (TPP) technique for the downstream process of inulinase obtained from Aspergillus niger. TPP is a simple non-chromatographic process used for purification and concentration of protein. The various conditions required for attaining efficient purification of inulinase were optimized. The optimum conditions for TPP were found to be 30% w/v ammonium sulfate saturation with 1.0:0.5 v/v ratio of t-butanol to crude extract at pH 4.0 and temperature 25 degrees C. The enzyme was purified by 10.2-fold using two-step TPP with an overall recovery of 88%. The enzyme's molecular mass was found to be 63.8kDa by SDS-PAGE analysis. Terminal hydrolysis fructose units from the inulin show that enzymes are exo-inulinase. The recovery of purified exo-inulinase achieved in this work shows the technical viability of enzyme purification by TPP.