Purification and characterization of exoinulinase from Pseudomonas putida isolated from agricultural waste materials


Inulinase(2,1-b-D-fructano -hydrolases EC is an enzyme catalyzing the hydrolysis of inulin into fructose and oligosaccharides, which are widely used as food additives. In thisstudy we report inulinase from Pseudomonas putida, as in the past decade there isn't any report on inulinase from this bacteria, especially purification and characterization of this enzyme. Pseudomonas putida wh2 gave the highest production level of inulinase, which purified to homogeneity by ammonium sulphate percipitation, gel filtration and ion-exchange chromatography with 109.1 fold of purification. The purified enzyme is a single peptide with approximate molecular mass of 72 kDa as assessed by SDS-PAGE. The enzyme is optimally active at 55 ̊c and pH 5, however it still possesses more than 70% of the maximal activity at pH ranging from 4.5 to 7.0, and it is stable at temperature up to 50 ̊c. TLC analysis of end product (enzyme) revealed that inulinase hydrolyzed inulin with a large amount of monosaccharides (fructose) and a trace amount of oligosaccharides, indicating that the purified inulinase had a high exoinulinase activity.