Abstract
The objective of this study was to create a fermentation process for the production of inulinase, an important enzyme with numerous applications in the food and pharmaceutical industries, using low-cost agricultural waste as substrates for Rhizopus oryzae NRRL 3563. High titer inulinase production in chicory roots by Rhizopus oryzae in a submerged culture was accomplished using a statistical experimental design. A two-level Plackett-Burman design followed by a three-level Box-Behnken design producing a high inulinase titer of 1085.11 U/mL, 2.83-fold the maximum level, was obtained in the screening experiment. The optimal levels were as follows: chicory root, 10 g/L; NaNO3, 5 g/L; and KCl, 0.2 g/L. The produced inulinase enzyme was purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.63-fold purification with 60% activity recovery. The enzyme had a molecular weight of approximately 130 KDa. The purified enzyme showed optimum activity at 50 degrees C and pH 6.0. The pH stability range was three to six and the temperature stability was up 70 degrees C. The purified inulinase could hydrolyze inulin and sucrose, but not cellobiose or soluble starch. Km and Vmax for inulin were determined to be 0.8 mg/mL and 50,000 U/mg, respectively. The two-level Plackett-Burman design was applied followed by a Box-Behnken model for optimization of fermentation conditions. Accordingly, the optimal combination of fermentation was a reaction time of seven hours, a temperature of 60 degrees C, and an enzyme concentration of 40,000 U/mL, which resulted in a 58.07% saccharification yield. The characteristics of the enzyme and its kinetic parameters suggested that it was highly effective in the fermentation of inulin and inulin-containing substrates. Additionally, it raises the potential of using inulinase enzymes in pharmaceutical and food industries.