Optimization of fermentation parameters for high-activity inulinase production and purification from Rhizopus oryzae by Plackett-Burman and Box-Behnken.

Abstract

The aim of study was to optimize fermentation parameters for inulinase production from Rhizopus oryzae by a statistical approach and to carry out purification of inulinase. Five isolated fungal strains were screen out inulin degradation by using Lugol's iodine solution. R. oryzae exhibited maximum zone of clearance around the colony and was used as an inulinase producer. The effect of carbon sources (inulin, glucose, maltose, sucrose, lactose, onion peel, stevia root, wheat bran) as medium component and fermentation parameters (temperature (25-45°C), initial pH (4-7), time (3-7days)) on inulinase production was investigated by Plackett-Burman Design. Wheat Bran (WB), temperature, pH, and incubation time were found to be significant for the production of inulinase (P < 0.05). Furthermore, Box-Behnken Design was employed to optimize fermentation conditions. The maximum experimental results for inulinase activity and specific activity were 348.36EU/mL and 3621.78EU/mg, respectively. The results were obtained at 5days of incubation time, 35°C of incubation temperature, initial pH of 5.5, and 2% (w/v) WB. Also, inulinase was purified by using ammonium sulfate precipitation, gel filtration chromatography with 2.19-fold and its molecular weight was found as 89.12kDa. The optimal pH and temperature of the purified enzyme were 4.0 and 60°C, respectively. Furthermore, the purified enzyme showed excellent stability at 60°C. In conclusion, the present study offers cost-effective method to produce inulinase from Rhizopus oryzae. Also, it can be suggested that the purified inulinase has strong potential for usage in production of fructose syrup and other industrial areas.