Production Of Naringinase By Submerged Fermentation With Penicillium Sp.1523 And Immobilization Of The Enzyme

Naringinase which is an enzyme complex consisting of a-L-rhamnosidase (EC 3.2.1.40) and flavonoid-β-D-glucosidase (EC 3.2.1.21) can be widely used in citrus debittering due to its hydrolysis ability of the primary bitter component naringin into rhamnose, glucose and tasteless naringenin in a stepwise catalysis. Compared with other debittering methods, this enzymatic process shows some advantages, such as mild work conditions, simple techniques, non-contaminant operation, keeping original nutritional value and so on. Moreover, naringinase can also be applied in pharmaceutics, cosmetics and food technology.At present the production level of naringinase is very low. Few reports can be available on commercial production of it, and the industrial application has been limited, so there must be a great practical significance if a research on improving production of naringinase is carried out.In this thesis Penicillium sp.1523 was chosen as starting strain, and the conditions of shaking-flask fermentation were optimized and processes of fermentation in a 5 L bioreactor was discussed, then the purification and characterization of naringinase was studied as well as immobilization of naringinase.The main results were taken as follows:Monofactorial experiments, Plackett-Burman design and response surface methodology were applied to optimize shaking-flask fermentation conditions of naringinase. The optimum carbon and nitrogen for Penicillium sp.1523 were obtained through monofactorial experiments, which were corn meal and bean cake powder respectively. So did the best inoculum age and inoculum size, which were 36 h and 5% (v/v) respectively. Through Plackett-Burman design seven related factors were evaluated, and the result revealed that corn meal, bean cake powder, and naringin had prominent effect on naringinase production. Then steepest ascent experiment and Box-Behnken were taken on the selected three factors to estimate optimum culture medium composition. The experimental results showed that the optimum culture medium for Penicillium sp.1523 was composed of 31.14 g/L corn meal,31.53 g/L bean cake powder,1.65 g/L naringin,1.00 g/L dipotassium hydrogen phosphate,0.10 g/L zinc sulfate,0.06 g/L heptahydrate magnesium sulfate, and 0.10 g/L calcium chloride. Under the optimized conditions, naringinase activity in broth reached 891.79±6.33 U/mL, which was consistent with the maximum predicted value. The research above provided a practical basis for fermentation scale-up later.Based on the results at shaking-flask fermentation level, scale-up studies were carried out in a 5 L bioreactor. The naringinase activity in crude broth reached 1229.40 U/mL in the optimized operating conditions, which was 41.0% higher than that in shaking-flask fermentation. The batch fermentation kinetics of naringinase produced by Penicillium sp.1523 was studied based on the experimental data from fermentation in a 5 L bioreactor. The kinetic models of the fermentation were established, which were shown as below:Penicillium sp.1523 growth model:Naringinase formation model:Substrate consumption model:The above models could fit the experimental data well. It might be helpful to understand and guide for further study of Penicillium sp.1523 fermentation process.Purification process of naringinase from Penicillium sp.1523 was explored using ultrafiltration, ammonium sulfate precipitation and Sephadex G-200 gel filtration chromatography methods, and the purification effects were identified by SDS-PAGE experiment at last. The purification protocol resulted in 11.99-fold purified naringinase with a final yield of 38.10%, and the specific activity reached 4478.85 U/mg. After successive purification two clear protein bands were obtained by SDS-PAGE, which indicated naringinase consisted of two subnits with molecular masses of 89 KDa and 72 KDa respectively.In order to improve naringinase catalytic performance, immobilization of naringinase using domestic TJS resin was studied. Monofactorial experiment and Box-Behnken design were adopted to optimize immobilization conditions. Then the data was analysed with SAS software version 8.0, and the optimum immobilization conditions were summed up:pH of immobilization system, immobilization temperature, the ration of carrier addition, and immobilized time were 6.5,26.6℃,0.79 g TJS resin per 5 mL enzyme solution, and 24 hours, respectively. Under these conditions, the specific activity of immobilized naringinase reached 3949.32 U/g, with an enzyme recovery of 44.79%, and the protein binding rate was 98.43%. The results above indicated that TJS resin was a good carrier for naringinase immobilization research.All of this study can provide a basic work for both the industrial production of naringinase and the practical application of this enzyme.