Fermentation, Purification And Characterization Of Hesperidinase Produced By Snow Rot Fusarium 6238

Citrus is one of the largest output fruits in the world, which has strong competitiveness in China. It has been widely acknowledged that China is one of the leading citrous producing countries, whose annual output of citrus petal cans is over 70% of the world production. With the development of orange juice and citrus petal cans processing production, large amounts of waste will be produced. The utilization rate of resources, as well as the economic benefits, would be greatly improved, if we recycle and reuse these waste. Flavonoid is a kind of important active substances in citrus, which, is relatively rich in citrus seeds and skins. Hesperidin is a kind of flavanone, which has the activity of antioxidant, antiallergic, anticancer, antibacterial, and the ability to strengthen the capillary toughness as well as lower blood pressure. With low so lubility in water, the hesperidin owns poor bioavailability.In this paper, we use Snow rot Fusarivm 6238 as the starting strain to produce hesperidinases, which will accelerate the biological transformation of hesperidin, and improve its pharmacological effects. Frstly, we studied the effects of fermentation medium and culture conditions on the production of hesperidinases. The results showed that hesperidinases were secondary metabolites, and the growth cycle was 72 h. The inducer hesperidin greatly helped the production of hesperidinases during the process that they were added into the culture medium in the inception phase. The medium composition and culture conditions were optimized in flask. The optimal conditions for producing hesperidinases were drawn as follows:glucose 40 g/L, yeast extract 5 g/L, inoculation amount 4%, potassium phosphate dibasic 2 g/L, the optimum temperature is 30℃, and the optimum pH is 6.5. Under such conditions, the hesperidinase activity would reach 1305 U/mL, increased by 2.2 fold compared to that before optimization.The study on fed-batch fermentation process of Snow rot Fusarium 6238 was conducted. The results showed that, if the initial sugar concentration was 20 g/L, and added the rest of the carbon source after fermentation for 36 h, the hesperidinases activity would increased by 19% than batch fermentation.The Snow rot Fusarium 6238 was treated by several mutation conditions to select the positive strain with high activity of enzyme. As a result, one strain, named UV-LiCl-39, was bred from the mixture of mutant strains, whose activity of enzyme was enhanced by 49%.The optimal hesperidinase catalytic temperature was 60℃, and it was stable at 55 ℃.80.52% enzyme activity was remained after incubating at 55 ℃ for 100 min. The optimal pH for hesperidinase was 4.0, which was stable from pH 3.0 to 5.0. Finally we did a preliminary study on the catalysis process of hesperidinases. The results showed that a-rhamnosidase could hydrolyse the bond between rhamnose and glucose, while the β-glucosidase plays a role in hydrolysing the bond between glucose and he speritin. After purification of a-rhamnosidase from the culture broth of Snow rot Fusarium 6238, molecular weight of a-rhamnosidase was measured to be about 66 kDa.