Study On Immobilization Of Naringinase By Polyamino Silica Gel

Naringin is the main bitterness component in citrus fruits.Naringinase is a complex enzyme consisting of both?-L-rhamnosidase and?-D-glucosidase activiyies and it can hydrolyze naringin to prunin in the first step and than to naringenin in the second step,which can decrease the bitterness of the citrus fruit juice.However,pure naringinase is expensive,poor in stability,and not easy to be recycled.In this thesis,the parameters of naringinase production by solid-state fermentation were first studied,and immobilized naringinase were prepared based on polyamino-silica(SiO₂-EDA)materials.Then,the immobilization process and the enzymatic properties of the immobilized enzymes were studied,and the assumed mechanism of naringin hydrolyzing process by the immobilized enzyme was further proposed.The effects of inoculum volumn,solid-liquid ratio,ratio of inducer to loosening agent,the variety and concentration of nitrogen source on nariginase production were analyzed and discussed during the solid fermentationprocess,and then orthogonal experiments were carried out to further optimized the experimental parameters..The order of influence degree for naringinase fermentation enzyme activity is inoculation volume>solid-liquid ratio>nitrogen source concentration>ratio of inducer to loosening agent.When the inoculation volume was 10%,the solid-liquid ratio of the medium was 1:2.5,and the concentration of(NH₄)₂SO₄ was 0.5 g/250 m L,and the ratio of inducing agent to loosening agent was 3:2.5,the naringinase activity reached 18697.49 U/g.As the SiO₂-EDA was modifed by ethylenediamine,it was indicated that 3%glutaraldehyde,50?,and 3 h was the optimized experiment conditions of aldhyde mofication.The optimal immobilization parameters were immobilization p H of 3.5,temperature of 30?,time of 3 h,initial enzyme activity of 200.75 U/m L,and the immobilized naringinase activity was up to 352.83 U/g.It was shown that the optimal reaction temperature for immobilized enzyme was 50?,and the optimal reaction p H was 8.0.At this time,the highest naringinase activity was 423.22 U/g,and there was still 71%enzyme activity left after stored at 4?for 30 days.Compared with free naringinase,the immobilized enzyme exhibited better manipulation,storage Stability and reuseability.At last,the the enzymolysis products were analyzed by liquid chromatography,it was indicated that at p H 8.0,there was only prunin in the hydrolysate.The hypothesis of the mechanism of hydrolyzing naringin by the immobilized naringinase was proposed,which provides a new direction for the preparation of prunin.