Study On Naringinase Immobilization On Magnetic Silica Materials

Naringinase,a complex enzyme composed ofα-L-rhamnosidase andβ-D-glucosidase,can hydrolyze bitterness like naringin and hesperidin in citrus fruits,which usually be used for debittering of citrus juice.In order to enhance the reusability and stability of naringinase and cut the cost of industrial production,naringinase is generally used for debittering of citrus juice after immobilization on some carriers.But in a real industrial production,the common materials used for immobilization often have some shortage,for instance rely on natural precipitation for solid-liquid separation,poor acid resistance and slowly separation speed.In this paper,novel magnetic silica polymer microspheres were prepared,then the effect of various technological parameter in the process of naringinase immobilization were studied,meanwhile the enzyme properties of immobilized naringinase were analyzed.Immobilized naringinase on sodium alginate and chitosan cross-linked by glutaraldehyde were prepared by embedding-crosslinking method and adsorption-coupling method,respectively.Then the optimal immobilization conditions were investigated.The results showed that the optimal specific activity of sodium alginate immobilized naringinase and chitosan immobilized naringinase were 55.23 U·g^-1 and 126.86 U·g^-1,respectively.The thermal stability of chitosan immobilized naringinase was obviously superior to of sodium alginate immobilized naringinase.Meanwhile,comparing with sodium alginate immobilized naringinase（34.19%）,chitosan immobilized naringinase showed better operational stability,which retained 74.32%residual activity after 5 consecutive operations.Consequently,chitosan was provided with better properties as a carrier for immobilization of naringinase.Magnetic silica chitosan（MSC）microspheres were prepared by chemical co-precipitation method,sol-gel method and emulsion cross-linking method,successively.After that,this microspheres were modified with epoxy group by reaction with epichlorohydrin.The optimal modification conditions of microspheres were obtained as follows:1.5 mol·L^-1 of NaOH,volume fraction of epichlorohydrin 50%,0.6 g·L^-1 of NaBH₄,and reaction at 40℃for 4 h.The epoxy density on MSC microspheres was 59.14μmol·g^-1 under the optimal conditions.The MSC microspheres modified with epoxy groups（named MSCE）were used for immobilization of naringinase.The optimal immobilization conditions were determined as follows:immobilization pH,temperature and time were 3.0,30℃and 4 h,and 57.48U·mL^-1 of crude naringinase broth activity.As a result,the binding efficiency,activity recovery rate and specific activity of the immobilized naringinase were 31.29%,88.92%and409.33 U·g^-1,respectively.Comparing with free naringinase,the immobilized naringinase showed better pH stability and thermal stability althought the optimal reaction pH and temperature were not change obviously,meanwhile it showed an excellent operational ability and storage ability.