The Preparation And Characterization Of Cellobiose 2-epimerase Hybrid Nanoflowers

Lactulose,a prebiotic,can be used as a functional food additive and a clinical drug for the treatment of hepatic encephalopathy,chronic constipation and other diseases.It has been widely used in food and medicine industry.Compared with chemical method of production,producing by enzymatic approach is not only energy-saving and eco-friendly,but also can reduce the generation of by-products.Therefore,this is a new trend of lactulose production.Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus(Cs CE)is currently the most efficient enzyme for producing lactulose.E161D/N365P,which is the site-directed mutagenesis strain of Cs CE,has better thermostability.However,the low reusability of free enzyme hinders its industrial application.This study was committed to the immobilization technology of E161D/N365P,using organic-inorganic hybrid immobilization approach to synthesize two nanoflowers EDNP@Co₃(PO₄)₂,EDNP@Ca₂P₂O₇ and food-grade cross-linked catalyst SA-EDNP@Co₃(PO₄)₂.The physical and chemical properties of these three immobilized enzymes were characterized,and their structures were analyzed by Field emission scanning electron microscope,Fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy and other technical methods.Their application in producing lactulose were further investigated.Firstly,EDNP@Co₃(PO₄)₂ was prepared by organic-inorganic hybrid immobilization technology.Under the optimal synthesized condition(0.1 mg/m L enzyme and 1.6 m M Co²⁺),the activity recovery rate could reach 62.88%,and the encapsulation yield was 57.22%.Adding 0.8 m M Co²⁺to the remaining solution which still contained residual enzyme after the first precipitation could further increase the total encapsulation yield to 65.82%,and at the same time,the nanoflower was structurally complete.Compared with the free enzyme,the thermostability of EDNP@Co₃(PO₄)₂ was slightly improved below 60?,and the optimum p H shifted to the acidic region,which could be beneficial to the clean production of lactulose.The nanoflower structure made EDNP@Co₃(PO₄)₂ better at kinetic parameters.Its catalytic efficiency?was increased by 1.48 times in comparison with free enzyme,and after 8 cycles of reactions,the enzyme activity still remained over 70%.When producing lactulose with lactose as the substrate(adding 3.5 U/m L EDNP@Co₃(PO₄)₂ biocatalysts),the conversion rate of lactulose could reach 53.18%after 6 hours of reaction.Secondly,on the basis of the immobilization approach for EDNP@Co₃(PO₄)₂,a food-grade cross-linked method was used as secondary immobilization to further improve its operational stability,so SA-EDNP@Co₃(PO₄)₂ was prepared.After 8 cycles,the residual enzyme activity was still 96.93%of the initial activity.Applying it to a low-temperature milk system at 8?,after reacting for 12 h,the lactose decomposition rate reached 44.65%,and the total conversion rate of lactulose and epilactose was 26.69%;at 55?,44.27%of lactose broke down after reacting for 1 h,and the total conversion rate of lactulose and epilactose was28.32%.Lastly,combing the immobilization approach of organic-inorganic hybridization,ion gelation method by chitosan was used to synthesize EDNP@Ca₂P₂O₇.Under the optimal preparation condition(protein concentration 0.15 mg/m L,sodium tripolyphosphate concentration 125 mg/m L,chitosan concentration 2.5 mg/m L),the enzyme encapsulation yield reached 97.23%,and the activity recovery rate was 88.63%.Compared with EDNP@Co₃(PO₄)₂ nanoflowers,the optimum temperature was 10?higher,the denaturation temperature T_m was increased by 1.29 times,and the maximum velocity V_max was slightly higher than that of the free enzyme.After 8 cycles of reactions at 70?,the residual activity was 58.19%of the initial activity.When producing lactulose with lactose as the substrate(adding 5 U/m L EDNP@Ca₂P₂O₇ biocatalysts),the conversion rate of lactulose could reach58.04%after 6 hours of reaction.