Study On The Synthesis Of Rebaudioside D Catalyzed By Double Enzyme Cascade

The excessive intake of high-calorie sweeteners such as sucrose has led to an increasing public health burden in many countries,which has prompted people to seek sugar-free and low-calorie natural sweeteners.Rebaudioside D（RD）derived from stevia is a natural sweetener with low calorie and high sweetness.However,the RD extracted from stevia has low content and many impurities,which cannot meet the market demand.In this study,glucosyltransferase（EUGT11）and sucrose synthase（SUS）cascade catalyzed RA to synthesize RD,which realized in-situ regeneration of glucosyluridine diphosphate（UDPG）.RD could be synthesized without exogenous addition of UDPG,and the cross-linking enzyme aggregation technology and magnetic nano-carrier were used to prepare immobilized enzyme,improving its reuse properties.The main studies are as follows:1.The transgenic engineering bacteria were constructed using genetic engineering techniques,and the target protein were expressed and purified.The glucosyltransferase EUGT11 and sucrose synthase SUS with catalytic activity were screened by HPLC analysis.The optimum temperature and p H of EUGT11 were determined to be 37℃and 9.0,respectively.SUS had an optimal temperature of 35℃and an optimal p H of 8.5.And RD was successfully synthesized by double enzyme cascade.2.The optimum conditions for the preparation of EUGT11-crosslinking enzyme aggregates（EUGT11-CLEAs）and SUS-crosslinking enzyme aggregates（SUS-CLEAs）were obtained by investigating the precipitation conditions and crosslinking conditions of proteins.The optimal conditions of EUGT11 were 50%ammonium sulfate precipitation and 0.25%glutaraldehyde crosslinking for 0.5 h.30%ammonium sulfate precipitated SUS and 0.15%glutaraldehyde was crosslinked for 1.25 h.The optimum temperature and p H of EUGT11-CLEAs were determined to be 45℃and 7.5,respectively.The optimum temperature of SUS-CLEAs was also 45℃,and the optimum p H was9.0.The resistance of the immobilized enzyme to temperature and storage time was improved in some extent,and the immobilized enzyme could be reused for some times.3.We prepared Uio-66,Fe₃O₄@Uio-66 and Fe₃O₄@Zr to immobilize the enzymes,and investigated the properties and stability of the immobilized enzymes.It was found that the immobilized enzymes had good p H stability and temperature stability.The repeated use effect of immobilized enzyme with Fe₃O₄@Uio-66 carrier was best,and the enzyme activity loss was least.SEM,TEM,FT-IR,XRD,BET,VSM,TG and TEM-EDS were used to characterize Fe₃O₄@Uio-66 before and after immobilization.We found that the immobilization process and the recycling process of immobilized enzyme had no effect on the carrier.The immobilization conditions,double enzyme ratio and substrate ratio were optimized to explore the kinetic parameters and reuse performance of immobilized enzyme.And we further optimized the conversion.The results showed that EUGT11 was immobilized with 25 mg for 40 min,SUS was fixed with 25 mg for 30 min to obtain the best immobilized enzyme.The best ratio of EUGT11 to SUS was 1:1,and the best ratio of RA,sucrose and UDP was 2:2:3.The activity of immobilized enzymes could still retain about80%of the initial activity after repeated use for 8 times.The conversion rate was higher when EUGT11 was added after SUS reaction 60 min.After 10 h of free enzymes reaction,the conversion rate reached 57%and did not change.However,the immobilized enzyme continued to react after reaching a similar conversion rate in 10 h,and the conversion rate was 84%after 24 h,which was significantly higher than that of free enzyme.