Co-immobilization Of Glycosyltransferases On Chitosan For Synthesis Of Rebaudioside M Via Cascade Biocatalysis

Stevia rebaudiana Bertoni,a perennial shrub of the Compositae family,originated in the virgin forests of South America.Steviol glycosides(SGs)are high-intensity sweetners extracted from the leaves of Stevia.Due to its pure nature,high sweetness,near-zero calories,high safety and good stability,SGs are known as the"third-generation healthy sugar source"and the "best natural sweetener".Among the many components of SGs,Rebaudioside M(Reb M)has received widespread attention because its taste is closest to white granulated sugar.However,because of the low content of Reb M in Stevia plant,the production cost is high with traditional extraction and separation methods.With the rapid development of synthetic biology in the 21st century,research on the heterogeneous synthesis of natural products by microorganisms has made continuous progress feasible,and the target-oriented catalytic synthesis of high-value SGs has gradually become a research hotspot.Chitin is a natural polymer compound with the second largest reserve,cellulose with the largest,and deacetylation of chitin produces chitosan.Abundant in functional groups such as amino and hydroxyl on the side chains,chitosan is suitable for chemical modifications and decorations and thus can be multifunctional chemically.Additionally,chitosan is biodegradable and environment-friendly,and therefore it is the ideal material for enzyme immobilization.In this paper,through optimizing the expression of glycosyltransferases that catalyzes Reb A to Reb D and Reb M.and then the directional catalysis of immobilized glycosyltransferase and co-immobilized glycosyltransferase to Reb A to Reb D and Reb M are systematically studied with chitosan as the glycosyltransferase carrier.The results show that the targeted catalytic efficiency is improved after the glycosyltransferases are immobilized.This study provides a new strategy and technical support for the biocatalytic process of high-value steviol glycoside components,and has important theoretical significance and industrial application value.The main research contents of this paper are as follows:Through the intracellular expression optimization,purification and biochemical characteristics analysis experiments of glycosyltransferases OsEUGT11 and SrUGT76G1 in Escherichia coli(E.coli),the expression of high-efficiency glycosyltransferase and optimized catalytic conditions were obtained.The optimal conditions for expression of glycosyltransferases OsEUGT11 and SrUGT76G1 in E.coli BL21(DE3)(pET28a-OsEUGT11)and E.coli BL21(DE3)(pET28a-SrUGT76G1)were determined,which were 25?,0.1mM IPTG induced for 12 h and 18?,0.1 mM IPTG induced for 16 h,respectively.The recombinant glycosyltransferases OsEUGT11 and SrUGT76G1 were purified by the 6His tag on the recombinase.When UDPG was used as the glycosyl donor,the purified OsEUGT11 could catalyze Reb A to Reb D,the conversion rate of Reb A could reach over 94%,and the yield of Reb D could reach over 90%;the purified SrUGT76G1 could catalyze Reb D to Reb M,the conversion rate of Reb D could reach over 88%,and the yield of Reb M could reach over 85%.After comparing the expression and purification characteristics of glycosyltransferase OsEUGT11 and SrUGT76G1 in E.coli and Pichia pastoris.it was decided to select glycosyltransferases OsEUGT11 and SrUGT76G1 expressed in E.coli for enzyme immobilization research.The above researches provide a theoretical and material basis for the immobilization of glycosyltransferases.The immobilization system and catalytic system of immobilized glycosyltransferases UGT1 and UGT2 were established.Using chitosan as an immobilization carrier,the glycosyltransferase OsEUGT11(UGT1)and SrUGT76G1(UGT2)expressed and purified in E.coli were immobilized on the surface of chitosan beads by cross-linking with glutaraldehyde,which successfully catalyzed Reb A to Reb D and Reb D to Reb M.Chitosan beads with a diameter of about 2.0 mm and a surface area of about 12.56 mm2 were prepared from a chitosan solution with a concentration of 4%(m/v).The conditions for the immobilization of glycosyltransferase on chitosan beads were experimentally optimized from the three dimensions of crosslinking concentration,crosslinking reaction time and enzyme load.The optimal glutaraldehyde crosslinking concentration was 0.1%(v/v),the optimal immobilization time was 8 hours,and the optimal loading of glycosyltransferase on 0.2 g chitosan beads was 0.05 mg.And the kinetic parameters of the enzyme reaction of the immobilized glycosyltransferases UGT1 and UGT2 were optimized and analyzed.The results showed that the optimal reaction temperature of UGT1 and UGT2 was 37?.the optimal pH value was 7.0,the catalytic rate of UGT1was higher than that of UGT2,the affinity between UGT2 and substrate was greater than UGT1,and the catalytic efficiency of UGT1 was higher than that of UGT2.The UGT1 and UGT2 could be reused and showed good stability.After 4 and 8 times of reuse,the relative activity of UGT1 was 60.6%and 48.5%,and that of UGT2 was 52.6%and 38.2%.UGT1 has higher stability than UGT2.On the basis of the above research,a mixed immobilized enzyme cascade reaction system and a co-immobilized enzyme cascade reaction system were established to convert Reb A to Reb M.The optimal ratio of UGT1 and UGT2 in the mixed immobilized glycosyltransferase catalytic system was 1:10,and the optimal ratio of UDPG to Reb A was 1:3.5.The activity of the mixed immobilized enzyme was 43%of the original enzyme activity after 5 cycles of use.It is determined that the optimal ratio of UGT1 and UGT2 in the catalytic system of coimmobilized glycosyltransferase was 1:10,the optimal ratio of UDPG and Reb A was 1:3,the optimal reaction temperature was 37?,and the optimal pH value was 7.0.When the feeding concentration of Reb A was 5g/L,the substrate conversion rate was 97.3%,and the Reb M yield was 72.2%.The yield of Reb M produced by the catalytic substrate Reb A of coimmobilized and mixed immobilized UGTs was compared.Reb A could be converted by 87.1%through biocatalysis of co-immobilized UGTs,which is 1.7 times higher than that of mixed immobilized UGTs.The yield of Reb M in the co-immobilization system was 75.4%,which was 3.2 times the yield of Reb M in the mixed-immobilization system.These results indicate that the co-immobilization system has higher catalytic efficiency.The reuse stability of the co-immobilization enzyme system was evaluated.After 4 and 8 uses,the activity of the coimmobilized enzyme remained at 72.5%and 53.1%,respectively,indicating that the coimmobilized enzyme system was highly stable.Besides reducing the total cost of the enzyme through multiple uses,the co-immobilized glycosyltransferase system also provides a support for the efficient bioproduction of Reb M.