Molecular Modification Of Bacillus Stearothermophilus NO2 CGTase And Preparation Of AA-2G

2-O-?-D-glucopyranosyl-L-ascorbic acid?AA-2G?is a glycosylated derivative of L-ascorbic acid.AA-2G can not only maintain stable properties,but also can produce L-ascorbic acid through the degradation by?-glucosidase,which can exert the normal physiological functions of L-ascorbic acid.AA-2G is an excellent substitute for L-ascorbic acid,thus it is widely used in cosmetics,medicine,food and feed additives.Cyclodextrin glycosyltransferase?CGTase,EC 2.4.1.19?is the most widely used and most studied enzyme among the enzymes used for synthesis of AA-2G.However,the preparation of AA-2G by CGTase has the problems of low conversion rate and low enzyme production,which results in high production cost of AA-2G and limits the wide application of AA-2G.In this research,Bacillus stearothermophilus NO2?Bs?CGTase was the main research object for the above problems.Based on sequence analysis and site-directed mutagenesis of CGTases,the effect of aromatic amino acids at the acceptor site on the preparation of AA-2G was explored,and molecular modification of non-aromatic amino acids at the acceptor site and amino acids at the donor site was performed.Meanwhile,isoamylase and CGTase are used to synergistically prepare AA-2G.Finally,molecular modification of CGTase was performed to improve the soluble expression of CGTase in Escherichia coli.The main results are as follows:?1?Effect of the aromatic amino acid at the acceptor site of CGTase on acceptor specificity.In the preparation of AA-2G,the by-product such as glucose and maltose may compete with L-ascorbic acid as acceptors,resulting in low conversion rate of AA-2G.In order to verify the effect of the aromatic amino acid at the acceptor site of CGTase on the specificity of the acceptor substrate and the conversion rate of AA-2G,sequence alignment analysis of CGTases were performed.It was found that the CGTase with high AA-2G conversion rate had amino acids at position 191 and 255?based on Bs CGTase?were both F,and CGTase with low AA-2G conversion rate had amino acids at position 191 and 255 were both Y.five mutants Bs F191Y,Bs F255Y,Bc Y195F,Pm Y195F,and Pm Y260F of CGTase of Bs CGTase,Bs CGTase and Pm CGTase were designed and validated,respectively.Under optimal conditions,the AA-2G yields of Bs F191Y and Bs F255Y were 34.3%and 7.9%lower than that of Bs CGTase,respectively.The AA-2G yield of Bc Y195F was 45.8%higher than that of Bc CGTase.The AA-2G yields of Pm Y195F and Pm Y260F were 36.9%and 12.6%higher than Pm CGTase,respectively.The experimental results show that the conversion rate of AA-2G was high when 191 and 255 were F,while the conversion rate of AA-2G was low when 191 and 255 were Y.Kinetic studies shown that when the 191 was F,the glucose specificity and maltose specificity were reduced,and the L-ascorbic acid specificity was improved;when the 255 was F of Bs CGTase and Pm CGTase,the glucose specificity was reduced,and the L-ascorbic acid specificity was improved.?2?Mutations in non-aromatic amino acids at the CGTase acceptor site were performed to improve L-ascorbic acid specificity.Three principles were used to select amino acids that may affect the acceptor specificity:located within 10?of the acid-base catalyst Glu 253,located at the binding site of the acceptor substrate,and the amino acids conservation were?30%.Eight amino acids were selected for saturation mutations.The mutants Bs K228R,Bs M230L and Bs K228R/M230L were obtained by screening.The optimal enzyme amount was 2000 U·g^-1maltodextrin,the optimal pH was 5.0,and the optimal temperature was 30°C for preparing AA-2G.To improve the production intensity,the concentration of maltodextrin was optimized with 250 g·L^-1 L-ascorbic acid.When 500 g·L^-1 maltodextrin was used as glycosyl donor,the AA-2G yield reached 210.3 g·L^-1,which was 48.9 g·L^-1 higher than that of wild type.Kinetic analysis showed that the L-ascorbic acid K_m of mutants Bs K228R,Bs M230L and Bs K228R/M230L were 20.3%,30.4%and 34.8%lower than that of wild-type,respectively,whereas the k_cat/K_m were 2.08-,2.06-and 2.69-fold higher than that of wild-type.Meanwhile,the k_cat/K_m of Bs K228R/M230L for glucose and maltose were 1.67-and 0.28-fold higher than that of wild-type,respectively.The results indicating that Bs K228R/M230L improved the L-ascorbic acid specificity as a acceptor.?3?Mutations at glycosyl donor site were performed to improve maltodextrin specificity.Eight amino acids were selected for saturation mutations,and mutants Bs S90D,Bs G176H and Bs S90D/G176H were obtained by screening.With 500 g·L^-1 maltodextrin and 250 g·L^-1 L-ascorbic acid as substrates,the AA-2G yield of Bs S90D/G176H reached 185.0 g·L^-1,which was 23.6 g·L^-1 higher than that of wild-type.Kinetic analysis indicating that the K_m of Bs S90D for maltodextrin was 12.5%higher than that of wild-type,the K_m of Bs G176H and Bs S90D/G176H for maltodextrin were 13.5%and 8.7%lower than that of wild-type,respectively,whereas the k_cat/K_m for maltodextrin of Bs S90D,Bs G176H and Bs S90D/G176H were 1.17-,1.30-and 1.47-fold higher than that of wild-type,respectively.Combining mutant of the donor site and the acceptor site,the Bs S90D/K228R/M230L with the highest AA-2G conversion rate was obtained.The AA-2G yield of Bs S90D/K228R/M230L reached 216.8 g·L^-1,which was6.5 g·L^-1 higher than that of Bs K228R/M230L and 55.4 g·L^-1 higher than that of wild-type.AA-2G was prepared by mixing isoamylase with Bs S90D/K228R/M230L.The concentration of maltodextrin was reduced to 350 g·L^-1,and the AA-2G yield could reach 208.4 g·L^-1.The cost of maltodextrin is reduced,and the viscosity of the solution and the concentration of by-products were reduced,which was beneficial to the later separation and purification.?4?Molecular modification was performed to improve the soluble expression of CGTase in E.coli,three mutants with increased enzyme activity were obtained through high-throughput screening.The site-directed mutations were used to identify the mutation sites affecting enzyme activity.The CGTase activities of Bs I631T,Bs I641T and Bs K647E were 1.7-,2.1-,and 2.2-fold higher than that of the wild-type(141 U·m L^-1).The CGTase activities of Bs I631T/I641T,Bs I631T/K647E,Bs I641T/K647E and Bs I631T/I641T/K647E were 1.9-,1.8,1.3-and 1.2-fold higher than that of the wild type,respectively,and there was no superposition effect.Through multiple sequence alignment of CGTases,K647 was found to be more conservative in the CGTase family.The Bc CGTase,Pm CGTase and Ag CGTase were introduced mutations at the corresponding sites to obtain three mutants Bc K654E,Pm K655E and Ag K656E.The CGTase activities of the three mutants were 62,39 and 113 U·mL^-1,which were 2.0-,1.5-and1.0-fold higher than those wild-types.Through structural modeling analysis and the surface negative charge analysis of four CGTases and their mutants,it was found that the introduction of mutations with improved negative charge in CGTase with low negative surface charge may increase the soluble expression of CGTase.The wild-type and Bs K647E were produced in a 3L fermenter.The CGTase activity of Bs K647E was 2150.6 U·mL^-1,which was 2.2-fold higher than that of the wild-type(960.3 U·m L^-1).The Bs K647E,Bs K228R/M230L and Bs S90D/K228R/M230L were combined,then Bs K228R/M230L/K647E and Bs S90D/K228R/M230L/K647E were produced in 3 L fermenter,respectively.The highest CGTase activity of Bs K228R/M230L/K647E and Bs S90D/K228R/M230L/K647E were2025.2 and 1344.5 U·mL^-1,respectively,which were 2.1-and 1.4-fold higher than that of the wild-type,respectively.Meanwhile,Bs K228R/M230L/K647E and Bs S90D/K228R/M230L/K647E were used for AA-2G preparation,and the combination mutants were found to be consistent with the AA-2G yield of Bs K228R/M230L and Bs S90D/K228R/M230L,respectively.The mutants with increased CGTase activity had no effect on the conversion rate of AA-2G.