Site-directed Mutagenesis Of Dextransucrase Based On Improved Transglycosylation And Product Specificity

Naturally occurring products have multiple physiological and pharmacological functions.However,some unmodified natural products have poor water solubility and low bioavailability,which limits their applications.Dextransucrase has successfully modified the structure of many natural compounds to improve the water solubility and stability,and gradually attracts more and more attention.However,the transglycosylation activity was very low.The application of this proces was limited.In this paper,dextransucrase mutant with high transglycosylation activity and product specificity was obtained by rational design using molecular biology technology with caffeic acid phenethyl ester(CAPE)as an acceptor substrate.Firstly,the double P473S/P856 S mutant of p ET-28a(+)/dex-YG from Leuconostocmesenteroides 0326 was used as a genomic DNA template for polymerase chain reaction(PCR).Dextransucrase mutant with high transglycosylation activity and product specificity was obtained by rational design using molecular biology technology with CAPE as an acceptor substrate.Under identical conditions,the conversion rates of diglycoside products(CAPE-2G)generated by the N555E(80.8%)and N555Q(84.5%)mutants were 3.30-and 3.46-fold higher than those generated by the original enzyme(24.4%).The conversion rates of monoglycoside products(CAPE-G)generated by the N555A(28%)and N555D(43%)mutants were 1.13-and 1.13-fold higher than those generated by the original enzyme.Secondly,the process was optimized: In the catalytic reaction of the N555 Q mutant,the optimal conditions for the formation of CAPE-2G were 0.5 U/m L,p H =4.5,30 ℃,and the yield of CAPE-2G could reach 98%;In the catalytic reaction of the N555 D mutant,the optimal conditions for the formation of CAPE-G were 4 U/m L,p H =5.5,35 ℃,sucrose 800 mmol/L and the yield of CAPE-G could reach 65.4%.Finally,Homology modeling and molecular docking combined with kinetic parameters were used to suggest a reasonable interpretation of the experimental results and analy the effects of different types of amino acids at N555 resite on the transglycosylation activity and product specificity.This study is expected to provide a reference for the investigation of the specificity of enzyme products.The strategy provides a method to improve the product specificity of transglycosylation and expand the applications of natural products.