Engineering Of Cyclodextrin Glucosyltransferase For The Synthesis Of Glycosylated Genistein

Genistein,widely used in the pharmaceutical and food fields,has multiple pharmaceutical activities.For example,genistein can be used for anti-cancer,anti-osteoporosis,prevention of cardiovascular disease,and alleviation of menopausal syndrome in women.However,low water solubility of genistein seriously limits its applications.Transglycosylation is regarded as an effective approach to improve the water solubility of genistein,and longer sugar chains usually lead to higher water solubility.In this study,cyclodextrin glycosyltransferase（CGTase）was engineered to enhanced the specificity of long chain glycosylated sophoricosides（LCGS）.The main results are as follows:1.cgt gene from Paenibacillus macerans JFB05-01（Gen Bank:JX412224）was cloned into p ET-20b（+）to construct recombinant plasmid p ET-20b（+）/cgt.CGTase activity with Omp A signal peptide increased by 29.41%and 2.2-fold than that with Pel B signal peptide and without signal peptide,respectively.The fermentation period was shortened to 40 h from 90 h using 75 mmol·L^-1 glycine and extracellular enzyme activity was increased by 16.29%using 5mmol·L^-1 magnesium ions.2.Optimization of the reaction system catalyzed by cyclodextrin glucosyltransferase for genistein glycosylation was investigated.The results showed that the optimal glycosyl donor and acceptor were water-soluble starch(40 g·L^-1 dissolved in PBS buffer,p H 6)and sophoricoside(1 g·L^-1 dissolved in DMSO),respectively.The volume ratio of sophoricoside/water-soluble starch was 2/6.The optimal reaction temperature and p H was 40°C and 6respectively,and the optimal reaction time was 16 h.3.Site directed saturation mutation of D182 was performed to improve the yield of the short chain glycosylated sophoricosides（SCGS）.The results showed that the conversion of variant D182C was increased by 13.42%than that of WT,and the major glycosylation products（sophoricoside mono-,di-,and tri glucosides）increased by 39.35%,56.05%,and64.81%,respectively.Enzymatic studies revealed that the cyclization,hydrolysis,and disproportionation activities of D182C were increased compared with WT.And the optimal p H and temperature were 6 and 40°C,respectively.Kinetic studies revealed that the K_m of D182C for substrates（glycosyl donor and acceptor）was reduced compared with WT,and the catalytic efficiency k_cat/K_m was significantly increased,which indicated that the affinity of D182C for substrates is greatly increased compared with WT.The homology modeling and molecular docking results suggest that the reason for the increased glycosylation efficiency of the mutant enzyme D182C may be related to the increased substrate force.4.The glycosylated derivative LCGS product specificity was improved by site directed saturation mutagenesis of the S77,Y195 sites.The conversion of variants S77N/Y195S and S77N/Y195I showed 13.86%and 11.85%more than that of WT.The proportion of LCGS increased by 15.27%and 29.96%,respectively.Enzymatic property studies revealed thatγ-cyclization and disproportionation activities of variants were increased compared with the wild type,with optimal p H and temperature of 6 and 40°C,respectively.Kinetic studies revealed that the K_m values of S77N/Y195S and S77N/Y195I for both substrates（glycosylation donor and acceptor）were decreased compared to WT,and the catalytic efficiency k_cat/K_m of variants was significantly increased,indicating that S77N/Y195S,S77N/Y195I displayed a higher affinity for the substrate than WT.Molecular docking and simulation results indicated that the reason for the increased glycosylation efficiency of S77N/Y195S and S77N/Y195I might be related to the increased substrate force and more flexible amino acids in the substrate binding site after mutation.