Study On Enzymatic Synthesis And Biological Activities Of Caffeic Acid Esters From Propolis Residues As Inhibitors Against Mulberry Ralstonia Solanacearum

Mulberry bacterial wilt is a kind of plant bacterial disease caused by Ralstonia solanacearum,which spreads rapidly with serious harm.It is difficult to prevent and control,result in devastating harm to silkworm production due to the lack of mulberry leaves.At present,the comprehensive control measures of mulberry wilt disease mainly include agricultural control,physical control,biological control and chemical control,but the current use of pharmaceuticals for chemical pesticides,toxic,pollute the environment,so it is urgent to discovery environment friendly,sustainable and cost-effective biopesticides with less toxic.Since ancient times,the antibacterial effect of propolis has been recognized,which is an important factor in the disease prevention and pest control for the natural defense system of honeycomb.At present,the propolis food and other products are often evaluated with the total flavonoid content as the main criteria for product quality,so a large number of caffeic acid and its esters with significantly antibacterial activity are still remained in the propolis residues.Therefore,the present study intends to use the propolis residue as a new feedstock to screen the inhibitors with high activity from the ester derivatives of caffeic acid and to develop natural and low-cost agents used in the control of mulberry bacterial wilt.The idea will make the propolis residues into high-value products,it not only saves the cost,but also protects the environment,which have important academic significance and economic value.Therefore,this study intends to find caffeic acid ester compounds with strong antibacterial activity from the propolis residues,construct a novel biphase system to improve the enzymatic synthesis efficiency,compare the whole genomes between two pathogens with sensitive difference,and explore the inhibition on important pests and natural enemies in the mulberry field.The main contents and conclusions are as follows:?1?In order to find antibiotic ingredients from propolis residues against five race strains of R.solanacearum,the grading extracts of propolis residues in accordance with solvent polarity were evaluated by the activity tracing method,the caffeic acid and its ester compounds were qualitatively and quantitatively analyzed by liquid chromatography combined with mass spectrum technology?LC-MS?,and then the selected monomers were further evaluated their inhibitory efficacy against R.solanacearum.The results showed that the different polar solvent extracts of propolis residues had certain inhibitory activity on the five races of R.solanacearum,in which the antibacterial activity of ethanol extract was significantly higher than others.The antimicrobial resistance of 0.1 mg/m L ethanol extract from propolis residues topathogen RS-5 was up to 91.61±4.75%.Using ESI-LC-MS technology,caffeic acid and its five esters were identified from the ethanol extract from propolis residues under full-scan mode,and the content of caffeic acid and caffeic acid phenylethyl ester?CAPE?were measured under selective reaction monitor?SRM?mode based on the characteristic ion pairs.Two high-activity monomer of caffeates including methyl caffeate?MC?and CAPE with EC50 of 310.0-1225.0 ?g/mL ? 165.2-752.7 ?g/m L were screened from the test caffeates,which made pathogen cells deformed,cell wall damaged and other serious injured.The migration ability and biofilm formation of pathogenic bacteria was greatly inhibited by them.Thus,MC and CAPE are expected to develop the precursor compounds of bacteriostatic agents of mulberry bacterial wilt,which provides a new idea for the further research and development of biocontrol agents of mulberry bacterial wilt from natural source.?2?In order to improve the solubility of substrate MC,trioctylphosphine oxide?TOPO?used as a complexing agent,a novel biphase system of TOPO-cyclohexane/[Bmim][Tf₂N] was established for enzymatic synthesis of CAPE with highly antibacterial activity based on biphase catalysis mechanism.By studying the regulating effect of the developed reversible complexation,the mechanism of enzyme-catalyzed reaction in two-phase system was revealed.This provides a new and green process for efficient biosynthesis of caffeates.In addition,in order to monitor the contents of MC and CAPE in the continuous process of lipase-catalyzed transesterification,Direct Analysis in Real Time-Mass?DART-MS?technique with a novel ion source was used to establish a rapid and simple quantitative method.The results indicated that the biphase of TOPO-cyclohexane/[Bmim][Tf₂N]?1:1,v/v?was firstly used to synthesize CAPE from MC and 2-phenylethanol catalyzed by Novozym 435 via transesterification.Because TOPO has a highly selective and reversible complex activity forcaffeates,such as MC and CAPE,the synthesis of CAPE could be achieved in the biphasic system with a higher yield and a lower cost.The MC conversion of 98.83±0.76% and the CAPE yield of 96.29±0.07% were obtained in the 25 g/L TOPO-cyclohexane/[Bmim][Tf₂N]?1:1,v/v?under the optimal conditions by response surface analysis.The reversible hydrogen bond interaction between TOPO and caffeates including MC and CAPE was confirmed by stoichiometry and FT-IR spectroscopy,which enhanced the synergetic effect of the enzymatic reaction coupled with separation.The MC concentration of 10 g/L was3.33-fold higher than that used before,so the overall cost is reduced greatly.Remarkably,the ratio of catalyst to donor was greatly decreased from 90:1 to 20:1,reducing the dosage of Novozym 435 to improve the technical feasibility in industry.Moreover,the results also show that a new DART-MS method for the determination of caffeic acid compounds in biocatalytic systems has been successfully established.The reactants were directly analyzed using DART-MS,and the corresponding ionization parameters were investigated.Special ions produced from MC?parent ion m/z 192.87 and product ion m/z 133.44?and CAPE?parent ion m/z 282.93 and product ion m/z 178.87?were determined using DART-MS2 in the negative ion mode.The peak areas of the select reaction monitoring?SRM?signals were calculated to develop the standard curves for quantitative analyses of the concentration.Reasonable linear regression equations of MC and CAPE were obtained in the range of 3.125 to50.000 mg/L,with linear coefficients?R2?of 0.9515 and 0.9973,limits of detection?LOD?of 0.005 and 0.003 mg/L,limits of quantification?LOQ?of 0.02 and 0.01mg/L,and recovery ranges of 92.50-97.11% and 90.11-97.60%,respectively.The results using DART–MS2were in good agreement with those using conventional High-Performance Liquid Chromatography with a UV detector?HPLC-UV?and were successfully applied to monitor the kinetics constants and mass transfer coefficients in a continuous-flow packed bed microreactor.Therefore,using the developed biphase syntem to synthesis CAPE from MC and PE using lipase-catalyzed transesterification would be the most effective procedure to the best of our knowledge,and the DART-MS2 method would be an efficient tool for analyzing caffeates in microfluidic biocatalysis with limited sample preparation and short operating time.?3?In order to investigate the sensibility difference between MC and CAPE,the complete genomic sequence analysis of two races including GIM1.71 and RS-5 were compared.The method of comparative genomics was used to analyze the differences in genomic structure between the two races,and to find a common gene that could lead to changes in drug resistance or sequence structure.Two races were sequenced using the latest third-generation sequencing technique.A total genome sequence of about 3.8 M including plasmid sequence were obtained from each strain.A total of about 5200 coding genes and about 70 noncoding RNAs?including sRNA and tRNA?were obtained in the genome of the coding gene,repeat and non-coding RNA by bioinformatics analysis.The function of the encoded gene was annotated by comparison with the Gene Ontology database.The biological pathways involved in coding genes were analyzed by the KEGG database annotation.A total of 390GIM1.71-specific genes and 76 genes shared by RS-5 and GMI1000 were found by comparison with the three races including GIM1.71,RS-5 and GMI1000,while GIM1.71 was deleted from the gene.33,697 and 44,368 SNP loci were identified in the GIM1.71 and RS-5 genomes by SNP and insert-deletion and genomic structural variation analysis.For the three genes,we identified 106 genes and only a small fragment insert occurred in the GIM1.71 strain.These findings will help to reveal the differences in phenylephrine tolerance between GIM1.71 and RS-5,providing a candidate for further in-depth analysis.?4?In order to investigate the inhibitory effect on the destructive mulberry pests and impact on the natural enemies of MC,and to evaluate the developed potential as novel mulberry pest inhibitors,we measured the effect of MC on the main fitness of Spodoptera litura,Diaphania pyloalis and Meteorus pulchricornis.The results indicated that the larvae of S.litura and D.pyloalis appeared significant antifeedant tendency towards 1.0% and 2.0% concentrations of MC solutions.The 2ndinstar of S.litura showed feeding choice tendency between 0.1% MC solutions and blank treatment,1.0% MC solutions and blank treatment,however,the larvae of D.pyloalis didn't show similar feeding choice tendency.When digesting the mulberry leaves contained 2.0% MC solutions,the increase of body weight slowed down significantly in the larvae of S.litura through long-term feeding test.Furthermore,the body weight of D.pyloalis larvae decreased to 37.67% and 30.00% of control when feeding the mulberry leaves contained 1.0% and 2.0% MC solutions after 7 days.Survival durations of the larvae of S.litura and D.pyloalis shortened significantly with the increase of MC concentrations.The survival durations of larvae of S.litura and D.pyloalis decreased by 24.6% and 57.38% after feeding 2.0% MC solutions respectively.There was no significant impact on the pupation rate of S.litura larvae caused by MC.The pupation rate of D.pyloalis larvae decreased by 24.6% and57.38% compared to the control after feeding 2.0% MC solutions respectively,and the eclosion rate was also decreased.When parasitized the larvae of S.litura which feeding different concentrations of MC solutions,the offspring lifespan,body size and other fitness indexes of M.pulchricornis didn't change significantly except that the number of cocoons decreased and development time prolonged in the 2.0% treatment.Therefore,MC had a significant impact on the pests while showed higher safety against parasitoid wasps among the tri-trophic levels.In summary,in order to provide new biocontrol agents for the prevention and treatment of mulberry bacterial wilt,and new ideas for comprehensive use of propolis residue,this study focused on the screen of active caffeates from propolis residue,the enzymatic synthesis of CAPE,the genome comparison of pathogens and the impact on mulberry insects of MC.