Study On The Biological Activity Of Endophytic Metabolites Of Toona Sinensis

Camphor wood,with hard texture,artistic grains and special fragrance,is a preferred timber for making furniture,which can repel insects and prevent mildew,with certain antioxidant capacity.Current studies on resistance of camphor are mainly centered in its chemical composition while neglecting the endophyte living inside it,which has resulted in insufficient development of its immense application value.Endosymbiontic theory has indicated that endophyte living in the plants can produce metabolites identical or similar to the host plant.This thesis isolates endophyte from camphor through tissue separation method and determines its antioxidant capacity by testing endophytic metabolites'ability of scavenging DPPH and OH free radicals.Its antibacterial ability is evaluated through agar disc diffusion and growth rate methods.Biologically active camphor endophyte is thus selected to identifyand analyze its activity mechanism.Through response surface method,this thesis optimizes the active substance in camphor endophyte and adopts methods including ammonium sulfate precipitation and gel filtration chromatography to separate and purify active substances.This thesis aims to study the relationship between biological activity and resistance mechanism of camphor in its endophyte and offers theoretical basis for the application of camphor and endophyte in the field of biological antiseptic treatment,pest control,and medical and health care.The results of the study are as follows:After selecting 15 strains of endophytic fungi from the roots and stems of camphora through tissue separation method,the activity test has found out that the metabolites of some endophytic bacteria boast good bioactivity.In the antioxidant test,metabolites of Endophyte R5 and Endophyte S6 extracted from acetic ether have strong ability of scavenging DPPH free radicals while metabolites of Endophyte R5and Endophyte S2 are better at scavenging OH free radicals.In the antibacterial test,the MIC of metabolites of Endophyte R5 extracted from methanol for staphylococcus aureus is 20mg/mL and the MIC for bacillus subtilis is 40mg/mL.Metabolites of Endophyte R5 extracted from methanol and Endophyte S5 extracted from ethyl acetatesignificantly inhibit the growth of penicillium while metabolites of Endophyte R7 and Endophyte R8 from ethyl acetate have more obvious antibacterial effect for aspergillus niger.It is found through morphological observation and molecular biology that trichoderma viride in trichoderma has the highest similarity with Endophyte R5.Through literature review,it is surmised that its resistance mechanism is related to the metabolite-beta glucosidase,which can encourage linalool precursor compounds mainly composed of terpene and aromatic ligands to produce volatile linalool in camphora.Linalool is good at repelling insects and preventing mildew.Besides,trichoderma green can also produce antibiotic products such as trichoderin and colloycin to inhibit the growth of pathogenic bacteria.Certain antioxidant capacity exhibited by the metabolites of trichoderma viride may be related to the production of polyphenols derivatives.Czapek's media is used as basal media to optimize and identify the perfect medium group,which contains 39.47g of lactose,3.96g of NaNO_3,0.53g of MgSO₄·7H₂O,1g of K₂HPO_4,0.5g of KCI and 0.01g of FeSO_4.Predicted enzyme activity was 1.873 U/mL while the actual activity is 1.825 U/mL.Based on this,fermentation conditions are optimized and it is concluded that the optimum fermentation condition is 50.83 mL of liquid quantity,5%of inoculum concentration,pH 5.06,and rotating speed at 140 r/min with temperature remaining at 30?.Under this condition,enzyme activity is 2.36 U/mL and it is tested to be 2.24 U/mL in the experiment,which has been improved by 40.88%before optimized.When 70%of ammonium sulphate is used to precipitate the crude enzyme solutionextracted from the fermentation medium of trichoderma viride,the enzyme specific activity is up to 1.27 U/mg,which is 2.19 times as much as that in crude enzyme solution.Sephadex G-100gel filtration chromatography is adopted to further purify enzyme to elicit 3 protein absorption peaks.Enzyme specific activity in the second absorption peak is 5.74 U/mg and the enzyme purity is 9.89 times higher.Purified samples only show one band after SDS-PAGE electrophoresis,which indicates good purification efficacy.The molecular weight is 68 KDa.