The Eucommia Protein Purification, Sequence Analysis And Antibacterial Activity

In this thesis,proteins of Eucommia bark and Eucommia rubber particle were isolated,and their amino acid sequences and antimicrobial activity were determined.The research results are mentioned below.A novel Eucommia antifungal peptide,named EAFP3,was isolated from the bark of E. ulmoides by the method of gel filtration and reversed-phase high-performance liquid chromatography.On the other hand,nine proteins with molecular mass of 14,16,18,22,24,28, 30,32 and 55 kDa,respectively,were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) from Eucommia rubber particle.Among the eleven proteins,only five proteins with molecular mass 4.16,4.2,8.3,16 and 18 kDa were identified and 14 peptide fragments were obtained through liquid chromatography mass/mass spectrometry(LC-MS/MS).The structure,sequence and function of these 14 peptide fragments were confirmed and accumulated in antimicrobial peptide database(APD).The results show that eleven peptides:-.LYQQLIAGITLNK.-,-.PCNAGLCCSIYGYCGSGNAYCGGNCR.-, -.LRLDGAMPPSYPSSWSVLPR.-,-.QIITHPSMDDYVRK.-,-.LPLGAGVNLSGGPVVK.-,-.L PVAVKTGAEVVYSK.-,-.ISTLNSHNLPILR.-,-.ISLTEGIRR.-,-.RHGNDHDVHPK.-,-.TNGP VVNLKAFLKK.-and-.GRSAPVLAATVAGDTR.-have the typical structural feature of antimicrobial peptide.Besides,the result of antifungal activity indicates that EAFP3 exhibits broad spectrum antifungal activity and can inhibit the growth of yeast,yeast-like,molds and phytopathogenic fungi. The IC₅₀ values of EAFP3 for Candida albicans,Aspergillus flavus and Botrytis cinere are 31.25, 31.25 and 62.5μg/mL.After heating at 100℃for 30 min,EAFP3 can keep its antifungal activity and thermal stability,and display a broad pH-activity,profile in the range from 5.8 to 8.0.The above results indicate that lead compounds for creating new biological pesticides and drugs as well as primer and probe sequences for cloning gene of antifungal protein could be obtained.