Isolation And Purification Analyze Of Intercellular Proteins Of Bitter Melon Leaves

Momordica charantia L. (Cucurbitaceae), a climber growing throughout the world, is commonly known in English speaking countries as "bitter gourd" or "bitter melon". It is used as vegetable as well as medicine. The health benefits of bitter gourd have been well documented, especially its anti-diabetic properties. It is also used as carminative, emmenagogue, in the treatment of colics, and as antiviral, anthelmintic, antimalarial, and antimicrobic remedy. This study presents an antifungal protein from bitter melon leaves. The intercellular solution of bitter melon leaves was obtained by vacuum method. Then we purified a single protein from intercellular of bitter melon leaves using three different chromatography ways. The antifungal protein was designated as Chit-Pr. And its N-terminal amino acid sequence was determined by automatic Edman degradation. We also tested the antifungal activity of Chit-Pr against several phytopathogenic fungi. Meanwhile, another antifungal peptide BTL was also purified from a Bacillus strain B-TL2 isolated from tobacco stem. The main results are as following:1. The purification procedure for the antifungal protein from intercellular liquid of bitter melon leaves consisted of ammonium sulfate precipitation, desalting by Bio-Gel P-6 column, cation exchange chromatography on CM-Sepharose Fast Flow column, size exclusion on HiLoad 16/60 Superdex column and reverse phase HPLC on SOURCE 5RPC column. After the last isolation step we obtained an antifungal protein, designated as Chit-Pr. 2. The antifungal protein Chit-Pr was displayed a single band, with a molecular mass of 25 000 Da, by SDS-PAGE. Its N-terminal amino acid sequence was determined as NH₂-VEYTITGNAGNTPGG using automatic Edman degradation. The BLAST result of the N-terminal amino acid sequence showed that it has highly similarity with plant pathogenesis-related protein from Atropa belladonna.3. Inhibition of Chit-Pr on mycelia growth and spore germination of several phytopathogenic fungi were tested. Chit-Pr can inhibit the mycelia growth of following fungi, Aspergillus niger, Fusarium oxysporum f.sp.vasinfectum, Cercospora personata, Alternaria alternat, Altenaria brassicae, Fusarium spp., Rhizoctonia solani and Mucor sp.. Chit-Pr also inhibits the spore germination of Cercospora personata and Altenaria brassicae.4. An antifungal peptide, designated BTL, was purified from a Bacillus strain B-TL2 from tobacco stem. The chromatographic procedure involved cation exchange on CM-Sepharose FF column and SOURCE 5RPC reversed-phase HPLC. The molecular mass of BTL is 2237.690 Da and its N-terminal amino acid sequence is: NH₂-KQQLATEAESAGPIL. The antifungal peptide BTL can inhibit the mycelia grow of following fungi, Bipolaris maydis, Aspergillus niger, Cercospora personata and Altenaria brassicae. BTL also shows thermal stability.