Isolation of a small chitinase-like antifungal protein from Panax notoginseng (sanchi ginseng) roots

An antifungal protein, with a molecular weight of 15 kDa and an N-terminal sequence analogous to those of chitinases, was first isolated from the Chinese medicinal material Panax notoginseng, using cation exchange chromatography and affinity chromatography. The protein was adsorbed on CM-cellulose, Affi-gel Blue Gel and Mono S. It exerted antifungal activity against Coprinus comatus, Fusarium oxysporum and Mycosphaerella arachidicola but not against Rhizoctonia solani. The protein was devoid of ribonuclease activity against yeast tRNA.     

Passiflin,a novel dimeric antifungal protein from seeds of the passion fruit

The intent was to isolate an antifungal protein from seeds of the passion fruit (Passiflora edulis) and to compare its characteristics with other antifungal proteins and bovine β-lactoglobulin in view of its N-terminal amino acid sequence similarity to β-lactoglobulin. The isolation procedure entailed ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on DEAE-cellulose, and FPLC-gel filtration on Superdex 75. The isolated 67-kDa protein, designated as passiflin, exhibited an N-terminal amino acid sequence closely resembling that of bovine β-lactoglobulin. It is the first antifungal protein found to have a β-lactoglobulin-like N-terminal sequence. Its dimeric nature is rarely found in antifungal proteins. It impeded mycelial growth in Rhizotonia solani with an IC₅₀ of 16 μM and potently inhibited proliferation of MCF-7 breast cancer cells with an IC₅₀ of 15 μM. There was no cross-reactivity of passiflin with anti-β-lactoglobulin antiserum. Intact β-lactoglobulin lacks antifungal and antiproliferative activities and is much smaller in molecular size than passiflin. However, it has been reported that hydrolyzed β-lactoglobulin shows antifungal activity. The data suggest that passiflin is distinct from β-lactoglobulin.     

First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species

An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 8.3 microM and 4.5 microM, respectively. It exhibited dose-dependent binding to lyso-alpha-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.     

Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris

An antifungal polypeptide, with a molecular mass of 28 kDa as judged by gel filtration and appearing as a single band with a molecular mass of 14 kDa in sodium dodecyl suflate-polyacrylamide gel electrophoresis, was isolated from fresh fruiting bodies of the mushroom Polyporus alveolaris. The antifungal polypeptide, designated as alveolarin, demonstrated an inhibitory action on mycelial growth in Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. Alveolarin was isolated with a procedure that entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75 by fast protein liquid chromatography.     

Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916

An antifungal protein, with a molecular mass of 41.9 kDa, and designated as bacisubin, was isolated from a culture of Bacillus subtilis strain B-916. The isolation procedure consisted of ion exchange chromatography on DEAE-Sepharose Fast Flow, and fast protein liquid chromatography on Phenyl Sepharose 6 Fast Flow and hydroxyapatite columns. The protein was adsorbed on all three chromatographic media. Bacisubin exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea. The IC50 values of its antifungal activity toward the last four fungal species were 4.01 microM, 0.087 microM, 0.055 microM and 2.74 microM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.     

Pleurostrin, an antifungal peptide from the oyster mushroom

A 7kDa peptide, with inhibitory activity on mycelial growth in the fungi Fusaerium oxysporum, Mycosphaerella arachidicola and Physalospora piricola, was isolated from fresh fruiting bodies of the oyster mushroom. The isolation procedure entailed extraction with an aqueous buffer, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. It demonstrated an N-terminal sequence different from known antifungal proteins and peptides.     

Sativin: a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon

An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani.     

Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage

An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The N-terminal amino acid sequence of the protein was highly homologous to that of plant cyclophilins and consequently the protein was denoted as C-CyP. To understand the antifungal activity of C-CyP, we isolated a cDNA encoding its gene from a Chinese cabbage leaf cDNA library. The Chinese cabbage genome bears more than one C-CyP gene copy and C-CyP mRNA is highly expressed in all tissues except the seeds. Recombinant C-CyP catalyzed the cis-trans inter-conversion of the Ala-Pro bond of the substrate, which indicates this protein has peptidyl-prolyl cis-trans isomerase activity. It also inhibited the growth of several fungal pathogens.     

Cicerarin,a novel antifungal peptide from the green chickpea

A peptide designated cicerarin, with an N-terminal amino acid sequence VKSTGRADDDLAVKTKYLPP dissimilar from known proteins and peptides and a molecular mass of 8kDa, was isolated from seeds of the green chickpea Cicer arietinum cv green chickpea. Cicerarin was isolated with a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10mM Tris-HCl buffer (pH 7.3). Cicerarin exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100 degrees C for 15min.     

Purification of allivin,a novel antifungal protein from bulbs of the round-cloved garlic

A novel antifungal protein, designated allivin, was isolated from bulbs of the round-cloved garlic Allium sativum var. round clove with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and FPLC-gel filtration on Superdex 75. Allivin possessed an N-terminal sequence demonstrating very little similarity to sequences of Allium sativum chitinases and ribosome inactivating proteins. Allivin exhibited a molecular weight of 13 kDa in gel filtration and SDS-polyacrylamide gel electrophoresis. It displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola. It inhibited translation in a cell-free rabbit reticulocyte system with an IC50 of 1.6 microM.