Isolation of the mustard Napin protein Allergen Sin a 1 and characterisation of its antifungal activity

Proteins and peptides belonging to the plant immune system can possess natural antibacterial, antifungal and antiviral properties. Due to their broad range of activity and stability, they represent promising novel alternatives to commonly used antifungal agents to fight the emergence of resistant strains. An isolation protocol was optimised to target proteins found in plants’ defence system, and it was applied to white mustard (Brassica hirta) seeds. Firstly, a ～14 kDa protein with activity against S. cerevisiae was extracted and purified; secondly, the protein was identified as the mustard Napin protein named Allergen Sin a 1. Napin is the name given to seed storage (2S) albumin proteins belonging to the Brassicaceae family. While several Napins have been described for their antimicrobial potential, Sin a 1 has been mainly studied for its allergenic properties. The antimicrobial activity of Sin a 1 is described and characterised for the first time in this study; it possesses antifungal and antiyeast in vitro activity, but no antibacterial activity was recorded. The yeasts Zygosaccharomyces bailii Sa 1403 and Saccharomyces cerevisiae DSM 70449 along with the filamentous fungi Fusarium culmorum FST 4.05 were amongst the most senstitive strains to Sin a 1 (MICs range 3–6 μM). The antimicrobial mechanism of membrane permeabilisation was detected, and in general, the antifungal activity of Sin a 1 seemed to be expressed in a dose-dependent manner. Data collected confirmed Sin a 1 to be a stable and compact protein, as it displayed resistance to α-chymotrypsin digestion, heat denaturation and insensitivity to pH variations and the presence of salts. In addition, the protein did not show cytotoxicity towards mammalian cells.     

桑氏链霉菌KJ07抗菌蛋白的分离纯化及部分特性

【目的】桑氏链霉菌(Streptomyces sampsonii)KJ07对无性阶段的杨树紫纹羽病菌(Rhizoctonia violacea)有较强的拮抗作用。为研究其抗菌物质,对其发酵液中主要抗菌物质进行分离纯化并明确其部分性质。【方法】采用硫酸铵分级沉淀、DEAE Sepharose Fast Flow离子交换层析、Sephadex G-50分子筛层析等方法进行分离纯化。【结果】获得单一抗菌活性蛋白,分子量约为28.4 k D。该抗菌蛋白抑菌谱较广,能使R.violacea菌丝畸形,菌丝隔膜不明显,细胞壁及胞内原生质开始降解,并产生黑色物质。稳定性试验显示抗菌蛋白最适温度为25°C,最佳p H为6.0,当温度≥60°C时,抑菌活性下降大于20%,当p H4.0或≥8.0时,抑菌活性下降大于12%。其活性还受金属阳离子影响,但对蛋白酶K不敏感。利用自动Edman降解法测得抗菌蛋白N端10个氨基酸序列,但通过NCBI BLAST程序未检索到与其相似性较高的已知抗菌蛋白。【结论】推测该抗菌蛋白可能是一种新的蛋白质。     

Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP)

A novel 6.6 kDa antifungal peptide (NFAP) from the culture supernatant of the mold, Neosartorya fischeri (anamorf: Aspergillus fischerianus), and its encoding gene were isolated in this study. NFAP is a small, basic and cysteine-rich protein consisting of 57 amino acid residues. It shows 37.9-50% homology to similar proteins described in literature from Aspergillus clavatus, Aspergillus giganteus, Aspergillus niger, and Penicillium chrysogenum. The in silico presumed tertiary structure of NFAP, e.g. the presence of five antiparallel β-sheet connected with filaments, and stabilized by three disulfide bridges, is very similar to those of the defensin-like molecules. NFAP exhibited growth inhibitory action against filamentous fungi in a dose-dependent manner, and maintained high antifungal activity within broad pH and temperature ranges. Furthermore, it exhibited relevant resistance to proteolysis. All these characteristics make NFAP a promising candidate for further in vitro and in vivo investigations aiming at the development of new antifungal compounds.     

Isarfelin, a peptide with antifungal and insecticidal activities from Isaria felina

Isarfelin, a peptide with inhibitory activity on mycelial growth in Rhizoctonia solani and Sclerotinia sclerotiorum and insecticidal activity toward Leucania separata, was isolated from the mycelia of Isaria felina. The IC₅₀ value of its antifungal activity against R. solani was 3.1 μg mL⁻¹. However, it was devoid of activity toward several bacterial species including Bacillus subtilis, E. coli and Staphylococcus aureus. The isolation procedure involved ethanol extraction, adsorption on YPR II macropore adsorption resin, ethyl acetate extraction, petroleum ether precipitation and recrystallization from ethyl acetate.     

Mungin, a novel cyclophilin-like antifungal protein from the mung bean

A protein designated mungin, isolated from mung bean (Phaseolus mungo) seeds, possessed activity against the fungi Rhizoctonia solani, Coprinus comatus, Mycosphaerella arachidicola, Botrytis cinerea, and Fusarium oxysporum. The 18-kDa protein also possessed a novel N-terminal sequence with similarity to cyclophilins. It exerts an inhibitory action against alpha- and beta-glucosidases suppresses [(3)H]thymidine in corporation by mouse splenocytes.     

Hypothetical protein AF2241 from Archaeoglobus fulgidus adopts a cyclophilin-like fold

AF2241 is a hypothetical protein from Archaeoglobus fulgidus and it belongs to the PFam domain of unknown function 369 (DUF369). NMR structural determination reveals that AF2241 adopts a cyclophilin-like fold, with a β-barrel core composed of eight β-strands, one α-helix, and one 3₁₀ helix located at each end of the barrel. The protein displays a high structural similarity to TM1367, another member of DUF369 whose structure has been determined recently by X-ray crystallography. Structural similarity search shows that AF2241 also has a high similarity to human cyclophilin A, however, sequence alignment and electrostatic potential analysis reveal that the residues in the PPIase catalytic site of human cyclophilin A are not conserved in AF2241 or TM1367. Instead, a putative active site of AF2241 maps to a negatively charged pocket composed of 9 conserved residues. Our results suggest that although AF2241 adopts the same fold as the human cyclophilin A, it may have distinct biological function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

First report of a novel plant lysozyme with both antifungal and antibacterial activities

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4kDa in SDS-polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355U/mg at pH 5.5 and 30 degrees C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 degrees C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.     

Isolation of pisumin,a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var macrocarpon

An antifungal protein with a novel N-terminal sequence GVGAAYGCFG and a molecular mass of 31 kDa was isolated from the legumes of the sugar snap pea Pisum sativum var. macrocarpon. The protein, designated pisumin, exhibited antifungal activity against Coprinus comatus and Pleurotus ostreatus and much weaker activity against Fusarium oxysporum and Rhizoctonia solani. Pisumin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 6 microM. Pisumin was similar to other leguminous antifungal proteins in that it was adsorbed on Affi-gel blue gel and CM-Sepharose.