Elsevier

Peptides

Volume 28, Issue 3, March 2007, Pages 553-559
Peptides

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

https://doi.org/10.1016/j.peptides.2006.10.009Get rights and content

Abstract

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 μM, 0.087 μM, 0.055 μM and 2.74 μM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.

Introduction

Antifungal proteins and peptides have been isolated from a great number of animals [1], [2], plants [7], [10], [31], bacteria [6], [34] and fungi [4], [9], [25]. They have been classified into many types [9], [22], [30], [31], such as PR-1proteins [1], [17], thaumatin-like proteins [5], [36], [20], glucanases [29], protease inhibitors [5], [40], ribosome inactivating proteins [14], [27], chitinases and chitinase-like proteins [42], cyclophilin-like proteins [37], [38], lipid transfer protein-like proteins [18], [35], and glycine/histidine-rich proteins[15].

The Bacillus subtilis species is widely used in the bio-control of plant diseases [3], [28], because they have a well developed secretory system producing structurally diverse secondary metabolites with a wide spectrum of antibiotic activity [7], [26]. Therefore, they have become very valuable for medical and agricultural applications. Additionally, B. subtilis strains synthesize non-ribosomally a variety of small antibiotic peptides (<2000 Da), such as iturin, surfactin, and fengycin [2], [24]. B. subtilis also secretes an abundances of proteins. However, little is known about the antifungal activity of these secreted proteins. We have succeeded in using B. subtilis strain B-916 to control rice false smut and rice sheath blight in Jiangsu Province. We have also found that the strain B-916 secretes proteins with high antifungal activity. This paper reports the isolation and characteristization of a novel antifungal protein secreted by B. subtilis B-916.

Section snippets

Isolation of antifungal protein

B. subtilis strain B-916 was cultured with shaking at 120 rpm in 8000 ml YPG medium (composed of 5 g yeast extract, 5 g tryptone, and 5 g glucose per 1055 ml water) for 48 h at 28 °C, and the supernatant was collected after centrifugation (9000 × g, 20 min). Solid ammonium sulphate was added to the supernatant to 95% relative saturation. The precipitate formed after standing at 4 °C overnight was collected by centrifugation (12,000 × g for 30 min), redissolved in 100 ml 25 mM Tris–HCl buffer (pH 8.01), and

Isolation of antifungal protein

Ion exchange chromatography of B. subtilis strain B-916 culture extract on a DEAE-Sepharose Fast Flow column produced an unadsorbed fraction (A) and two adsorbed fractions (B and C) (Fig. 1A). Of these fractions, only fraction B showed high antifungal activity. Hydrophobic interaction chromatography (HIC) of fraction B on Phenyl Sepharose 6 Fast Flow yielded an unadsorbed fraction D and an adsorbed fraction E. Antifungal activity was detected only in fraction E (Fig. 1B). Fraction E was

Discussion

This paper reports a novel antifungal protein, designated as bacisubin, from B. subtilis strain B-916. The molecular mass of bacisubin was different from those of other reported antifungal proteins from B. subtilis, such as bacillomycin D synthetase C (309.04 kDa), putative sensor kinase (53.38 kDa), BamD (44.94 kDa), endo-1-4-β-glucanase (46.60 kDa), bacillomycin D synthetase B (607.23 kDa), YnfF (45.39 kDa), putative response regulator (27.74 kDa), endo-1-4-β-xylanase (54.26 kDa), bacillomycin D

Acknowledgments

This research was supported by state 973 program (2006CBb101900), the Jiangsu Provincial Natural Science Foundation (BK2005108) and the Opening Foundation of the State Key Laboratory for Biology of Plant Disease and Insect Pests (PD2). The authors are grateful to Dr. Li Shifang and Dr. Lu Meiguang for their technical help. We also thank Miss Fion Yung for her skilled secretarial assistance.

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