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Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14.

ABSTRACT: BACKGROUND:Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis. RESULTS:An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 8 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using a Niaffinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 demonstrated broad spectrum antifungal activity against different varieties of fungi such as Fusarium oxysporum, Verticillium dahliae, Bipolaris papendorfii, and Fusarium proliferatum. rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. Meanwhile, rF2 maintained its activity under treatment by proteinase K and trypsin and over a wide pH range from 5 to 10. CONCLUSIONS:A novel antifungal protein, F2, was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to verify its antifungal activity against R. cerealis and the validity of the gene encoding F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to resist fungal infections caused by R. cerealis.

SUBMITTER: Zhang X

PROVIDER: S-EPMC7684727 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14.

Zhang Xuechao X Guo Xiaojun X Wu Cuihong C Li Chengcui C Zhang Dongdong D Zhu Baocheng B

Microbial cell factories 20201123 1

<h4>Background</h4>Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis.<h4>Results</h4>An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography ...[more]

PMID: 33228718

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Project description:BackgroundChitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated beta-(1,4)-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures.However, the chitosanases used so far in transformation and transfection experiments show optimal activity at high temperature, which is incompatible with most transfection and transformation protocols. Thus, there is a need for chitosanases, which display activity at lower temperatures.ResultsThis paper describes the isolation of a chitosanase-producing, cold-active bacterium affiliated to the genus Janthinobacterium. The 876 bp chitosanase gene from the Janthinobacterium strain was isolated and characterized. The chitosanase was related to the Glycosyl Hydrolase family 46 chitosanases with Streptomyces chitosanase as the closest related (64% amino acid sequence identity). The chitosanase was expressed recombinantly as a periplasmic enzyme in Escherichia coli in amounts about 500 fold greater than in the native Janthinobacterium strain. Determination of temperature and pH optimum showed that the native and the recombinant chitosanase have maximal activity at pH 5-7 and at 45 degrees C, but with 30-70% of the maximum activity at 10 degrees C and 30 degrees C, respectively.ConclusionsA novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10 degrees C to 30 degrees C, temperatures that are preferred in transfection and transformation experiments.

Dataset Information

Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14.

Publications

Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14.

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