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.

Metabolomic and Transcriptome Analysis of the Inhibitory Effects of Bacillus subtilis Strain Z-14 against Fusarium oxysporum Causing Vascular Wilt Diseases in Cucumber

“…Bacillus is an ideal biocontrol microorganism for the scientific study of plant disease management. 7,8 B. subtilis BS06 isolated from soybean rhizosphere can be used to effectively control F. oxysporum (FO) and significantly reduce FO infection of soybean roots. 9 In recent years, the biological control of FO was accomplished with new strains of Bacillus and enhanced control mechanism.…”

“…They secrete a variety of secondary metabolites and significantly inhibit the occurrence of plant diseases without adversely affecting human or animal health or polluting the environment. Bacillus is an ideal biocontrol microorganism for the scientific study of plant disease management. , B. subtilis BS06 isolated from soybean rhizosphere can be used to effectively control F.…”

Compound fermentation supernatants of antagonistic bacteria control Rhizoctonia cerealis and promote wheat growth

“…Biological control for plant pathogens is associated with the synthesis of bioactive substances, such as active protein, HCN and hydrolytic enzymes, considered as traits defining biocontrol ability (Liu et al 2014). Zhang et al (2020) purified an antifungal protein F 2 isolated from the culture supernatant of B. subtilis Z-14 against R. cerealis. B. subtilis V26 produced a variety of hydrolytic enzymes and volatile substances in the report of Ben Khedher et al (2021).…”

“…It is reported that B. cereus 0-9 could reduce the disease incidence of wheat sheath blight (Xu et al 2014). Bacillus could produce antimicrobial proteins that altered the cell wall structure of mycelium and inhibit the mycelial growth of R. cerealis (Zhang et al 2020).…”

In Vitro Antagonistic Effects of Some Bacillus Spp. On the Growth and Mycotoxin Production of Toxigenic Aspergilli

“…(Foysal et al, 2018;Morita et al, 2019;Zhang et al, 2020). Biological control of fungal growth and food deterioration by antagonist bacteria such as Bacillus spp.…”