title: Purification and characterization of a potential antifungal protein from Bacillus subtilis E1R-J against Valsa mali.
abstract: In order to identify the antagonistic substances produced by Bacillus subtilis E1R-J as candidate of biocontrol agents for controlling Apple Valsa Canker, hydrochloric acid precipitation, reverse phase chromatography, gel filtration, and ion exchange chromatography were used. The purified fraction EP-2 showed a single band in native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fraction EP-2 was eluted from native-PAGE and showed a clear inhibition zone against V. mali 03-8. These results prove that EP-2 is one of the most important antifungal substances produced by B. subtilis E1R-J in fermentation broth. SDS-PAGE and Nano-LC-ESI-MS/MS analysis results demonstrated that EP-2 was likely an antifungal peptide (trA0A086WXP9), with a relative molecular mass of 12.44 kDa and isoelectric point of 9.94. The examination of antagonistic mechanism under SEM and TEM showed that EP-2 appeared to inhibit Valsa mali 03-8 by causing hyphal swelling, distortion, abnormality and protoplasts extravasation. Inhibition spectrum results showed that antifungal protein EP-2 had significantly inhibition on sixteen kinds of plant pathogenic fungi. The stability test results showed that protein EP-2 was stable with antifungal activity at temperatures as high as 100 °C for 30 min and in pH values ranging from 1.0 to 8.0, or incubated with each 5 mM Cu(2+), Zn(2+), Mg(2+), or K(+). However, the antifungal activity was negatively affected by Proteinase K treatment.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
authors.forename: N N
authors.initials: NN
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
authors.forename: X
authors.initials: X
authors.lastname: Yan
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
authors.forename: X N
authors.initials: XN
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
authors.forename: H J
authors.initials: HJ
authors.lastname: Niu
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
authors.forename: Z S
authors.initials: ZS
authors.lastname: Kang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China. huanglili0001@hotmail.com.
authors.forename: L L
authors.initials: LL
authors.lastname: Huang
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001203:Ascomycota / Q000187:drug effects*; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D007526:Isoelectric Point; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D001426:Bacterial Proteins; D010455:Peptides
keywords: Antifungal substances; Apple Valsa Canker (AVC); Biocontrol agents (BCAs); Endophytic Bacillus subtilis