Han Dongmei1, Ban Huifang, Yu Ziquan, Yu Ziniu, Sun Ming. 1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. hdm00@webmail.hzau.edu.cn
Abstract
OBJECTIVE: Amorphophallus soft rot disease, caused by Erwinia carotovora, affects Amorphophallus industry development. We identified and characterized protein APn5 against Erwinia carotovora, isolated from Bacillus subtilis strain BSn5. METHODS: Protein APn5 was purified from BSn5 culture by ammonium sulfate precipitation with 30% relative saturation and ultrafiltration. Inhibition activity was tested by agar well diffusion assay. Protein APn5 was treated at different temperatures, pH conditions and proteinase. The growth curve of BSn5 was examined; meanwhile, the inhibition activity of supernatant of BSn5 culture in different growth phase was tested. Amino acid sequence of protein APn5 was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadrupole-time-of-flight (Q-TOF). RESULTS: Protein APn5 showed a narrow inhibition spectrum, mainly strongly inhibiting the growth of a few plant pathogenic bacteria. Protein APn5 was sensitive to high temperature and alkaline pH, and partial sensitive to trypsin, proteinase K and pronase E. During strain BSn5 growth phase, the inhibition activity was unstable, which would gradually lose on stationary phase and disappeared finally. CONCLUSION: On the basis of the difference on inhibition spectrum and characteristics, protein APn5 was suggested as a novel antimicrobial protein.
OBJECTIVE:Amorphophallus soft rot disease, caused by Erwinia carotovora, affects Amorphophallus industry development. We identified and characterized protein APn5 against Erwinia carotovora, isolated from Bacillus subtilis strain BSn5. METHODS: Protein APn5 was purified from BSn5 culture by ammonium sulfate precipitation with 30% relative saturation and ultrafiltration. Inhibition activity was tested by agar well diffusion assay. Protein APn5 was treated at different temperatures, pH conditions and proteinase. The growth curve of BSn5 was examined; meanwhile, the inhibition activity of supernatant of BSn5 culture in different growth phase was tested. Amino acid sequence of protein APn5 was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadrupole-time-of-flight (Q-TOF). RESULTS: Protein APn5 showed a narrow inhibition spectrum, mainly strongly inhibiting the growth of a few plant pathogenic bacteria. Protein APn5 was sensitive to high temperature and alkaline pH, and partial sensitive to trypsin, proteinase K and pronase E. During strain BSn5 growth phase, the inhibition activity was unstable, which would gradually lose on stationary phase and disappeared finally. CONCLUSION: On the basis of the difference on inhibition spectrum and characteristics, protein APn5 was suggested as a novel antimicrobial protein.