Extraction, Isolation and Identification of Antimicrobial Substances from Bacillus amyloliquefaciens CMN1308

Authors

  • Yingyou FANG Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)
  • Linling LI Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)
  • Yongliang ZHENG Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)
  • Honghui YUAN Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)
  • Xuehua ZHANG Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)
  • Shuiyuan CHENG Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)
  • Hua CHENG Huanggang Normal University, Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratory, Huanggang, 438000 (CN)

DOI:

https://doi.org/10.15835/nbha45110654

Abstract

Four separation methods of antimicrobial substances produced by CMN1308 (Bacillus amyloliquefaciens) were evaluated and selected according to number of antimicrobial substances and its activity in vitro. The results showed that extraction by acid precipitation of the fermentation supernatant of CMN1308 was the best with a diameter of inhibition zone of pathogen fungi P. expansum of 12.3 mm in a laboratory bioassay. Applying a silica thin layer chromatography (TLC), SDS-PAGE and other separation technologies we isolate antimicrobial substances, and the separated band were cut off for mass spectrometry analysis. The TLC of crude extract of CMN1308 show a topical band corresponding with the surfactin standard (Rf value =0.75), proved that the strain CMN1308 can produce this surface active compound. The mycoprotein extracted from CMN1308 was separated by Tricine-SDS-PAGE modified with the addition of urea in the separation gel. After mass spectrometric analysis and protein characterization, the isolated mycoprotein showed a maximum ion peak at M/Z of 2679 and molecular weight of 29.5 kDa, matching with protein flagellin. The extracellular antimicrobial protein of strain CMN1308 display four bands after urea-Tricine-SDS-PAGE, but after mass spectrometry analysis only two bands were identified. Band “A” with a maximum ion peak at M/Z of 1926 and molecular weight of 49.8 kDa, aligned with NCBI database, matching with DLDH (dihydrolipoamide dehydrogenase enzyme). Band “D” show the maximum ion peak at M/Z of 2936 and molecular weight of 22.4 kD, matching with a chitin binding protein. Thus, the strain CMN1308 has the potential to be developed as a commercial biological control agent for chestnut common pathogenic fungi.

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Published

2017-06-10

How to Cite

FANG, Y., LI, L., ZHENG, Y., YUAN, H., ZHANG, X., CHENG, S., & CHENG, H. (2017). Extraction, Isolation and Identification of Antimicrobial Substances from Bacillus amyloliquefaciens CMN1308. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 45(1), 308–315. https://doi.org/10.15835/nbha45110654

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Section

Research Articles
CITATION
DOI: 10.15835/nbha45110654

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