TY - JOUR AU - FANG, Yingyou AU - LI, Linling AU - ZHENG, Yongliang AU - YUAN, Honghui AU - ZHANG, Xuehua AU - CHENG, Shuiyuan AU - CHENG, Hua PY - 2017/06/10 Y2 - 2024/03/29 TI - Extraction, Isolation and Identification of Antimicrobial Substances from Bacillus amyloliquefaciens CMN1308 JF - Notulae Botanicae Horti Agrobotanici Cluj-Napoca JA - Not Bot Horti Agrobo VL - 45 IS - 1 SE - Research Articles DO - 10.15835/nbha45110654 UR - https://notulaebotanicae.ro/index.php/nbha/article/view/10654 SP - 308-315 AB - <p>Four separation methods of antimicrobial substances produced by CMN1308 (<em>Bacillus amyloliquefaciens</em>) were evaluated and selected according to number of antimicrobial substances and its activity <em>in vitro</em>. 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 <em>P. expansum</em> 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 ā€œAā€ 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 ā€œDā€ 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.</p> ER -