Chemicals
All chemicals were analytical grade. Bovine serum albumin was from Sigma. Low-molecular weight protein standards were purchased from TaKaRa Biotechnology (Dalin) Co., Ltd. (China), DEAE-Sephadex Fast Flow was obtained from GE, SephadexG-100 from Pharmacia.
Microorganisms and culture conditions
B. subtilis B25 was grown in fermentation medium containing (g/L): glucose, 8.0; peptone, 8.0; yeast extract, 1.0; beef extract, 3.0; NaCl, 10.0; The pH of the medium was adjusted to 7.0 before autoclaving.
Cultivation was performed using 250 ml Erlenmeyer flasks containing 100 ml medium for 48 h at 28°C. As inoculation, 2% (v/v) bacteria grew in Luria-Bertani broth (g/L): peptone, 10.0; yeast extract, 5.0; and NaCl, 10.0; pH 7.0.
Fusarium oxysporum f.sp.cubense was isolated from wilted banana vascular and stored at 4°C in PDA and it was used as the indicator.
B25 was isolated from banana rhizosphere soil and identified as Bacillus subtilis on the basis of its morphological, biochemical and physiological characteristics, and 16S r DNA sequence. It’s capability of controlling Fusarium wilt in green house and in the field was evaluated (Liu et. al. 2011). Also, the characteristics of the active substance, such as precipitated quality, molecular mass, heat-tolerance were test (Lin et. al. 2013).
Assay of of antifungal activity
B25 was assayed for its antagonistic activity in vitro against Fusarium oxysporum f.sp.cubense, Corynespora cassiicola, Alternaria solani, Botrytis cinerea, Colletotrichum gloeosporioides and Aspergillus niger via duel culture method. A fungal disc of five day old mycelial mat of 1 cm diameter was placed in the centre of the petri plate containing PDA (potato dextrose agar). And the 24 hr old Bacillus B25 culture was single streaked at a distance of approx. 3 cm from the fungal disc. The plate was incubated at 28°C and observed after every 24 hrs for any inhibition of mycelial growth.
The antifungal activity against FOC of precipitated protein and all fractions acquired in the procedure of separation was tested by agar-diffusion method. A fungal disc was placed in the centre of PDA. Plate. Wells of 0.5 cm diameter were made at a distance of approx. 3 cm from the fungal disc. And tested substances were discharged in the wells. The Plate was incubated at 28°C and observed for any inhibition of mycelial growth.
Production and purification of antibiotics
A loop of B25 cells from a slant culture of fresh nutrient agar was inoculated to a 250 ml flask containing 100 ml LB broth (pH 7.0). The flask was incubated on a rotary shaker at 200 rpm for 9 ~ 12 h at 28°C. This fresh culture was inoculated to fermentation broth, each 2 ml. These flasks were incubated under the same conditions as described above for 48 h. Culture supernatants obtained after removed the cells by centrifugation at 6000 r/min for 20 min for further study.
The proteins were precipitated from the supernatant at 70% (w/v) (NH4)2 SO4 saturation. The latter was added in small portions with constant stirring for 30 min. The stirring was continued for 1 h, and the mixture was kept overnight at 4°C. The precipitate was collected by centrifugation at 10000 r/min for 30 min, dissolved in a 1/20 (v/v) phosphate buffer (0.02 mol/L, pH 6.8), and dialyzed for 48 h with 8 changes in the same buffer (500 ml each) to remove ammonium sulfate. The dialysates were condensed to yield precipitated proteins, which were further purified by column chromatography. A part of the precipitated proteins were dissolved in phosphate buffer (0.02 mol/L, pH 6.8), and the antifungal activity of the precipitated proteins was tested against Fusarium oxysporum by agar-diffusion method.
Chromatography was carried out on a DEAE-sepharose fast Flow ion exchange column previously equilibrated with 0.02 mol/L phosphate buffer (pH 6.8). The column was eluted with NaCl in phosphate buffer (0.02 mol/L, pH 6.8) to desorb the absorbed proteins (fractions I, II, III, IV). Each fraction was dialyzed in distilled water to remove NaCl, and then adjusted to the same concentration with phosphate buffer. Anti-fungal activity was tested against Fusarium oxysporum by agar- diffusion method. Fraction III was subsequently chromatographed on SephadexG-100. The column was eluted with 0.02 mol/L phosphate buffer (pH 6.8) to collect one main protein peak (P1) and another small protein peak (P2). The antifungal activity of the two fractions was tested. The protein concentration was determined using the method of Bradford (1976), with bovine serum albumin as a standard.
NanoLC-ESI-MS/MS analysis
The antifungal protein was separated by SDS-PAGE, analyzed by NanoLC-ESI-MS/MS, (ProtTech.Inc).