Study On The Character Of Antagonistic Protein Of Bacillus Cereus J₁ And Improvement Of Antagonistic Bacteria

Bacterial wilt of ginger is a destructive soil-borne disease which occurs generally andfrequently during the production. It is a kind of bacterial wilt diseases caused by thepathogen of Ralstonia solanacerum. The Bacillus cereus isolated from the soil of bacterialwilt exhibited desirable antagonism to the disease. This bacterium shall be studied furtherdue to its strong antagonism to bacterial wilt of ginger.Bacillus cereus J₁ was fermented for 55 h at 30℃with shacking (120r/min), and theantagonistic protein was isolated from this ferments by ammonium sulfate (60ï¼…saturation). The armulated column method was then performed to observe its antagonismspectrum against 8 bacteria. Results showed that the antagonistic protein exhibited strongantagonistic activity against Fusarium. oxysporum f. sp. niveum and Fusarium. oxysporum f.sp. Zeae, and the width of antagonism belt reached to 10-15 mm. In addition, it exhibitedmoderate antagonism to the other 6 pathogens such as Colletotrichum gloeosporioides.The antagonistic protein was treated with various temperatures, pH values and lightingconditions. Results revealed that its antagonism activity maintained well at 40-80℃andlost completely at 100℃in 30 min after heating. The protein showed activity between pHvalues of 4 and 8, and the optimal pH value was 7.0. The antagonistic activity of strain J₁was not changed under fluorescent light or ultraviolet drastically, and not changed in lightcompared to in darkness. This confirmed that the protein was stable in different lightingconditions. However the protein lost its activity by treating with 37℃for 90 min,indicating the active substance was hydrolyzed enzymatically.The Econo-Pac Q ion exchange column was balanced to pH 8.0 with 200 mM Tris-HC1.Proteins were eluted against the balanced solution (2mL/min) to isolate the antagonisticprotein with NaCl gradients (0-1M). The molecular weight of purified protein was 45 kDaby bioassay and SDS-PAGE analysis.Strain J₁ was improved by various inducements. The desirable inducement was achieved when treated with ultraviolet for 60s-90s and under the condition of 80ï¼…-90ï¼…of mortality.Takamasa mutation frequency was reached with low dosage of Ethyl sulfonate (EMS) for alonger time, so several strains with high production were obtained. J₁ was improved thebest by the combination of UV (60s) and LiC1 (1.2ï¼…), while it was improved the worstwith acridine orange because there was no positive mutates induced. The improved strainEMS-2 was obtained through multiple screening, and the protein efficiency ratio of itsferments was 22.79ug/mL, 30.38ï¼…higher than that of non-improved J₁.EMS-2 was improved further through protoplast, and 0.5mg/mL of lysozyme was thebest concentration for making protoplast when it was treated at 30℃for 30 min. Resultsshowed that mortality of the EMS-2 protoplast exposed to UV for 60 s was far smaller thanthat of J₁, beyond the expectation of this experiment. This might be contributed to theprotective mechanism of hyperosmotic solution. The positive mutation rate reached thebest when exposed to UV for 50 s, indicating UV could induce protoplast at low dosage.The improved strain UP-9 was subjected to inducement further with EMS. The resultindicated that EMS could result in the damage of protoplasts significantly, and the positivemutation efficiency reached to the peak of 31.3ï¼…in 35 min after inducement.The improved strain EP-5 was eventually obtained through multi-generation screeningof strain J₁. The protein efficiency rate of this strain was 46.73mg/mL, 1.65 times higherthan that of J₁ ferments.