Studies On Fermentation, Purification And Biology Activity Of Marine Bacillus S-12-86 Lysozyme

Many antibacterial and antiviral substances possessed special function could be obtained from marine microorganism. Our research group had gotten marine Bacillus S-12-86 from the sea mud sample collected from the East Sea of China which produced lysozyme. Based on the research, the project studies mainly focused on fermentation, purification and biology activity of marine Bacillus S-12-86 lysozyme(MBL). The results showed as:The culture medium and fermentation conditions of marine Bacillus S-12-86 were studied. Marine Bacillus S-12-86 could utilize maltose, starch, glycerol and glucose as carbon sources but not mannitol or sucrose; it also could utilize beef extract, yeast extract, tryptone and ammonium sulfate as nitrogen sources, not potassium nitrate, ammonium chloride, or urea. Beef extract was the best the carbon source. Zn2+, Mn2+ and Cu2+ inhibited the growth or enzyme production of the strain. Fe2+ had no effect on the growth of the strain, but inhibited the enzyme production. Na+ and K+ had no effect on the growth or enzyme production. Ca2+ had little effect on the enzyme production but promoted the growth of the strain obviously. Mg2+ had good effect on both the strain growth and enzyme production. The optimum fermentation conditions for marine Bacillus S-12-86 were incubation of 24 h, incubation temperature at 30 degrees Celsius, inoculum's level 4%(v/v), medium volume 10%(v/v) and initial pH value 8.0. The results showed that the optimum culture media components were glucose 10.0 g/L,tryptone 5.0g/L,MgSO4 5.0 g/L,CaCl2 2.0 g/L. Comparison of MBL production before optimization (14454.4 U/mL) with that after optimization (25636.8 U/mL), MBL in optimized process increased by about 75.4%. The pilot production experiment indicated that the enzyme production was 26697.87 U/mL. The results indicated that the fermentation conditions selected from flask could be applied to the pilot production. Comparison of the culture conditions of marine Bacillus S-12-86 with those of Streptomyces albus G, Streptomyces griseus P-51, Bacillus subtilis77, et al, we could find that marine Bacillus S-12-86 was easily cultured with greater enzyme production and lower production cost. We could concluded that marine Bacillus S-12-86 had the potential to be used at large scale in practice.A novel bacteriolytic enzyme was purified from the crude culture of MBL by ammonium sulfate precipitation, Sephadex G-100 chromatography and CM-Sephadex A-50 ion-exchange chromatography. The molecular weight was 16.0 kD. The optimal pH and temperature for the Micrococcus lysodeikticus was 8.0 and 35℃,respectively. Zn2+ and Cu2+ could stimulate the activity, whereas Mn2+or Ag+ inhibited the lytic activity. Ba2+,Li+,Ca2+,Na+,K+ and Fe3+ had no effect on the enzyme activity. MBL was stable in some chemicals and thickening agents. Containing 0.1g/L surfactant, the relative enzyme activity was improved for 15% or 21% by Alkyl poly glycosides (APG) or cationic Alkyl poly glycosides(cAPG) respectively, decreased 15% by Sodium dodecyl sulfate (SDS), and had no change in Tween 20 or Tween 80 solution. The activity of liquid enzyme had more loss at room temperature than that at low temperature, lyophilized powder of the enzyme at room temperature or low temperature had significant differences at the enzyme activity. Three results showed that lyophilized powder was fitter to be saved than that in liquid for MBL.MBL had a broad antimicrobial spectrum against standard strains including G+, G- and fungi, et al. MBL influenced bacteria on the logarithmic growth prophase. Observation under the transmission electron microscope revealed that the cytoplasm of E. coli was concentrated, cell membrane was separated from cell and part of cell wall was disappeared, then the cell was broken down into cavity by MBL for 12 h. While the cytoplasm of S.aureaus was asymmetrical and serious contracted with fuscous conglobation, and the cell wall became thinner, cell membrane was separated from cell wall and part of cell wall was dim. The cell wall of Candida albicans was distortional seriously, and the cytoplasm was asymmetrical. There were many cavities in the cytoplasm. The breakage effect of MBL on the inner structure of Candida albicans became stronger as the time running. The bacteria inhibition concentration was between 0.25 mg/mL~4.00 mg/mL and the bactericidal concentration was between 0.25 mg/mL~8.00 mg/mL.We studied the anti-microbial activity of MBL and its Combinations with APG. The method was used to test the antibacterial effects of it , APG and those of the compound solution of them respectively. The results showed that the solution with concentration of more than 5.0 mg/mL MBL had effective anti-microbial activity. The solution containing 0.25 mg/mL ~1.0 mg/mL APG was almost ineffective on anti-microbial activity. 5.0 mg/mL MBL plus with available 1.0 mg/mL APG(Compound Enzyme Prepration, CEP) could kill Escherichia coli, Staphylococcus aureus or Candida albicans. CEP was placed in culture incubator for two weeks at 54℃, and then the rates of killing was no changed. We could conclude that CEP killed bacteria effectually, and the capability of CEP in killing bacteria was stable.To develop an new anti-PRV medicine from MBL, the anti-PRV effect of MBL in PK-15 cell culture was observed by means of the inhibition of cytopathic effect. The results showed that in PK-15 cell culture, MBL was found to be an inhibitor of PRV in a concentration-dependent manner. The median toxic concentration (TC50) of MBL was 100.0μg /mL, the median effective concentration (EC50) of MBL was 0.46μg /mL, the selectivity index (TI=TC50/ EC50) is 217. In time of addition experiment, MBL inhibited the effect of PRV in PK15 cell when it was added at 0 h, 2 h, 4 h, 6 h, and 8 h after virus infection. We could conclude that in PK-15 cell culture, MBL was found to be an inhibitor of PRV; In addition, MBL has good inhibiting effect on NDV in vivo.