Study On Purification And Partial Properties Of Antimicrobial Substance From Brevibacillus Laterosporus S62-9

In recent years, the rapidly arise of resistant organism has been caused by unreasonable application of antibiotics. The researches of new type of antimicrobial substance which can control drug-resistant bacteria become a focus. According to our previous experimental studies, Brevibacillus laterosporus S62-9 strain showed a strong activity to inhibit the growth of many kinds of microbiology. The inhibition mechanism is producing antimicrobial substance.In this paper, we took Brevibacillus laterosporus S62-9 strain preserved in the enzyme engineering laboratory as the fermentation strain.The purification procedure of the crude fermentation solution was elucidated by research on rotary evaporator concentrating, ammonium sulfate precipitation, dialysis desalting, ion-exchange chromatography on DEAE-cellulose 52 and the Sephacryl S-100 chromatography. Subsequently, the purified antimicrobial substance was identified as single chromatographic peak by capillary electrophoresis (CE). It was showed that the antimicrobial substance was pure through differential scanning calorimetry (DSC) analysis. Furthermore, the properties of the antimicrobial substance were studied. All these results lay a foundation for further study of characteristics, structure and antimicrobial mechanism of the antimicrobial substance, and provided later scale production a technology platform.To gain physical and chemical properties of the antimicrobial substance produced by Brevibacillus laterosporus S62-9 strain, the effects of heat, pH and proteases were studied. The result showed that the antimicrobial substance were thermal stable, about complete activity remained after heating at 20°C~100°C for 20 min. Under 121°C for 20 min the activity only decreased 11.08% compared with the activity of 20°C. On the other hand, the antimicrobial substance still retains 95% of initial activity when autoclaved at 121°C for 2 h. After the treatment in pH 3~12 for 1 h, the antimicrobial activity decreased slightly between pH 4~6, which showed a strong and stable antimicrobial activity between pH 3~12. The antimicrobial substance was tolerant to protease K, trypsinase, acid protease, and neutral protease.To estimate the antimicrobial effect of the antimicrobial substance to different kinds of microbiology, we use agar drilling diffusion method to detect the inhibitory activity. As a result, the antimicrobial substance showed a very wide inhibititory spectrum, as it could inhibit both typical Gram- bacteria and some Gram+ bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Escherichia coli, Proteus mirabilis, and Salmonella typhimurium. In addition, it could also inhibit some fungi such as Aspergillus Niger, Pichia pastoris and Candida albicans.Structures of the antimicrobial substance were studied through ultraviolet spectrum (UV) and infrared spectrum (IR). The results showed the antimicrobial substance might be a kind of peptides.In all, this study provides a theoretical basis for the continuous researchs of the antimicrobial substance and the potential application on industrial producting.