Purification, Characterization And Antagonism Of Extracellular Protease From Bacillus Subtilis

Some bacteria in the genus Bacillus, which are both resistant to adverse environment and usually against bacterial and fungal pathogens, are among the dominant microorganisms in the soil and plant microecological systems. The biocontrol mechanisms of Bacillus include antagonism, competition and induced plant systemic resistance. The action of fungal cell-wall degrading enzymes such as chitinase, protease andβ-1.3-glucanase were considered as one of the main mechanisms involved in the antagonistic process. The role of chitinase andβ-1.3-glucanase in biocontrol against fungal pathogens has so far constituted the primary research. Bacillus spp. is one of the important sources of protease applied in industry. However, little is known about the role of protease in biocontrol produced by Bacillus strains displaying evident biocontrol activity.In order to further reveal the importance of extracellular protease in biocontrol, Bacillus strains, which were potential biocontrol agent against a broad spectrum of plant pathogenic fungi with the ability to produce extracellular protease, were isolated and identified. Subsequently, the purification and biochemical characterization of an extracellular protease isolated from Bacillus strain were studied. Additionally, the antifungal activity of the purified protease was tested. The main results and conclusions are as follows:1. 118 isolates were obtained from the healthy wheat and peanut rhizosphere at Taian, Qingdao, Shandong Province of China. Strain T2, a potential biocontrol agent against a broad-spectrum plant pathogenic fungi with the ability to produce extracellular protease, was selected using agar medium containing skimmed milk and in vitro inhibitory tests. According to the characteristics of morphology, physiology, biochemistry tests and the comparison of 16S rDNA sequence, strain T2 was identified as Bacillus subtilis.2. When grown in fermentation medium, B. subtilis T2 secretes several kinds of extracellular protease according to the result of SDS-PAGE with gels containing gelatin as protease substrate. The fermentation condition optimization for protease indicated that the optimum temperature and time were 37℃and 60 h. A kind of protease was purified by using ammonium sulfate precipitation following by DEAE Sepharose Fast Flow chromatography, SP Sepharose Fast Flow chromatography and Sephadex G-75 gel filtration chromatography. It appeared as a single band corresponding to molecular weight(MW)of approximately 29.0 kDa on SDS-PAGE with silver staining.3. The optimum temperature for protease activities is 60℃and optimum pH is 8.0. The protease was more stable when temperature was lower than 50℃and pH ranged from 5.0 to 7.5. In order to ascertain the nature of the protease concerned, the protease was assayed against a range of substrates to assess substrate specificity and the effect of different metal ions and modifiers. According to the results, the high or proper activities were revealed against the casein and also the substrate azocasein, whereas little activity against the BSA and Albumin Egg. It was stimulated by Na⁺, K⁺, Pb²⁺ and EDTA, partly inhibited by Ca²⁺, Ba²⁺, Al³⁺, Zn²⁺, Mn²⁺, Hg²⁺ and SDS, and strongly inactivated by PMSF, manifested the characteristics of serine protease.4. The crude antagonistic proteins obtained by precipitating the cell-free culture of B. subtilis T2 with 80% saturated (NH4)2SO4 could inhibit the conidia germination and hyphal growth of F. oxysporum f.sp. vasinfectum. It remained certain inhibitory effect after treated at 100℃for 30 min, whereas it was still lower than that of the former. This result indicates that both the thermostable antimicrobial proteins and the hydrolytic enzymes participate in the antagonistic process. Subsequently, an in vitro assay showed that the purified protease had inhibitory effect on conidia germination and hyphal growth of F. oxysporum f.sp. vasinfectum, which the swelling, distortion hyphae and shorten germination tubes were observed under light microscope. The results suggest the importance of extracellular protease produced by Bacillus subtilis T2 in antagonism of plant pathogenic fungi.