The Preparation, The Study On Action Mechanism And Exploration On Structural Modification Of Bacilysin

A bacterium strain isolated from the East China Sea, China, which can dramatically inhibit the growth of Candida albicans. Based on sequencing, it was identified as Bacillus amyloliquefaciens ZJU-2011. Fermentation process optimization was achieved in 5 L fermenter and the effect factors were optimized, and under these conditions, the antifungal activity was improved to 6.9 ×103 U/mL. Afterwards fermentation process was scaled up in 3T fermenter, and the antifungal activity was further improved to 8.5× 103 U/mL.On these basis, separation and purification of the antifungal targets was carried out. The broth was firstly pretreated by ethanol precipitation and macroporous resin adsorption, and 84.6% impurities were removed. After that, reversed-phase chromatography was applied and two crude components were obtained. The obtained products were further processed with gel filtration chromatography, finally two antifungal components with high-purity were produced. Based on MS and NMR analysis, they were identified as bacilysin and chlorotetaine respectively. Then a seperation process of bacilysin based on low-pressure chromatography was developed, which has a high recovery rate (-76%) and the purity was 95%.In vitro bacteriostasis testing of bacilysin showed that it displayed strong inhibitory effect against the tested strains with the MICs ranging from 0.9 to 7.8 μg/mL. Especially it showed best inhibitory effect against Candida krusei. Median lethal dose test revealed that the LD50 for rat tested was 135.3 g(bacilysin)/kg meaning that the acute toxicity of it was quite low. In vitro metabolism of bacilysin with liver mocrosomes was carried out and the metabolic model was determined.Glucosamine-6-phosphate synthase (antifungal target of bacilysin, Gfa) from Candida albicans was heterogeneously expressed in E, coli(BL21(DE3)). Compared with the C-His tagged Gfa, it was found that His tags at N terminal would greatly decrease its aminotransferase activity. Similarly, stronger inhibitory effect of bacilysin against C-His tagged Gfa was detected. It was found that the reaction between bacilysin and Gfa was reversible belonging to linear mixed inhibition. On these basis, it was concluded that bacilysin could directly react with Gfa in a reversible way.Molecular simulation was applied to study the antifungal mechanism of bacilysin. Firstly, the transport process of bacilysin and anticapsin was studied. Through docking, it was found that bacilysin could be docked into the active site of the transporter in a reasonable conformation, however anticapsin could not. In the molecular dynamic process, we discovered that the interaction of bacilysin and the transporter was stronger to form a stable complex than that of anticapsin. Especially, in the complex of bacilysin and transporter, the salt bridge between Glu403 and -NH2 of bacilysin was detected, which is the most important factor involved in the transport of peptides. Secondly, the action mechanism of anticapsin and bacilysin with Gfa was investigated. In the docking process, both anticapsin and bacilysin could be docked into the active site of Gfa in a relatively reasonable conformation. However, through dynamic analysis, it was revealed that when bacilysin interacted with Gfa, they would form a more stable complex. Moreover, the -SH of Cys would interact with the carbonyl group of bacilysin to form C-S group. Given these, it was concluded that bacilysin could be transported across the membrane in a high-efficiency way, which lead to its stronger antifungal effect than anticapsin.A replicative plasmid (pBAC-CE) was constructed for the mark-free gene knockout of bacD of ZJU-2011. The preparation of Bacillus amyloliquefaciens ZJU-2011 competent cell and the electrotransformation process were optimized. The optimum conditions were determined as follows:OD6000.8-0.9, electric field intensity 2.5 kV, resistance 200 Ω and recovery time 4 h. Under these conditions, the competent cells were transformed with SacI-linearized pBAC-CE. After twice homologous recombinations, the engineered Bacillus amyloliquefaciens ZJU-2011 with bacD knocked out, named ZJU-2011(1) was obtained. Through fermentation and product separation, the existence of anticapsin was confirmed.