Production Improvement Of Novel Antimicrobial Glycopeptide Sublancin And Its Bioactivity Study

Antibiotic residues resultant from abuse and excessive use of antibiotics has cause some serious problems on human health and livestock development. Study on antibiotic alternative has attracted more and more attention from relative researcher. Natural antimicrobial peptides(AMPs) and its derivative chimeric AMPs has several merits, including a wide spectrum activity of antivirus, antifungi, antibacteria and inhibiting cancer cell. In addition, they have a fast metabolism, no residue and no damage on cell integrity and bioactivity in animal or human body. Thus, study on AMPs has a very important significance. Research on and development of AMPs products can promote some fundamental changes in several application fields such as in food processing, clinical medicine, transgene technology of animal and plant and so on, and AMPs products have a prospective application in future.Different antimicrobial activities exist among AMPs from different sources regarding to their various structure. Antimicrobial glycopeptide sublancin was primarily obtained from Bacillus subtilis 168 strain, and has a post–translational modification of S–linked glycosylation and two disulfide bond structure. Hence, sublancin is difficult to be chemically synthesized based on its complicated structure. It is a feasible way to improve sublancin productivity and to obtain a large quantity of sublancin by recombinating chromosome of B. subtilis strains. In this study, B. subtilis 1A747 strain that is a derivative of B. subtilis 168 strain and harbors the same genes responsible for mature sublancin synthesis was used as a original strain for improving sublancin productivity.To improve the productivity of sublancin, B. subtilis 1A747 strain(named after B. subtilis ZQSY strains) was recombinated at chromosome level, and response surface methodology was used to optimize the parameters of medium component and culture conditions of recombinant B. subtilis ZQSY strain in shake-flask level. The investigations of antimicrobial activity of purified sublancin on several commonly seen pathogenic bacteria and conditional stability of sublancin were also carried out. Subsequently, potential risks of sublancin on animal body through oral administration were evaluated by using acute toxicity test and bioavailability on Kunming mice that were used as experimental animal models. The main results as below:1. Three genes(cluster) of sun I, sun A and sun T–bdb A–sun S–bdb B, which are responsible of biosynthesizing mature sublancin, were chemically synthesized. And three strong characteristic promoters of P43, Pglv and Plux S were placed before 5’ regions of these three genes(cluster), respectively, and the resultant plasmid p DM036 was developed based on above genes(cluster) and their promoters. Following that, recombinant B. subtilis 1A747 strain(named B. subtilis ZQSY strain) that was capable of improving sublancin productivity was obtained, by homologous recombination of p DM036 into SPβ genome in B. subtilis 1A747 strain. The fermentation of recombinant B. subtilis ZQSY was carried out in shake-flask level, and the supernatant of fermentation was detected by tricine–SDS–PAGE. The result of tricine–SDS–PAGE showed that a band at position of 3.8 k Da was present in lane where the supernatant sample was analyzed, and this outcome was further confirmed by western blotting analysis. The result of reversed-phase high–performance liquid chromatography(RP–HPLC) demonstrated that 642 mg sublancin was obtained from 1 L fermentation culture in shake–flask level.2. The medium components and culture conditions were optimized, on the basis of recombinant B. subtilis 168 strains, to further improve the productivity of sublancin. At first, Plackett–Burman design as a module from software Design Expert 8.0.5b was used to selected important variables that had a significant effect on sublancin productivity biosynthesized by recombinant B. subtilis ZQSY. The result showed that the important factors who had a significant effect(P < 0.05) on sublancin productivity were corn powder, soybean meal and culture temperature. After steepest ascent experiment, the optimal levels of 3 important factors determined by Box–Behnken Design(BBD) as a method of response surface methodology were those: corn powder 28.46 g/L, soybean meal 23.05 g/L and culture temperature 30.9°C. under these medium components and culture condition, the theoretical predicted value of maximum productivity of sublancin was 887.62 mg/L, which was in agreement with 896.8 mg/L obtained from practical fermentation culture of recombinant B. subtilis ZQSY strain under calculated optimal levels of 3 important variables. With a 5 L bioreactor and the optimal levels obtained from shake–flask condition, the productivity of sublancin was further improved to 968 mg/L.3. Purification of sublancin and in vitro antimicrobial activity were performed. After a series of purification process including filtration and reversed–phase HPLC, sublancin was purified and determined by using Electro–spray Ionization Mass Spectrometry. The purified sublancin showed a different antimicrobial activity towards 9 clinical bacterial strains such as Staphylococcus aureus and Enterococcus faecalis, especially against methicillin–resistant S. aureus with a minimum inhibitory concentration(MIC) of ≤ 0.4 ± 0.21 mg/L. The results of stability analysis displayed that temperatures as high as 60 oC for 30 min could slightly affect the antimicrobial activity of sublancin; however, temperatures above 80 oC for 30 min could significantly decrease the antimicrobial activity of sublancin. p H ranging from 4.0 to 9.0 had almost no influence on the antimicrobial activity of sublancin, but some activity loss was observed when p H was below 4.0. Under the challenge of human neutrophil elastase, Pseudomonas aeruginosa elastase, S. aureus V8 protease pepsin and trypsin, sublancin maintained a majority of antimicrobial activity.4. The potential risk caused by sublancin on animal or human body was primarily evaluated by using Kunming mice as animal model. The result of oral acute toxicity test of sublancin on Kunming mice(half male and half female) showed that oral single dose of 5000 mg/kg/d could not cause any damage to the tested viscera of Kunming mice, and this dose of sublancin could not elicit measured blood biochemical parameter changes over the normal range. The result of bioavailability demonstrated that sublancin could not enter into Kunming mice body in its intact form. These results suggested that it was safe when sublancin was orally administrated into gastrointestinal tract of Kunming mice.