| Bacilli are species of Gram-positive bacterium broadly distributed in nature, among which, as to be the predominant flora in soil and plant micro-ecosphere, Bacillus subtilis and Bacillus amyloliquefaciens can produce several kinds of antimicrobial substance, some of which have been applied in plant protection and with potential value for clinic use. Fengycin was a kind of lipopetptide antimicrobial substance produced by many Bacilli strains; showed strong inhibitory activity on growth of mould and with a broad application prospect as preservative for food and forage industry using. Owing to the complexity of fengycin synthetase complex, in this study, based on the development of molecular biology, studies amied on the specific A domain from fengycin synthetase was carried out in vitro. One of adenylation domain nucleic acid fragment of fengycin synthetase operon from genome of Bacillus subtilis strain was obtained through PCR amplification, this target adenylation domain fenE2-A showed multi-substrate selectivity during fengycin biosynthesis process. Recombinant cloning plasmid pMD19-E2A was constructed to preserve gene fragment; expression vector pET-23a-E2A and pET-32a-E2A was constructed to realize gene prokaryota expression in Escherichia coli; relation between gene expression quantity and inducing conditions was investigated, attempts on refolding of gene expressing inclusion body was explored; besides, relevance of lipopeptide tolerance and it’s antimicrobial effects was surveyed, providing guidance for lipopeptide fermentation productivity improvement. This thesis mainly drew conclusions as follows:1. Fengycin synthetase encoding gene from Bacillus subtilis was analyzed by molecular biology software Vector NTI, nucleic acid sequence encoding fenE2-A domain from fengycin synthetase open reading frame was ascertained by sequence alignment. Using genome from Bacillus subtilis fmbj as template, target gene fragment fenE2-A was cloned and then ligated to pMD19-T plasmid to form pMD19-E2A, and then transformed into Escherichia coli DH5a preserved under cold storage. 2. Recombinant expression plasmid pET-23a-E2A and pET-32a-E2A was constructed by inserting target DNA fragment fenE2-A into expression vector pET-23a and pET-32a after digested by the same restriction endonuclease, incubated under IPTG addition and low temperature, gene expressing products was analyzed by SDS-PAGE, protein electrophoresis result showed that product encoded by fenE2-A mainly exist in precipitate, indicating that target gene was expressed as the form of inclusion body which become an obstacle to detect activity of heterogenic expression protein directly. The optimum cultivating conditions was ascertained as following:inoculated at1%inoculation after incubating for150min till culture OD600reached1approximately, then IPTG was added at a final concentration of10Mg/mL to initiate inducing, temperature was fixed at37℃and inducing time was ascertained for4h; Using degeneration buffer to dissolve inclusion body for2h, dissolved protein concentration was0.906mg/mL, through refolding process,53.88%of inclusion body protein regained solubility.3. Based on gene recombinant and combinatory biosynthesis, works with the expectations to replace fenE2-A gene fragment with another A domain that also showed relaxing substrate specificity of the same synthetase complex was proceeded. Gene fragment up and down the under-replaced gene was cloned, termed U and D respectively, through three rounds of SOE-PCR, three gene fragments was ligated together to form fusion fragment UED without any restriction nucleotide base importing. Recombinant integration vector pUS19-UED was constructed using UED ligate to linear pUS-19. Based on the plasmid pUS19-UED, a recombinant plasmid pUS19-UED-CrmR was constructed which possess Chloromycetin resistance screening label.4. Through antibiotic tolerance degree regulation towards antibiotic producing strain, influence of crude lipopeptide extracts on the growth of lipopeptide producing Bacilli was investigated; relevance between lipopeptide tolerance and strains’ antimicrobial effects was surveyed. Conclusions showed that lipopeptide extracts can inhibit growth of lipopeptide producing Bacilli strain; the more resistant to lipopeptide, the stronger antimicrobial activity it shows toward indicator bacterium, lipopeptide extracts in which with surfactin concentration of93mg/L can take on apparently negative effects on producing strain, colony with high lipopeptide tolerant ability is of23.2%higher surfactin fermentation productivity compared to low lipopeptide tolerance colony. |
PDF Full Text Request