The Breeding For High-Yield Subtilosin A Strain With Genome Shuffling And Structural Identification Of AurantininB

SubtilosinA is a kind of lantibiotics with special structure. A covalent bond is formed between the sulfur atoms of cysteine, two phenylalanine and the alpha position of threonine in the molecule. SubtilosinA is divided into the new fourth sub-categories of the first kind of bacteriocin with has a wide range of antimicrobial activity, not only inhibiting gram positive bacterial including Bacillus anthracis, Bacillus cereus, bacillus thuringiensis, Streptococcus agalactiae, Listeria monocytogenes, but also having certain effect on gram negative bacterial, such as Gardnerella vaginalis and Shigella sonnei, Enterobacter aerogenest. SubtilosinA also has good stability for pH, heat and UV. Therefore,has potential application prospects in food, biological control and pharmaceutical manufacture.But so far, the fermentation yield of SubtilosinA is still at a relatively low level, consequently limit its application to some extent. SubtilosinA-producing strain named Bacillus subtilis A1B2-2 preserved in our lab has a lower yield. In this study, SubtilosinA fermentated from Bacillus subtilis A1B2-2 was isolated and purified. Then the quantitative detection method for SubtilosinA was established by HPLC and the biological titer of SubtilosinA was determinated. Genome shuffling was used to improve the yield of SubtilosinA of Bacillus subtilis A1B2-2. A novel substance which can inhibit Gram-positive bacteria was found and identified. The main results are described as below.1. Quantitative detection method for SubtilosinA was established and the biological titer of SubtilosinA was determinated. SubtilosinA was preliminary separated and purified from the fermentation broth of Bacillus subtilis njaA1B2-2 by combination of purification steps including acid precipitation, extraction with methanol, Sephadex LH-20 column chromatography and semi-preparative HPLC. Then the quantitative detection method for purified SubtilosinA was established in HPLC, and the standard curve was y =0.0609x-5.2240. The method had a good linear relationship when content was 3.75-600 mg/L. The correlation coefficient reached to 0.9982. The detection limit was 9.375 mg/L. The limit of quantification was 218ng. The precision was 1.65%, and the purity of the sample was above 95%. The standard curve of nisin titer was determined the standard curve was y=109.87x-301.01. The biological potency of SubtilosinA was determinated as 9.93×106U.2. High-yield SubtilosinA strains were obtained by physical and chemical mutagenesis. nja 4-43 was selected out by undertaking gamma ray mutagenesis on Bacillus subtilis A1B2-2 preserved by our laboratory. Under the optimal conditions for protoplast formation and regeneration, which is high salt as the washing system, 0.1mg/ml lysozyme processing 40min, and then coating on the NB regeneration medium prepared with SMM, the protoplast formation rate reached 95.49%, and the regeneration rate was 89.25%. UV mutagenesis, HN02 mutagenesis and composite mutation of HN02 and UV were carried on nja 4-43 protoplast. Then nja N2-99、nja Z1-53 and nja Hl-65 were selected out with higher yield of SubtilosinA.3. Genome shuffling was carried out to improve the yield of SubtilosinA. Four high-yield SubtilosinA strains, nja 4-43、nja N2-99、nja Z1-53 and nja H1-65,were used as parent strains, and recursive protoplast fusion was launched, with inactivating the parent strain as the detection method for fusant. The UV-inactivated optimum conditions were 18W,15cm,25min. The heat-inactivated optimum conditions were 100℃,19 min. Inactivated protoplasts was then placed in RAD-BIO electroporation instrument and set the pulse voltage 150V, pulse width 100ms, number 9, interval 10s. After standing for lmin, when arranged in clustes, set the protoplast electroporation conditions as below:pulse voltage 1200V, pulse width 0.5ms, pulse number 2, a pulse interval of 5s. The regeneration rate was 1.39×10-3. After two rounds of genome shuffling, high-yield SubtilosinA strain R2-264 was selected out, and its production reached to 17.59mg/L,3.8 times high than the original strain.4. Identification of a new antimicrobial substance. A new substance was found when the crude protein of Bacillus subtilis njaA1B2-2 was separated and purified. The precise molecular weight of this substance was determinated as 780.4858 by HR-ESI-MS analysis. By consulting relevant literatures, this substance was suggested maybe as AurantininB. The ultraviolet absorption peak of this substance were 239,279,288,306,318 nm, consistent with AurantininB, and the coincidence degree of fragment peaks of this substance and AurantininB was high through MS2 and MS3 analysis. Finally, this unknown antimicrobial substance was determinated as AurantininB by the assay of infrared spectroscopy.