Breeding Of High-Yield Strains Of Bacillomycin D And Fermentation Process

Bacillus subtilis is a FDA approved and recognized "GRAS" strain.Its metabolism secretes many kinds of bioactivators,such as antibiotics,enzymes,which are harmless to environment and organism.Bacillomycin D is an antibacterial lipoprotein belonging to the iturin family,which is catalyzed by Bacillus subtilis non ribosomal enzyme.It is a surface-active amphipathic cyclic lipopeptide composed of a hydrophilic cyclic heptapeptide and a hydrophobic ?-amino aliphatic chain.Because of its characteristics of safety,non-toxicity,broad antimicrobial spectrum,and being easily biodegradable,it has broad application prospects in food,grain preservation,and pharmaceutical development.The yield of antimicrobial peptides from wild type bacteria is low,and the cost of existing media is relatively high,which resulted in too low profits for production and cannot be applied to industrial production.This study used Bacillus subtilis fmbJ as the research object,and the strain was transformed.The mutants with high yield and stable inheritance were obtained through mutagenesis and genome shuffling.Through optimizing its industrial fermentation medium,we can further improve the yield of antimicrobial peptide Bacillomycin D,so as to achieve industrial application.1.The Bacillus subtilis fmbJ was treated by UV(ultraviolet),MNNG(1-Methyl-3-nitro-1-nitrosoguanidine),and ARTP(atmospheric and room temperature plasmas)mutagenesis to select high-yield strains of the antibacterial peptide Bacillomycin D.By calculating the fatality rate and positive mutation rate,the mutation conditions were as follows,UV treatment:UV for 20 W,20 cm,60 s;MNNG treatment:1.2 mg/mL,37?for 10 min;ARTP treatment:incident power was 200 W,reflected power was 40 W,ventilation was 10 SLM,,irradiation distance was 10 mm,20? for 7 s.Through three kinds of mutagenesis treatments,7 mutant strains with higher Bacillomycin D yield than the original strain were obtained,with an average increase of 27.7%.2.The preparation,regeneration,inactivation and fusion conditions of protoplasts of Bacillus subtilis finbJ were studied,and Bacillomycin D high-yield strains were selected by genome shuffling.The optimal conditions for protoplasts preparation were as follows:cell culture time for 8 h,0.07 mg/mL lysozyme,enzymolysis time about 70 s;The optimized conditions for protoplasts UV inactivation were:UV for 20 W,irradiation distance was 20 cm,irradiation time for 100 min;The optimized conditions for thermal inactivation were:water bath at 85? for 50 min;The optimized conditions for protoplast fusion were as follows:40%PEG6000,37? for 10 min.Under the above protoplast treatment conditions,five high-yield recombinant strains of F131,2F14,2F82,5F1 and 5F5 were obtained through genome shuffling of the 6 strains of Bacillomycin D high-yield strains screened from the previous mutation,and the yield of Bacillomycin D reached to 441.77 mg/L,487.62 mg/L,458.03 mg/L,500.55 mg/L and 509.05 mg/L,respectively.The yield of the highest Bacillomycin D was 1.84 times higher than that of original strain.3.In order to reduce the cost of antibacterial peptide production in fermentation industry and to improve the yield of antibacterial peptide Bacillomycin D,Plackett-Burman design was utilized to evaluate the significant nutritional factors which affect the yield of Bacillomycin D in the original medium 6010 on the basis of the single factor experiment.And the industrial culture medium was optimized by the Central Composite Design response surface method.The results showed that the most three significant factors were maltose syrup,urea and MgSO4·7H2O.The optimum production was obtained when the cells grown in media containing:maltose syrup 46 g/L,urea 0.72 g/L,MgSO4·7H2O 0.35 g/L,corn steep liquor 16 mL/L,(NH4)2SO4 3 g/L,K2HPO4 7 g/L,MnSO4·H2O 0.05 g/L.After optimization,the yield of Bacillomycin D fermented by Bacillus subtilis M3 64 reached(620.2±13.9)mg/L,which increased by 50%compared to the non-optimized medium and 2.6 times compared to the Landy fermentation medium.The yield of Bacillomycin D in fermenter was(890.6 ± 20.1)mg/L,which is 1.14 times compared to the original 6010 medium.