Screening Of High Yield Antibacterial Peptides Mutants From Bacillus Subtilis HS-A38 And Action Mechanism Of Antibacterial Peptides

Due to the widespread use of antibiotics,pathogenic microorganisms are resistant to antibiotics,and the abuse of antibiotics makes this situation more serious.Therefore,research and development of new antibiotics and antibacterial compounds have become an urgent need today.The active antibacterial products of Bacillus subtilis are mostly small molecular antimicrobial peptides.Because of its broad spectrum of antibacterial properties,low drug resistance,high thermal stability and low side effects,it has attracted the attention of researchers as an alternative to antibiotics.In this study,B.subtilis HS-A38 isolated from the Bohai Sea in Dalian was used as the original strain to produce antimicrobial peptides and study the antimicrobial mechanism of the antimicrobial peptide against Vibrio parahaemolyticus.The B.subtilis HS-A38 was amplified by PCR and obtained 5 antibacterial peptide genes fragments.The gene fragment was ligated into the cloning vector pM18-T and transferred into Escherichia coli DH5?using CaCl₂ transformation method for sequencing.By aligning sequences in NCBI database,it was confirmed that the B.subtilis HS-A38 genome contains five antibacterial peptide genes encoding Iturin,Surfactin,Fengycin,Bacilysin and Bacilillin.In order to obtain high-yield strains,mutagenesis screening was performed using a variety of mutagenesis methods,and primary strains and rescreening of superior strains were performed by the Oxford cup method.After three generations of mutagenesis,a mutant strain mutHS-301 with good antibacterial effect and stable genetic properties was finally obtained.The antibacterial activity of the mutant strain mutHS-301 against Staphylococcus aureus was19.31%higher than that of the original strain,and the antibacterial activity against V.parahaemolyticus was 21.37%higher than that of the original strain.The finished antibacterial peptides was obtained from the fermentation broth.The yield of the mutant strain mutHS-301 was higher than that of the original strain HS-A38.The antimicrobial peptides were used to study the treatment of the indicator bacteria V.parahaemolyticus by antibacterial peptides produced via the mutant strain mutHS-301,cell proliferation before and after treatment,cell membrane permeability,cell morphology,intracellular DNA synthesis and changes of cell in protein synthesis.The results showed that the antimicrobial peptides affect the metabolic activity by destroying the cell membrane and influencing the synthesis of intracellular proteins,thereby inhibiting the growth of V.parahaemolyticus.The study provides theoretical basis and technical support for the development and utilization of antimicrobial peptides and biological control of marine V.parahaemolyticus.diseases.