Physicochemical Properties And Antibacterial Mechanism Of Prokaryotic Expression Pediococcus Acidilactici R-4 Bacteriocin PA-1

The pedA gene of Pediococcus acidilactici R-4 was amplified and recovered by polymerase chain reaction,ligated with pMD19-T vector,and transferred into E.coli DH5α receptor cells to achieve mass production of bacteriocins and to detect their physicochemical properties and inhibition mechanism.The cloned pedA gene was extracted and ligated with the expression vector pET-32a(+)to form the recombinant plasmid pET-32a-pedA,which was then transferred into E.coli BL21(DE3)receptor cells,and the protein size of Pediococcus acidilactici R-4 bacteriocin PA-1 expressed intracellularly in E.coli was 26 kDa after IPTG induction.The protein was purified using Ni-NTA column and identified as Pediococcus acidilactici R-4 bacteriocin PA-1 with His-tag by Western blot,and the antibacterial effect and protein activity was determined to be high using Staphylococcus aureus as the indicator bacterium.Pediococcus acidilactici R-4 bacteriocin PA-1 concentration was maximum at 0.245 mg/mLwhen induced using Amp mass concentration of 80 μg/mL,OD600 of 0.6,induction temperature of 37℃,IPTG concentration of 0.8 mM,and 8-hour induction culture.Staphylococcus aureus was used as an indicator organism to determine the physicochemical parameters of pure Pediococcus acidilactici R-4 bacteriocin PA-1 by acted at 40～121℃ for 20 min,in pH 2 to 12,ultraviolet irradiation 0～10h and catalase acted for 2h,the separately bacteriostasis ranges were 14.7～15.6 mm,14.0～16.5 mm,15.1～15.8 mm and 14.9 mm.However,after 2 hours of pepsin and trypsin,bacteriostatic effect was lost.This indicated that Pediococcus acidilactici R-4 bacteriocin PA-1 was stable to high temperature,strong acid,ultraviolet and catalase,however,weak sensitivity to strong bases and pepsin and trypsin inactivate it.The minimum inhibitory concentration of Pediococcus acidilactici R-4 bacteriocin PA-1 on Staphylococcus aureus was 0.025 mg/mL;it had inhibitory effect on Staphylococcus aureus growth;as the mass concentration of Pediococcus acidilactici R-4 bacteriocin PA-1 increased,the cell morphology of Staphylococcus aureus was gradually lost,the permeability of cell wall and cell membrane was gradually increased,and the decomposition of DNA increased The effect of varied concentrations of Pediococcus acidilactici R-4 bacteriocin PA-1 on Staphylococcus aureus DNA release was variable,1×MIC and 2×MIC increased the release of DNA with the increase of action time.The release of DNA at 3×MIC concentration reached the maximum within 1 h and then decreased slightly.The inhibition of Staphylococcus aureus biofilm formation increased from 43.68%to 71.70%as the concentration of Pediococcus acidilactici R-4 bacteriocin PA-1 was increased.It was shown that Pediococcus acidilactici R-4 bacteriocin PA-1 on Staphylococcus aureus was by disrupting its cell membrane,DNA and inhibiting biofilm formation,thus the bacterium showed morphological changes,breakage,some of the bacterium had crumpling and appeared to dry out leading to death.