| Spirulina belongs to a family of aquatic photoautotrophic prokaryotes known as cyanobacteria. Spirulina Platensis has unique advantage to be samples for preparation of bioactive peptides due to its simple short breeding period, high reproduction and high protein content.This study has demonstrated that an antibacterial peptide SP-1 has been isolated and characterized from the alkaline protease and papain hydrolysate of S. platensis, and further investigated the antibacterial mechanism of SP-1 against Escherichia coli.The main research results are as follows:(1) Antibacterial peptide SP-1 has been isolated from the alkaline protease and papain hydrolysate of Spirulina platensis through a series of chromatographic methods including Sephadex G-25 chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), and Superdex 75 10/300 GL chromatography. Its amino acid sequence was determined via liquid chromatography-tandem mass spectrometer (LC-MS/MS). SP-1 is a cationic peptide which contains 18 amino acid residues (KLVDASHRLATGDVAVRA) with the molecular mass of 1878.97 Da.AGAR diffusion method was used to determine SP-1’s antibacterial activities against gram-negative bacteria and gram-positive bacteria. The minimum inhibitory concentration (MIC) of SP-1 was 8 mg/mL for Escherichia coli and 16 mg/mL for Staphylococcus aureus. No hemolysis was observed up to a peptide concentration corresponding to eight times the MIC. The hemolysis result showed that the peptide was safe and nontoxic to normal mammalian cells with high specificity.(2) Circular dichroism spectra of SP-1 was recorded in buffer and in the presence of SDS micelles. SP-1 was relatively unstructured in aqueous solution (random coil content 58.1%). However, when it binded to a membrane or in membrane-mimicking hydrophobic environment, it could convert to a largely P-sheet structure (β-sheet content 57.2%).The mechanism of the antibacterial peptide SP-1 against E. coli was studied. The results showed that SP-1 penetrated the cell membrane of E. coli and accumulated in the cytoplasm immediately after being added to the cells. SP-1 can increase permeability of the bacterial outer and inner cell membrane and interact with the bacterial membrane, causing membrane permeabilization in a time-dependent manner, the damage of cell membrane integrity and leakage of intracellular ions (K+), which suggested that bacterial membrane was the first target site of antibacterial peptide SP-1. The intracellular action of e SP-1 on E. coli was studied. Gel retardation experiment results showed that SP-1 had effect on E. coli genomic DNA in a concentration-depended manner, which suggested that SP-1 can entered and accumulated in the E. coli cells.The action target was DNA. After E. coli cells treated with SP-1, the function of gene transcription was suppressed, causing the significant increase of the percentage of E. coli cells in phase I (or in phase Gl) which indicated that SP-1 first inhibited the synthesis of RNA, then further inhibited the synthesis of DNA and eventually resulted in the death of bacteria.The current research has demonstrated that a novel cationic antibacterial peptide named SP-1 was isolated and purified from Spirulina platensis hydrolysates. The antibacterial mechanism analysis showed that SP-1 first destroyed cell membrane and after penetrating the impaired membrane barrier, SP-1 interacted with DNA and RNA, influencing DNA replication and inhibiting cellular functions. Multiple pressures resulted in cell death. The results provided a solid foundation for antibacterial drug and natural preservative development and application. This study also provided new resources and approach for the development and utilization of novel antibacterial peptides. In the near future, the antibacterial peptide of Spirulina platensis may have the potential to be widely used in the food, pharmaceutical, and cosmetic industries. |
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