New functions of degradation products of Bacillus subtilis ggt protein and identification of antimicrobial peptides
A Bacillus subtilis, antimicrobial peptide technology, applied in the field of plant protection and biological control
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Embodiment 1
[0034] Purification and mass spectrometry identification of Bacillus subtilis GGT protein.
[0035] Inoculate Bacillus subtilis BU412 into YME liquid medium (maltose 10 g / L, yeast extract powder 4 g / L, glucose 4 g / L, pH 7.5) for overnight culture, centrifuge at 12000g to collect supernatant, use 80% sulfuric acid The protein fraction was obtained by ammonium precipitation, and the precipitate was dissolved with 20mM Tris-HCl (pH7.5) buffer solution, filtered through a 0.22 μm filter membrane, and anion exchange chromatography was performed using an AKTA protein purifier, and the purification column used was GE’s Q hp anion exchange column (column volume is 5mL). Use 20mM Tris-HCl (pH7.5) buffer to load and equilibrate, perform 0-100% linear gradient elution with 0.5M NaCl, 20mM Tris-HCl buffer, and collect protein fractions P1-P3 according to the absorption peak ( figure 1 ), using the Oxford cup method to determine the antibacterial activity of each isolated component agai...
Embodiment 2
[0038] Prediction of antimicrobial peptides from Bacillus subtilis GGT proteins.
[0039] The amino acid sequence of GGT (Genbank accession number is KIU12867.1) in CAMP R3 The online prediction of the antimicrobial peptide region on the database found that multiple polypeptides of GGT may constitute antimicrobial peptides, combined with the secondary structure and positioning of these polypeptides in the three-dimensional structure of the GGT protein ( Figure 6 ), from which 3 peptides (Table 1) were selected for online analysis in the APD database. The results showed that all three peptides could form α-helices with hydrophobic surfaces, which may have the activity of antimicrobial peptides. According to the number of amino acids in the three polypeptide chains, they were named LT30, AE26 and IF20, respectively. In addition, the three-dimensional structure of the GGT protein was queried from the PDB database and the three peptide chains were spatially positioned using Py...
Embodiment 3
[0043] Synthesis and activity detection of predicted antimicrobial peptides.
[0044] Three candidate antibacterial peptides of the GGT protein of Bacillus subtilis were synthesized by solid-phase peptide synthesis technology. The N-terminus of the synthesized peptides was acetylated and the C-terminus was amidated, with a purity of more than 95%. Peptides were dissolved in 20 mM Tris-HCl (pH 8.0) buffer at a final concentration of 50 mM. The antibacterial activity of these three polypeptides against Streptomyces scabies was determined by the Oxford cup method. The results showed that the polypeptide LT30 had no inhibitory effect on Streptomyces scabica, while AE26 and IF20 had inhibitory effects on Streptomyces scabica, and the effect of IF20 was better than that of AE26 ( Figure 7 ).
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