| Congresso Brasileiro de Microbiologia 2023 | Resumo: 981-1 | ||||
Resumo:To address the increasing challenge presented by antibiotic-resistant bacteria, there has been a surge of interest in novel and effective enzyme-based therapies. Endolysins are enzymes produced by bacteriophages that can degrade the bacterial cell wall, leading to cell lysis and the release of progeny phages. Therefore, endolysins are a promising strategy to tackle the current antibiotic resistance crisis. This study provided a comprehensive understanding of the biochemical and kinetics parameters of a new endolysin to further application as antibacterial and antibiofilm agent. The phage vB_PaM_USP_2 genome was recently elucidated (GenBank accession MT491205.1) and the bioinformatic analysis had predicted a putative endolysin. The gene that code for the endolysin was cloned [pET-28(+)] and used to transform Escherichia coli CD43 (DE3) strain. Expression was induced with 0.1 mM of IPTG and the protein extracts were purified in a nickel affinity chromatography. A synthetic peptide sequence was used as a substrate to measure the endolysin activity through a fluorescence resonance energy transfer (FRET) assay. The effect of pH on the activity of the endolysin was determined at different pH values, employing acetate (pH 4.5 and 5.0), MES (pH 5.5, 6 and 6.5), HEPES (pH 7, 7.5 and 8), BICINE (pH 8.5 and 9) and CAPS (pH 9.5, 10 and 10.5) buffer. The influence of temperature was investigated in the range of 25°C to 50°C in HEPES buffer (50 mM, pH 7). In parallel, the effect of anionic (SDS), cationic (CTAB) and nonionic surfactants (Tween 20 and Triton X) were investigated at percentages of 0.1, 0.25, 0.5, 0.75 and 1% in HEPES buffer (50 mM, pH 7) at 40ºC. The effect of metal ions on the activity of the endolysin was determined by incubating the enzyme with AlCl3, LiCl, MnCl2, KCl, MgSO4, CaCl2, BaCl2, ZnSO4, CoCl2, NaCl, CuCl2,NiSO4 and Al2(SO4)3 in HEPES buffer (50 mM, pH 7) at 40ºC. The new endolysin was characterized as an endopeptidase. The maximum activity of the bacteriophage endopeptidase was observed at pH and temperature 7.0 and 40°C, respectively. The recombinant endopeptidase was sensitive to an increase in surfactant concentration, and it was widely inactivated by 0.1% CTAB. Curiously, the activity of the endolysin was increased in the presence of AlCl3, LiCl, MnCl2 and Al2(SO4)3. The inhibition was noticed in the presence of CaCl2, BaCl2, ZnSO4, CoCl2, NaCl, CuCl2 and NiSO4. The recombinant endopeptidase might represent potential to be used for the development of antibacterial and antibiofilm therapies targeting, mainly, multidrug resistant strains. Palavras-chave: Endolysin, Bacteriophage, Biochemical characterization, Enzyme kinetics Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - (2018/09757-0; 2021/00510-5; 2022/01992-6). | |||||