title: Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323.
abstract: Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.
authors.affiliation: Institute of Medical Science, Tzu Chi University, Taiwan.
authors.forename: Chia-Ann
authors.initials: CA
authors.lastname: Yang
authors.forename: Chi-Hua
authors.initials: CH
authors.lastname: Cheng
authors.forename: Shu-Ying
authors.initials: SY
authors.lastname: Liu
authors.forename: Chaur-Tsuen
authors.initials: CT
authors.lastname: Lo
authors.forename: Jeng-Woei
authors.initials: JW
authors.lastname: Lee
authors.forename: Kou-Cheng
authors.initials: KC
authors.lastname: Peng
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000898:Antibiosis; D055162:Biocatalysis; D002463:Cell Membrane Permeability / Q000187:drug effects; D016384:Consensus Sequence; D053938:DNA Fragmentation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017382:Reactive Oxygen Species / Q000378:metabolism; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D050830:L-Amino Acid Oxidase