The purpose of this study was to evaluate the inhibitory mechanism of cell-free supernatants of Lactobacillus plantarum (LCFS) on Proteus mirabilis and estimate the expression of histamine synthesis-related genes. The minimum inhibitory concentration (MIC) of the LCFS and antibacterial curve assay was determined by the tube double-dilution method. The antibacterial mechanism was explored by cell integrity, morphology, and structure of P. mirabilis . Further, the expression of the histidine decarboxylase ( hdcA ) gene and histidine/histamine antiporter ( hdcP ) gene of P. mirabilis and the intracellular and extracellular contents of histidine and histamine were determined. The MIC of the LCFS was 12.5 mg/mL, and the antibacterial curve assay showed an inhibited growth of P. mirabilis . The increase in the Alkaline Phosphatase (AKP) content, membrane permeability, and potential indicated the destruction of the cytoplasm, cell membrane, and cell wall of P. mirabilis . The results of SEM and TEM revealed that the LCFS disrupted the normal morphology and structure of the cell. Moreover, differences in the extracellular and intracellular histidine and histamine content between the MIC group and the control group was consistent with the decreased expression of hdcA and hdcP genes in the MIC group observed in the previous antimicrobial studies. Hence, the inhibitory mechanism of LCFS on histamine formation was clarified at cellular and molecular levels.
l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination ofl-amino acids into α-keto acids, which are widely used in the pharmaceutical, food, chemical, and cosmetic industries. However, the substrate specificity of available LAADs is limited, and most substrates are concentrated on several bulky or basicl-amino acids. In this study, we employed a LAAD fromProteus mirabilis(PmiLAAD) and broadened its substrate specificity using a semi-rational design strategy. Molecular docking and alanine scanning identified F96, Q278, and E417 as key residues around the substrate-binding pocket of PmiLAAD. Site-directed saturation mutagenesis identified E417 as the key site for substrate specificity expansion. Expansion of the substrate channel with mutations of E417 (E417L, E417A) improved activity toward the bulky substratel-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward variousl-amino acid substrates. The variant PmiLAADE417Kshowed remarkable catalytic activity improvement on seven substrates (l-Ala,l-Asp,l-Ile,l-Leu,l-Phe,l-Trp, andl-Val). The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions. These PmiLAAD variants with a broadened substrate spectrum can extend the application potential of LAADs.Keywords:l-amino deaminase;amino acids;keto acids;substrate spectrum;substrate channel
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