Substrate Specificity and Peptide Motif Preferences of β-Lytic and L5 Proteases from Lysobacter spp. Revealed by LC–MS/MS Analysis

β-Lytic protease (Blp) and protease L5 are enzymes from Lysobacter bacteria with distinct proteolytic and bacteriolytic activities. To characterize their substrate specificity, we employed liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis following hydrolysis of fractionated protein mixtures. Heatmaps and sequence logos revealed a pronounced specificity of Blp towards glycine and lysine residues, while L5 preferentially cleaved non-polar residues such as methionine, phenylalanine, and leucine. Notably, proline was frequently observed at the P2 position in L5 substrates. Comparative analysis with trypsin revealed that L5 generated significantly shorter peptides, whereas Blp produced fragments similar in length to tryptic peptides. These findings indicate different cleavage preferences and suggest potential applications for these enzymes in proteomic analysis.