Subcellular Localization of β-Galactosidase Protein from Probiotic Limosilactobacillus fermentum LF08 Strain: Probability of Cell Wall Association

Lactic acid bacteria exhibit high adaptability to their environment due to their wide variety of enzymes. Despite extensive knowledge of bacterial cell walls, the subcellular localization of β-galactosidase in many probiotic lactic acid bacteria, including Limosilactobacillus fermentum, remains unclear. Determining the cellular localization of such enzymes may improve insight into bacterial metabolic mechanisms and support the development of efficient downstream processes, as well as applications. In this study, three cell disruption strategies (mechanical homogenization and chemical disruption with different agents) were applied to assess the subcellular localization of β-galactosidase from the Ll. fermentum LF08 strain. Enzyme activity was measured in a ferment broth, a supernatant and cell-associated fractions. No and very low β-galactosidase activity was detected in the ferment broth and the supernatant, respectively, when either chemical or mechanical treatment was applied, whereas the main enzyme activity was assayed in the cell suspension fraction. Combined lysozyme and CTAB treatment resulted in a 21.4-fold increase in β-galactosidase activity in the supernatant fraction (2.14 U/mL), compared with CTAB treatment alone (0.10 U/mL). Bioinformatic analyses provided additional significant information to propose the potential cell wall association (maybe the outer side of the cell wall) of the subcellular localization of β-galactosidase. This feature may support understanding of the interactions between probiotic bacteria and host tissues, as well as the development of probiotic immobilized cell systems for applications such as the elimination of lactose, designing novel functional foods.