Enhanced production, activity and deregulation of β-galactosidase in a new mutant strain Bifidobacterium longum BIM B-813 D: Characterization and industrial potential

Abstract

This study aimed to engineer a Bifidobacterium longum strain with enhanced production of the industrially relevant enzyme β-galactosidase. Initial screening of 12 bifidobacterial strains identified B. longum Cf as a high-activity candidate (340 Miller units), though its enzyme synthesis was strongly suppressed by glucose (98%). The parent strain's specific growth rate was 0.36 h−1, with a maximum β-galactosidase synthesis rate of 0.11 U•mg−1•h−1. Chemical mutagenesis with ethyl methanesulfonate and subsequent selection on glucose medium containing X-Gal yielded a deregulated mutant, B. longum BIM B-813 D. The mutant demonstrated constitutive β-galactosidase synthesis, exhibiting a 6.5–7-fold increase in specific activity compared to the parent strain, achieving 2500 Miller units. Enzyme production in the mutant was no longer inhibited by glucose, indicating a fundamental change in genetic regulation of the β-galactosidase operon. Whole-genome sequencing of BIM B-813 D revealed a single circular chromosome containing the β-galactosidase-related genes bgal_small_N, lacZ1, bgaB1, bgaB2, bgaB3 and lacZ2, and an ISL3 insertion sequence located in the regulatory region of lacZ1, which likely underlies the altered expression control. Analyses showed that the strain retains a typical B. longum growth profile, produces at least three intracellular β-galactosidase isoforms under both glucose and lactose conditions, and exhibits a broad pH (5.0–7.0) and temperature (up to 50 °C, with residual activity at 4 °C) activity range. The developed strain represents a promising food-grade biocatalyst for the efficient production of β-galactosidase, with direct applications in the manufacture of low-lactose dairy products and prebiotic galactooligosaccharides.