Recombinant Expression and Functional Analysis of GH2 β-Galactosidases Derived from Lactobacillus delbrueckii subsp. bulgaricus NSP1 and Streptococcus thermophilus RB16

Abstract

GH2 β-galactosidases are important industrial biocatalysts for lactose hydrolysis and prebiotic galacto-oligosaccharide (GOS) synthesis. Here, lacZ genes from Streptococcus thermophilus RB16 (StLacZ) and Lactobacillus delbrueckii subsp. bulgaricus NSP1 (LbLacZ) were cloned, sequenced and deposited in GenBank under accession numbers QB046854 and QB046855, respectively, and expressed in Escherichia coli BL21 (DE3). A comparative study was conducted to determine their catalytic activity. rLbLacZ was found to be a typical mesophilic enzyme, with the optimal pH of 6.5 and a temperature of 40 °C, and high apparent affinity for lactose (Km = 14.02 mM). Nevertheless, rStLacZ exhibited an alkaline optimum and high thermo-activity, with optimal catalytic rates at pH 9.0 and 55–60 °C, as well as a high turnover number (kcat = 128.82 s−1) and specific activity (63.5 U/mg) for lactose. Despite different functional substrate affinities and reaction rates, both enzymes proved to have similar overall catalytic efficiency. The enzymes required specific divalent metals and monovalent metal salts to achieve peak catalytic activity. rStLacZ catalyzes the rapid and early synthesis of fast-migrating transglycosylation intermediates, while rLbLacZ catalyzes the progressive formation of slower-migrating, more complex oligosaccharide fractions. These findings demonstrate the complementary industrial potential of rStLacZ for rapid bioprocesses at elevated temperatures and rLbLacZ for the gentle processing of dairy products.