In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus

beta-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of beta-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the beta-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other beta-galactosidases of this species. The molecular model revealed that the new beta-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The beta-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme's temperature optimum at 55 degrees C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, beta-Gal produced a large amount of GOS with DP3 containing beta-(1 -> 3) and beta-(1 -> 4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 degrees C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of beta-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

Automated summary

Beta-galactosidase is an enzyme with dual activity that can eliminate lactose in milk and produce prebiotic galactooligosaccharides (GOS). This study characterized a beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus 43, which showed amino acid replacements compared to other enzymes of the species. The new enzyme acted as a tetramer and exhibited high expression efficiency when cloned into E. coli BL21(DE3). The enzyme had optimal activity at 55 degrees C and pH 6.5, and its activity was positively influenced by Mg2+, Mn2+, and Ca2+. It produced a large amount of GOS with beta-(1 -> 3) and beta-(1 -> 4) linkages, and showed good stability at 55 degrees C.