An acid-tolerant and cold-active β-galactosidase potentially suitable to process milk and whey samples.

A novel β-galactosidase gene (gal) was cloned from an aquatic habitat metagenome. The analysis of its translated sequence (Gal) revealed its phylogenetic closeness towards Verrucomicrobia sp. The sequence comparison and homology structure analysis designated it a member of GH42 family. The three-dimensional homology model of Gal depicted a typical (β/α)8 TIM-barrel containing the catalytic core. The gene (gal) was expressed in a heterologous host, Escherichia coli, and the purified protein (Gal) was subjected to biochemical characterization. It displayed β-galactosidase activity in a wide range of pH (2.0 to 9.0) and temperature (4 to 60 °C). The heat exposed protein showed considerable stability at 40 and 50 °C, with the half-life of about 100 h and 35 h, respectively. The presence of Na, Mg, K, Ca, and Mn metals was favorable to the catalytic efficiency of Gal, which is a desirable catalytic feature, as these metals exist in milk. It showed remarkable tolerance of glucose and galactose in the reaction. Furthermore, Gal discerned transglycosylation activity that is useful in galacto-oligosaccharides' production. These biochemical properties specify the suitability of this biocatalyst for milk and whey processing applications. KEY POINTS: • A novel β-galactosidase gene was identified and characterized from an aquatic habitat. • It was active in extreme acidic to mild alkaline pH and at cold to moderate temperatures. • The β-galactosidase was capable to hydrolyze lactose in milk and whey.