Construction and Evaluation of Peptide-Linked β-Galactosidase Heterodimers.

BACKGROUND

β-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry.

OBJECTIVE

We describe the recombinant expression of an unstudied, heterodimeric β-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated.

METHODS

The heterodimeric subunits of the β-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits.

RESULTS

The co-expression in two independent expression vectors only resulted in low β-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the β-galactosidase activity significantly. The recombinant β-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-β-D-galactoside.

CONCLUSION

The usability of the recombinant L. brevis β-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused β-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.