Construction and Evaluation of Peptide-Linked Lactobacillus brevis β-Galactosidase Heterodimers

Abstract

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. Method: 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.

Engineering Bifunctional Galactokinase/Uridyltransferase Chimera for Enhanced UDP-d-Xylose Production

“…Crucial to the efficacy of these chimeric proteins is the correct specification of the linker peptides connecting the enzyme units�their type, length, and amino acid composition being imperative factors in maintaining both activity and stability. 14,15 Nevertheless, to date, no single fusion protein or naturally occurring bifunctional enzyme has been described for the direct conversion of D-xylose to UDP-D-xylose (Figure 1c). In the present study, we combined two assembly orientations and four linker peptides with different lengths and compared the catalytic performance of the eight resulting chimeric fusion enzymes.…”

“…Such engineered fusion enzymes not only underscore their potential for large-scale production but also highlight their capacity for synthesizing analogs and establishing novel metabolic pathways to support glycosylation reactions. Crucial to the efficacy of these chimeric proteins is the correct specification of the linker peptides connecting the enzyme unitstheir type, length, and amino acid composition being imperative factors in maintaining both activity and stability. , …”

“…Crucial to the efficacy of these chimeric proteins is the correct specification of the linker peptides connecting the enzyme units—their type, length, and amino acid composition being imperative factors in maintaining both activity and stability. 14 , 15 …”