Sugars as Acceptors for the Transferase Activity of Beta-galactosidases

ß-Galactosidases (b-gal; EC 3.2.1.23) are found widespread in nature. They catalyze the hydrolysis of lactose or related compounds in hydrolysis mode. An attractive biocatalytic application is found in the transgalactosylation potential of these enzymes, which is based on the catalytic mechanism of ß-galactosidase. Galacto-oligosaccharides (GOS) are the products of transgalactosylation reactions when using lactose or other structurally related galactosides as the substrate. ß-Galactosidases have also been used to produce hetero-oligosaccharides (HOS). Mannose, fructose, N-acetylneuraminic acid, glucuronic acid and a number of aromatic compounds have been shown to act as galactosyl acceptor for ß-galactosidases. The choice of suitable acceptor and enzyme allows the formation of tailor-made HOS of high interest for applications in the food industry. The proposed research project aims at studying in detail ß-galactosidases from probiotic strains of lactic acid bacteria and bifidobacteria, namely Bifidobacterium longum biovar infantis ATCC 15697, B. breve ATCC 15700, Lactobacillus reuteri (from our culture collection), Lactobacillus bulgaricus ATCC 11842 and Streptococcus thermophilus DSM 20259, with respect to their propensity to transfer galactosyl moieties onto certain sugar acceptors. These results will be valuable to obtain novel HOS with potentially extended functionality in addition to GOS. Some of the newly formed sugar structures are expected to resemble human milk oligosaccharides (HMO) structures, which are of great interest as HMO are known to possess several biological functions including as prebiotics for stimulating the growth of intestinal bacteria associated with health/well-being, as a mechanism to protect infants against exogenous infections, and as receptor analogues to block the infection of pathogenic bacteria. The main scientific objective is to deepen the understanding of the relative extent of galactosyl transfer of a specific ß-galactosidase to its substrate (lactose), to the primary reaction products glucose and galactose and to other galactosyl acceptors such as N-acetylglucosamine, N-acetylgalactosamine and L-fucose, thus the prediction of product formation can be made. To accomplish the objective of the project, the following tasks are envisaged: (i) cloning and expression of the selected ß-galactosidases for the overproduction of recombinant enzymes, (ii) characterization of the successfully overexpressed recombinant ß-galactosidases pertaining to properties that are relevant to the application of the enzymes in biocatalytic processes, (iii) detailed investigations of the transgalactosylation activity and the analysis of intermolecular galactosyl transfer of the selected enzymes to the substrate and the test galactosyl acceptors under defined, initial-velocity conditions, (iv) analysis of the novel oligosaccharides formed using capillary electrophoresis (CE), high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as NMR.

Certain oligosaccharides are considered to be beneficial for human and animal hosts due to their ability to stimulate selectively growth and/or activity of one or a limited number of bacteria in the colon. They are classified as prebiotics, new functional food ingredients that are of considerable interest. The prebiotic compounds are typically oligosaccharides of various compositions, and galacto-oligosaccharides (GOS) are non-digestible carbohydrates meeting the criteria of prebiotics. GOS are of special interest to human nutrition because of the presence of structurally related oligosaccharides together with different complex structures in human breast milk. Furthermore, galactose-containing hetero-oligosaccharides (HOS) have attracted an increasing amount of attention because they are structurally more closely related to human milk oligosaccharides (HMO). GOS are the products of transgalactosylation reactions catalyzed by ?-galactosidases when using lactose or other structurally related galactosides as the substrate. ?-Galactosidases have also been used to produce HOS. Mannose, fructose, N-acetylneuraminic acid, glucuronic acid and a number of aromatic compounds have been shown to act as galactosyl acceptor for ?-galactosidases. The choice of suitable acceptor and enzyme allows the formation of tailor-made HOS of high interest for applications in the food industry.In this project we investigated the detailed kinetics of various ?-galactosidases from lactobacilli and bifidobacteria pertaining to this transferase reaction onto different galactosyl acceptors. We have quantified this transferase ability, and used this quantification to select an appropriate enzyme for the biocatalytic conversion of lactose and N-acetyl-D- glucosamine, or N-acetyl-D-galactosamine, or L-fucose to novel hetero-oligosaccharides, which might be of interest because of its potentially extended functionality in addition to GOS. Although the feasibility of the transferase reaction onto different galactosyl acceptors has been shown previously, no study had dealt with a more detailed biochemical characterization of this reaction. The main scientific significance of this work was to deepen the understanding of the relative extent of galactosyl transfer of a specific ?-galactosidase to these alternative sugar acceptors as well as to its substrate lactose and its primary products D-glucose and D-galactose. Thus a prediction of product formation can be made for the transferase reaction from a given sugar composition. The insights into the structures and the production of GOS and HOS certainly results in the enhancement of the production of these novel oligosaccharides.