Unravelling the carbohydrate specificity of MelA from Lactobacillus plantarum WCFS1: An α-galactosidase displaying regioselective transgalactosylation

Biosynthesis of Nondigestible Galactose-Containing Hetero-oligosaccharides by Lactobacillus plantarum WCFS1 MelA α-Galactosidase

“…The efficient hydrolytic and transgalactosylation activities of MelA toward single melibiose (Galα(1 → 6)-Glc) acting as both the donor and acceptor have been recently demonstrated. 17 In the current study, transgalactosylation activity of the MelA protein was studied using starting equimolar binary mixtures of melibiose and several carbohydrates with different glycosidic linkages. In the case of fucose, only α-GOS derived from melibiose acting both as the donor and acceptor was found throughout the enzymatic reaction, whereas HOS derived from fucose could not be detected, thereby revealing its inability to act as the galactosyl acceptor under the studied conditions (data not shown).…”

“…MelA α-galactosidase from L. plantarum WCFS1 was overproduced in Escherichia coli and purified as previously indicated. 17 The total activity of MelA was determined as the amount of released p-nitrophenolate (pNP) from pnitrophenyl-α-D-galactopyranoside at 420 nm using an extinction coefficient of ε = 9310 M −1 cm −1 in a microplate spectrophotometer PowerWave HT (Bio-Tek, USA). MelA expressed an α-galactosidase activity of 624 units per milligram (U mg −1 ), where one unit (U) is defined as the amount of MelA required to release 1 μmol of pNP per minute in 10 mM of p-NP-galactopyranoside, at 30 °C in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT.…”

“…Transgalactosylation reactions were carried out using binary mixtures of melibiose (donor): fucose, lactose, lactulose, cellobiose, isomaltose, or isomaltulose (acceptors) (20:20, respectively, expressed in g 100 mL −1 ) and 59.3 U of MelA mL −1 of solution. 17 Reactions were carried out in triplicate (n = 3) at 22 °C in 50 mM of MOPS buffer with 20 mM of NaCl at pH 7 and monitored by GC-FID after taking aliquots at time intervals of 0, 2, 8, 24, 32, and 48 h. The enzymatic reactions were stopped by heating at 100 °C for 5 min.…”

“…24,25 A recent study has addressed the biochemical characterization and carbohydrate specificity of MelA, a cold-active α-galactosidase from L. plantarum WCFS1 belonging to the glycoside hydrolase (GH) family 36, demonstrating a unique preference for the transfer of galactosyl residues from melibiose, RFOS, or nonfructosylated α-GOS to the C6-hydroxyl group of galactose units to elongate the chain of the acceptor substrates. 17 Despite this great diversity in accommodating specific α-GOS compounds acting both as donor and acceptor substrates, studies about acceptor specificity of MelA from L. plantarum WCFS1 using melibiose as a galactosyl donor and a variety of potentially suitable acceptors are scarce 18 and their resistance to intestinal digestion and potential prebiotic effect have never been studied. This approach could be a promising strategy for obtaining novel tailored HOS of high interest and added value for potential applications in the food and/or nutraceutical sectors.…”

Hydrolysis of Lactose and Transglycosylation of Selected Sugar Alcohols by LacA β-Galactosidase from Lactobacillus plantarum WCFS1

“…plantarum strain whose genome was totally sequenced , and is one of the best-studied lactobacilli due to its high genetic variation in genes involved in hydrolysis, synthesis, or modifications of carbohydrates. , Diverse glycosidase activities have been described in L. plantarum, such as α- and β-glucosidase, , α- − and β-galactosidase, β-fructofuranosidase, or α-rhamnosidase …”