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The -galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic -galactosidases and -galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L-1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with -(1¿6) regioselectivity from 40 mm 4-nitrophenol -d-galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4-nitrophenol -d-Galp-(1¿6)-d-Galp, -d-Galp-(1¿6)--d-Galp-(1¿6)-d-Glcp and -d-Galp-(1¿6)--d-Galp-(1¿6)-d-Glcp-(1¿ß2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol -d-galactopyranoside (40 mm), -(1¿6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39–58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 -galactosidase as the template and superimposition of melibiose from the complex with human GH27 -galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred -galactosyl to 6-OH of the terminal residue in the -linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the ß-linked lactose, lactulose and cellobiose in 28–38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. -d-Galp-(1¿6)-d-Manp; -d-Galp-(1¿6)-ß-d-Glcp-(1¿4)-d-Glcp; -d-Galp-(1¿6)-ß-d-Galp-(1¿4)-d-Fruf; -d-Galp-(1¿6)-d-Glcp-(1¿1)-d-Glcp; and -d-Galp-(1¿6)--d-Glcp-(1¿3)-d-Fruf.