A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing b-glucose 1-phosphate (b-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and b-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di-and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm b-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be a-Glcp-(1 fi 4)-Glcp (maltose), a-Glcp-(1 fi 4)-GlcNp (maltosamine), a-Glcp-(1 fi 4)-GlcNAcp (N-acetyl maltosamine), a-Glcp-(1 fi 4)-Manp, a-Glcp-(1 fi 4)-Xylp and a-Glcp-(1 fi 4)-l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.Abbreviations ABC, ATP-binding cassette; GH, glycoside hydrolase family; HPAEC-PAD, high-performance ion-exchange chromatography equipped with a pulsed amperometric detector; MalE, maltodextrin-binding protein; MalF and MalG, maltodextrin ABC transport permease proteins; MalL, oligo-1,6-glucosidase; MalN, neopullulanase; MalP, Lactobacillus acidophilus NCFM maltose phosphorylase; MalR, transcriptional regulator of the LacI-GalR family; MsmK, maltodextrin import ATP-binding protein; b-Glc 1-P, b-glucose 1-phosphate.

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The a-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic a-galactosidases and a-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 gAEL )1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with a-(1 fi 6) regioselectivity from 40 mm 4-nitrophenol a-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenolrespectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol a-d-galactopyranoside (40 mm), a-(1 fi 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 a-galactosidase as the template and superimposition of melibiose from the complex with human GH27 a-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 a-galactosyl to 6-OH of the terminal residue in the a-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the b-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,Abbreviations AglC, a-galactosidase from Aspergillus nidulans; GalA, a-galactosidase from Thermotoga maritima; GH, glycoside hydrolase family; HPAEC-PAD, high-performance anion-exchange chromatography equipped with pulsed amperometric

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