Journal article
Aspergillus nidulans α-galactosidase of glycoside hydrolase family 36 catalyses the formation of α-galacto-oligosaccharides by transglycosylation : Transglycosylation by A. nidulansα-galactosidase
The α-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic α-galactosidases and α-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 α-(1→6) regioselectivity from 40 mm 4-nitrophenol α-d-galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4-nitrophenol α-d-Galp-(1→6)-d-Galp, α-d-Galp-(1→6)-α-d-Galp-(1→6)-d-Glcp and α-d-Galp-(1→6)-α-d-Galp-(1→6)-d-Glcp-(α1→β2)-d-Fruf (stachyose), respectively.
Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol α-d-galactopyranoside (40 mm), α-(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 α-galactosidase as the template and superimposition of melibiose from the complex with human GH27 α-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 α-galactosyl to 6-OH of the terminal residue in the α-linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the β-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. α-d-Galp-(1→6)-d-Manp; α-d-Galp-(1→6)-β-d-Glcp-(1→4)-d-Glcp; α-d-Galp-(1→6)-β-d-Galp-(1→4)-d-Fruf; α-d-Galp-(1→6)-d-Glcp-(α1→α1)-d-Glcp; and α-d-Galp-(1→6)-α-d-Glcp-(1→3)-d-Fruf.
Language: | English |
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Publisher: | Wiley |
Year: | 2010 |
Pages: | 3538-3551 |
ISSN: | 17424658 , 1742464x , 14321033 and 00142956 |
Types: | Journal article |
DOI: | 10.1111/j.1742-4658.2010.07763.x |
ORCIDs: | Abou Hachem, Maher , Duus, Jens Øllgaard and Svensson, Birte |
acceptor specificity carbohydrate structural analysis transglycosylation α-galacto-oligosaccharides α-galactosidase
Amino Acid Sequence Aspergillus nidulans Biocatalysis Carbohydrate Conformation Cloning, Molecular Escherichia coli Glycosylation Hydrogen-Ion Concentration Hydrolysis Kinetics Models, Molecular Oligosaccharides Phylogeny Recombinant Proteins Temperature a-galacto-oligosaccharides a-galactosidase acceptor specificity alpha-Galactosidase