Premium
Aspergillus nidulans α‐galactosidase of glycoside hydrolase family 36 catalyses the formation of α‐galacto‐oligosaccharides by transglycosylation
Author(s) -
Nakai Hiroyuki,
Baumann Martin J.,
Petersen Bent O.,
Westphal Yvonne,
Hachem Maher Abou,
Dilokpimol Adiphol,
Duus Jens Ø.,
Schols Henk A.,
Svensson Birte
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07763.x
Subject(s) - xylobiose , melibiose , chemistry , monosaccharide , rhamnose , stereochemistry , galactose , biochemistry , xylan , polysaccharide , enzyme , maltose
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 m m 4‐nitrophenol α‐ d ‐galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4‐nitrophenol α‐ d ‐Gal p ‐(1→6)‐ d ‐Gal p , α‐ d ‐Gal p ‐(1→6)‐α‐ d ‐Gal p ‐(1→6)‐ d ‐Glc p and α‐ d ‐Gal p ‐(1→6)‐α‐ d ‐Gal p ‐(1→6)‐ d ‐Glc p ‐(α1→β2)‐ d ‐Fru f (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 m m ) and the donor 4‐nitrophenol α‐ d ‐galactopyranoside (40 m m ), 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 m m ), 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 ‐Gal p ‐(1→6)‐ d ‐Man p ; α‐ d ‐Gal p ‐(1→6)‐β‐ d ‐Glc p ‐(1→4)‐ d ‐Glc p ; α‐ d ‐Gal p ‐(1→6)‐β‐ d ‐Gal p ‐(1→4)‐ d ‐Fru f ; α‐ d ‐Gal p ‐(1→6)‐ d ‐Glc p ‐(α1→α1)‐ d ‐Glc p ; and α‐ d ‐Gal p ‐(1→6)‐α‐ d ‐Glc p ‐(1→3)‐ d ‐Fru f .