Aspergillus nidulansα‐galactosidase of glycoside hydrolase family 36 catalyses the formation of α‐galacto‐oligosaccharides by transglycosylation

Abstract

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

Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

“…Tetrameric assemblies based on the structure of AgaSK could be constructed for Aga1 and Aga2 and revealed that the active site pocket, similarly to that of Lactobacillus brevis and L. acidophilus NCFM ␣-galactosidases (25), is forged by the tight association of adjacent monomers, suggesting cooperation between subunits, although this needs to be further confirmed. Family GH36 members carry out hydrolysis with a net retention of the anomeric configuration, which is why enzymes from this family have been shown to possess transglycosylation activity (38). This property is essential for the synthesis of oligosaccharide structures which cannot be synthesized by classical chemistry (51).…”

α-Galactosidase/Sucrose Kinase (AgaSK), a Novel Bifunctional Enzyme from the Human Microbiome Coupling Galactosidase and Kinase Activities

“…Family GH36 members originate from bacteria, fungi, and plants and carry out hydrolysis with a net retention of the anomeric configuration, which is why many enzymes from this family have been shown to possess transglycosylation activity (21)(22)(23)(24)(25)(26)(27). For their ability to hydrolyze ␣-galactosides non-digestible by humans and to synthesize diverse oligosaccharides, GH36 ␣-galactosidases have potential applications for the production of prebiotics.…”

High-level expression of a novel α-galactosidase gene from Rhizomucor miehei in Pichia pastoris and characterization of the recombinant enyzme

“…Many microorganisms, plants, and animals produce a-galactosidases with multiple subunits [13,27]. Most of the GH family 36 a-galactosidases exist as tetramers with subunit molecular mass of $80 kDa [1,12,14,15]. In this study, RmgalB had a relative molecular mass of 83.1 kDa in SDS-PAGE and a native molecular mass of 317.7 kDa (Fig.…”

“…Many a-galactosidases from fungi such as Bispora sp. [18], Clostridium josui [26] and Neosartorya fischeri [27] belong to GH family 27 while several a-galactosidases from fungi such as R. miehei [15], Aspergillus nidulans [14], Gibberilla sp. [13], Absidia corymbifera [12] belong to GH family 36.…”

“…Due to this, there is an increased interest in producing the enzyme by recombinant methods. Although many a-galactosidase genes have been cloned from mesophilic fungi [4,[12][13][14], there are few reports on the a-galactosidase genes from the thermophilic fungi such as T. lanuginosus and Rhizomucor miehei [14,15]. The methylotrophic yeast Pichia pastoris has been increasingly exploited for protein production due to the various advantages [16].…”