Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity

Adiphol Dilokpimol, Hiroyuki Nakai, Charlotte H. Gotfredsen, Martin J. Baumann, Natsuko Nakai, Maher Abou Hachem, Birte Svensson (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review


The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (kcat/KM) towards β-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer β-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-β-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.
Original languageEnglish
Pages (from-to)1720-1729
JournalBiochimica et Biophysica Acta
Issue number12
Publication statusPublished - Dec 2011
MoE publication typeA1 Journal article-refereed


  • Aspergillus nidulans/chemistry
  • Carbohydrate Sequence
  • Cloning, Molecular
  • Glycosylation
  • Hydrolysis
  • Isoenzymes/chemistry
  • Mannosidases/chemistry
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligosaccharides/metabolism
  • Protein Conformation
  • Recombinant Proteins/chemistry
  • Substrate Specificity


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