A novel Colletotrichum graminicola raffinose oxidase in the AA5 family

Martina Andberg*, Filip Mollerup, Kirsti Parikka, Sanna Koutaniemi, Harry Boer, Minna Juvonen, Emma Master, Maija Tenkanen, Kristiina Kruus

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

27 Citations (Scopus)
129 Downloads (Pure)


We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on D-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family.

Original languageEnglish
Article numbere01383-17
JournalApplied and Environmental Microbiology
Issue number20
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed


  • Carbohydrate
  • CAZy AA5
  • EC 1.1.3.-
  • Galactose oxidase
  • NMR
  • Nuclear magnetic resonance


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