Abstract
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 language | English |
---|---|
Article number | e01383 |
Number of pages | 17 |
Journal | Applied and Environmental Microbiology |
Volume | 83 |
Issue number | 20 |
Early online date | 2017 |
DOIs | |
Publication status | Published - 2017 |
MoE publication type | A1 Journal article-refereed |
Keywords
- carbohydrate
- CAZy AA5
- EC 1.1.3.-
- galactose oxidase
- NMR
- nuclear magnetic resonance