Abstract
| 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 |
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Keywords
- carbohydrate
- CAZy AA5
- EC 1.1.3.-
- galactose oxidase
- NMR
- nuclear magnetic resonance
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A novel Colletotrichum graminicola raffinose oxidase in the AA5 family. / Blomster Andberg, Martina; Mollerup, Filip; Parikka, Kirsti; Koutaniemi, Sanna; Boer, Harry; Juvonen, Minna; Master, Emma; Tenkanen, Maija; Kruus, Kristiina.
In: Applied and Environmental Microbiology, Vol. 83, No. 20, e01383, 2017.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - A novel Colletotrichum graminicola raffinose oxidase in the AA5 family
AU - Blomster Andberg, Martina
AU - Mollerup, Filip
AU - Parikka, Kirsti
AU - Koutaniemi, Sanna
AU - Boer, Harry
AU - Juvonen, Minna
AU - Master, Emma
AU - Tenkanen, Maija
AU - Kruus, Kristiina
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - carbohydrate
KW - CAZy AA5
KW - EC 1.1.3.-
KW - galactose oxidase
KW - NMR
KW - nuclear magnetic resonance
UR - http://www.scopus.com/inward/record.url?scp=85030697826&partnerID=8YFLogxK
U2 - 10.1128/AEM.01383-17
DO - 10.1128/AEM.01383-17
M3 - Article
VL - 83
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 20
M1 - e01383
ER -