A novel Colletotrichum graminicola raffinose oxidase in the AA5 family

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

Research output: Contribution to journalArticleScientificpeer-review

4 Citations (Scopus)

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 languageEnglish
Article numbere01383
Number of pages17
JournalApplied and Environmental Microbiology
Volume83
Issue number20
Early online date2017
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Fingerprint

Glomerella graminicola
Colletotrichum
Raffinose
raffinose
substrate preference
Oxidoreductases
Galactose Oxidase
galactose
Uronic Acids
uronic acids
mass spectrometry
disaccharide
bioinformatics
acid
Melibiose
activity pattern
formation mechanism
aldehyde
melibiose
trisaccharides

Keywords

  • carbohydrate
  • CAZy AA5
  • EC 1.1.3.-
  • galactose oxidase
  • NMR
  • nuclear magnetic resonance

Cite this

Blomster Andberg, Martina ; Mollerup, Filip ; Parikka, Kirsti ; Koutaniemi, Sanna ; Boer, Harry ; Juvonen, Minna ; Master, Emma ; Tenkanen, Maija ; Kruus, Kristiina. / A novel Colletotrichum graminicola raffinose oxidase in the AA5 family. In: Applied and Environmental Microbiology. 2017 ; Vol. 83, No. 20.
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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.",
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author = "{Blomster Andberg}, Martina and Filip Mollerup and Kirsti Parikka and Sanna Koutaniemi and Harry Boer and Minna Juvonen and Emma Master and Maija Tenkanen and Kristiina Kruus",
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language = "English",
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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 journalArticleScientificpeer-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

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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 -