Characterisation of a 1,4-β-fucoside hydrolase degrading colanic acid

René Verhoef, Gerrit Beldman, Henk A. Schols, Matti Siika-aho, Marjaana Rättö, Johanna Buchert, Alphons G.J. Voragen (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

13 Citations (Scopus)

Abstract

A novel colanic acid-degrading enzyme was isolated from a mixed culture filtrate obtained by enrichment culturing of a compost sample using colanic acid as carbon source. The enzyme was partially purified resulting in a 17-fold increase in specific activity. Further purification by Native PAGE revealed that the enzyme is part of a high-molecular weight multi protein complex of at least six individual proteins. The enzyme showed a temperature optimum at 50 °C while after 5 h at 50 °C and pH 7 still 70% of the total activity was left. The pH optimum was found to be pH 7. Analysis of the degradation products showed that the enzyme is a novel 1,4-β-fucoside hydrolase that liberates repeating units of colanic acid with varying degrees of acetylation. Km and Vmax of the enzyme were determined against the native substrate as well as its de-O-acetylated and depyruvated forms. Compared to the native substrate the affinity of the enzyme for the modified substrates was much lower. However, for the de-O-acetylated sample a dramatic increase in catalytic efficiency was observed. The native form of the substrate showed substrate inhibition at high concentrations, probably due to the formation of nonproductive substrate complexes. Removal of the acetyl groups probably prevents this effect resulting in a higher catalytic efficiency.
Original languageEnglish
Pages (from-to)1780 - 1788
Number of pages9
JournalCarbohydrate Research
Volume340
Issue number11
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

Hydrolases
Enzymes
Substrates
Native Polyacrylamide Gel Electrophoresis
Acetylation
colanic acid
Purification
Proteins
Soil
Carbon
Molecular Weight
Molecular weight
Degradation
Temperature

Keywords

  • Colanic acid
  • Exopolysaccharide (EPS)
  • Enzymes
  • Fucoside hydrolase
  • Paper industry
  • Biofouling

Cite this

Verhoef, R., Beldman, G., Schols, H. A., Siika-aho, M., Rättö, M., Buchert, J., & Voragen, A. G. J. (2005). Characterisation of a 1,4-β-fucoside hydrolase degrading colanic acid. Carbohydrate Research, 340(11), 1780 - 1788. https://doi.org/10.1016/j.carres.2005.06.003
Verhoef, René ; Beldman, Gerrit ; Schols, Henk A. ; Siika-aho, Matti ; Rättö, Marjaana ; Buchert, Johanna ; Voragen, Alphons G.J. / Characterisation of a 1,4-β-fucoside hydrolase degrading colanic acid. In: Carbohydrate Research. 2005 ; Vol. 340, No. 11. pp. 1780 - 1788.
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Verhoef, R, Beldman, G, Schols, HA, Siika-aho, M, Rättö, M, Buchert, J & Voragen, AGJ 2005, 'Characterisation of a 1,4-β-fucoside hydrolase degrading colanic acid', Carbohydrate Research, vol. 340, no. 11, pp. 1780 - 1788. https://doi.org/10.1016/j.carres.2005.06.003

Characterisation of a 1,4-β-fucoside hydrolase degrading colanic acid. / Verhoef, René; Beldman, Gerrit; Schols, Henk A.; Siika-aho, Matti; Rättö, Marjaana; Buchert, Johanna; Voragen, Alphons G.J. (Corresponding Author).

In: Carbohydrate Research, Vol. 340, No. 11, 2005, p. 1780 - 1788.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Characterisation of a 1,4-β-fucoside hydrolase degrading colanic acid

AU - Verhoef, René

AU - Beldman, Gerrit

AU - Schols, Henk A.

AU - Siika-aho, Matti

AU - Rättö, Marjaana

AU - Buchert, Johanna

AU - Voragen, Alphons G.J.

PY - 2005

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N2 - A novel colanic acid-degrading enzyme was isolated from a mixed culture filtrate obtained by enrichment culturing of a compost sample using colanic acid as carbon source. The enzyme was partially purified resulting in a 17-fold increase in specific activity. Further purification by Native PAGE revealed that the enzyme is part of a high-molecular weight multi protein complex of at least six individual proteins. The enzyme showed a temperature optimum at 50 °C while after 5 h at 50 °C and pH 7 still 70% of the total activity was left. The pH optimum was found to be pH 7. Analysis of the degradation products showed that the enzyme is a novel 1,4-β-fucoside hydrolase that liberates repeating units of colanic acid with varying degrees of acetylation. Km and Vmax of the enzyme were determined against the native substrate as well as its de-O-acetylated and depyruvated forms. Compared to the native substrate the affinity of the enzyme for the modified substrates was much lower. However, for the de-O-acetylated sample a dramatic increase in catalytic efficiency was observed. The native form of the substrate showed substrate inhibition at high concentrations, probably due to the formation of nonproductive substrate complexes. Removal of the acetyl groups probably prevents this effect resulting in a higher catalytic efficiency.

AB - A novel colanic acid-degrading enzyme was isolated from a mixed culture filtrate obtained by enrichment culturing of a compost sample using colanic acid as carbon source. The enzyme was partially purified resulting in a 17-fold increase in specific activity. Further purification by Native PAGE revealed that the enzyme is part of a high-molecular weight multi protein complex of at least six individual proteins. The enzyme showed a temperature optimum at 50 °C while after 5 h at 50 °C and pH 7 still 70% of the total activity was left. The pH optimum was found to be pH 7. Analysis of the degradation products showed that the enzyme is a novel 1,4-β-fucoside hydrolase that liberates repeating units of colanic acid with varying degrees of acetylation. Km and Vmax of the enzyme were determined against the native substrate as well as its de-O-acetylated and depyruvated forms. Compared to the native substrate the affinity of the enzyme for the modified substrates was much lower. However, for the de-O-acetylated sample a dramatic increase in catalytic efficiency was observed. The native form of the substrate showed substrate inhibition at high concentrations, probably due to the formation of nonproductive substrate complexes. Removal of the acetyl groups probably prevents this effect resulting in a higher catalytic efficiency.

KW - Colanic acid

KW - Exopolysaccharide (EPS)

KW - Enzymes

KW - Fucoside hydrolase

KW - Paper industry

KW - Biofouling

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DO - 10.1016/j.carres.2005.06.003

M3 - Article

VL - 340

SP - 1780

EP - 1788

JO - Carbohydrate Research

JF - Carbohydrate Research

SN - 0008-6215

IS - 11

ER -