Characterisation of a 1,4-beta-fucoside hydrolase degrading colanic acid.
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 degrees C while after 5h at 50 degrees C and pH7 still 70% of the total activity was left. The pH optimum was found to be pH7. Analysis of the degradation products showed that the enzyme is a novel 1,4-beta-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.
Verhoef
René
R
Wageningen University, Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Bomenweg 2, 6703 HD Wageningen, The Netherlands.
Beldman
Gerrit
G
Schols
Henk A
HA
Siika-aho
Matti
M
Rättö
Marjaana
M
Buchert
Johanna
J
Voragen
Alphons G J
AG
D000838:Anions; D002236:Carbohydrate Conformation; D002384:Catalysis; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D011134:Polysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D005644:alpha-L-Fucosidase / Q000737:chemistry*
D000838:Anions; D011134:Polysaccharides; C004275:colanic acid; D006026:Glycoside Hydrolases; C028219:beta-D-fucosidase; D005644:alpha-L-Fucosidase