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Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer

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Author: Groot, · Kosters, H.A. · Jongh,
Institution: TNO Kwaliteit van Leven
Source:Biotechnology and Bioengineering, 4, 97, 735-741
Identifier: 240053
doi: doi:10.1002/bit.21264
Keywords: Nutrition · Food technology · Circular dichroism · Deglycosylation · Ovalbumin · PNGase-F · S-ovalbumin · Size exclusion chromatography · Thermostability · Carbohydrate moiety · Deglycosylation · Ovalbumin · Thermostability · Carbohydrates · Differential scanning calorimetry · Heat treatment · pH effects · Reaction kinetics · Size exclusion chromatography · Thermodynamic stability · Proteins · glycopeptidase · ovalbumin · s ovalbumin · unclassified drug · article · circular dichroism · deglycosylation · differential scanning calorimetry · enzyme binding · gel permeation chromatography · mass spectrometry · protein conformation · protein denaturation · thermostability · Animals · Chickens · Chromatography, Gel · Circular Dichroism · Enzyme Stability · Glycosylation · Heat · Isoelectric Focusing · Mass Spectrometry · Molecular Weight · Ovalbumin · Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase · Protein Conformation · Spectrometry, Fluorescence


To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 ± 0.4°C). After heat treatment of the processed protein at 55°C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients. © 2006 Wiley Periodicals, Inc.