Peroxidase-mediated cross-linking of bovine a-lactalbumin

W.H. Heijnis

Research output: Thesisinternal PhD, WU


The research presented in this thesis aimed at controlling the horseradish peroxidase-catalyzed cross-linking of bovine α lactalbumin and the implications of this cross-linking for the foam stabilizing properties. Attention is also given to microreactors and their potential to control the enzymatic cross-linking of proteins.
The proportion of cross-linked α lactalbumin dimers, oligomers and polymers could be directed by variations in ionic strength, pH, H2O2, and temperature.
Covalent α lactalbumin dimers were proteolytic digested. FTMS analysis of the peptide mixture resulted in the unambiguous identification of a Tyr18 Tyr50 dityrosine cross-link. Structural modeling of the α lactalbumin dimer indicated that favorite electrostatics direct the selectivity of the cross-linking reaction and, hence, the formation of an intermolecular cross-link. The formation of the Tyr18-Tyr50 cross-link suggests that further cross-linking of α lactalbumin dimers enables the formation of linear polymers.
A microreactor system was set up to obtain control over the reaction conditions to cross-link proteins. The enzymatic cross-linking of α lactalbumin was analyzed as a function of enzyme and substrate(s) feed. The increase in absorption at 318 nm due to dityrosine formation was found to be directly correlated to the decrease in monomeric α lactalbumin and was shown to be a good tool to monitor the cross-linking reaction.
The α lactalbumin oligomers produced were investigated for their foam stabilizing properties. Cross-linked α lactalbumin oligomers did not stabilize foams, whereas α lactalbumin polymers acted as an anti-foam, destabilizing other protein films.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Gruppen, Harry, Promotor
  • van Berkel, Willem, Co-promotor
  • Wierenga, Peter, Co-promotor
Award date10 Dec 2010
Place of PublicationS.l.
Print ISBNs9789085858324
Publication statusPublished - 10 Dec 2010


  • alpha-lactalbumin
  • peroxidase
  • foaming
  • foams
  • enzymatic cross-linking


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