Applied Biochemistry and Biotechnology

, Volume 166, Issue 7, pp 1736–1746 | Cite as

Lysozyme Immobilized on Micro-Sized Magnetic Particles: Kinetic Parameters at Wine pH

  • Katia LiburdiEmail author
  • Raffaello Straniero
  • Ilaria Benucci
  • Anna Maria Vittoria Garzillo
  • Marco Esti


In order to use lysozyme as an anti-microbial agent during the winemaking process, hen egg-white lysozyme (LYZ) was covalently immobilized on two different micro-size magnetic particles (tosyl-activated and carboxylated, TSA and CA, respectively). A cell suspension of Oenococcus oeni, an oenological strain involved in the winemaking process, was utilized as LYZ substrate. Both a kinetic study and a study of the stability of free and immobilized LYZ were performed in McIlvane buffer at pH 3.2, that is the average minimum pH value in wine. The activity and kinetic parameters measured for the free LYZ at pH 3.2 are lower than those reported at the optimum pH (4.5); however the residual activity at pH 3.2 is sufficient to be of interest for further immobilization and applications in winemaking. All kinetic parameters of both biocatalysts (LYZ-CA and LYZ-TSA) are altered after immobilization, probably due to the structural modifications in the active site caused by covalent attachment to the supports. The half-life calculated at 25 °C was 39 h for free LYZ, while it increased to 280 and 134 h for LYZ-TSA and LYZ-CA, respectively. This result indicates that immobilization improves the enzyme stability and that LYZ can be utilized in wine applications in its immobilized forms. In addition, LYZ-TSA seems to be the best biocatalyst for further applications in winemaking.


Hen egg-white lysozyme Micro-size magnetic particles Oenococcus oeni Enzyme immobilization Kinetic parameters Stability 



Optical density at 360 nm


Enzyme units corresponding to 0.001/min OD360 nm decrease


Maximal initial enzyme activity


Michealis–Menten constant


Turnover number


Specificity constant (k cat/K M)


Specific activity of free lysozyme


Specific activity of immobilized lysozyme


Relative activity


Lysozyme activity measured at the t time


Initial lysozyme activity


Inactivation constant


Reaction time


Dry weight



The research was supported by financial backing of the Italian Ministry of Agriculture, Food and Forestry.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Katia Liburdi
    • 1
    Email author
  • Raffaello Straniero
    • 1
  • Ilaria Benucci
    • 1
  • Anna Maria Vittoria Garzillo
    • 2
  • Marco Esti
    • 1
  1. 1.Department for Innovation in Biological, Agro-food and Forest Systems (DIBAF)University of TusciaViterboItaly
  2. 2.Department of Ecology and Biology (DEB)University of TusciaViterboItaly

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