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Catalase Stability in Amorphous and Supercooled Media Related to Trehalose- Water- Salt Interactions

  • Patricio R. Santagapita
  • M. Florencia Mazzobre
  • M. Pilar BueraEmail author
ORIGINAL ARTICLE
  • 31 Downloads

Abstract

Sugars are the most common excipients added to pharmaceutical and biotechnological formulations as protein protectants due to their adequate physicochemical properties, low toxicity, high purity and low cost. However, a second excipient is generally required to extend their protection in supercooled media. The effect of electrolytes is of special interest because of their universal presence in biological systems, their major influence on water structure, and their capability to interact with biomolecules. The purpose of the present work was to analyze the effect of different salts on the stability of catalase in amorphous (glassy and supercooled) trehalose matrices. Trehalose-Mg2+ system was better than trehalose alone to protect the enzyme both during freeze-drying and later storage at low RH (22%). The stabilizing effect observed for certain salts in these systems was not related with an increase of the Tg value. Under conditions at which trehalose crystallizes (43 %RH), salts (especially Mg2+) were detrimental since the enzyme became confined in a salt-concentrated region. Protein denaturation and aggregation were analyzed through differential scanning calorimetry in order to correlate activity changes with physical changes. Trehalose systems without salt and Mg2+-containing systems showed almost no aggregation after denaturation, in agreement with the thermal stability of the enzyme. Thus, the two major parameters related to enzyme stability in freeze-dried non-crystallized systems are: enzyme characteristics (type, quaternary structure) and salt-protein specific interactions.

Keywords

Enzyme stability Trehalose Salts Crystallization Freeze-drying Catalase 

Notes

Acknowledgments

The authors acknowledge the financial support of ANPCYT (PICT 2012-3070; PICT 2013-1331), CIN-CONICET (PDTS 2015 n° 196) and UBA (Project UBACyT 20020170100459BA). All authors are members of CONICET.

Compliance with Ethical Standards

Conflict of Interest

none.

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Facultad de Ciencias Exactas y Naturales, Departamentos de Industrias y Química Orgánica & CONICET-Universidad de Buenos Aires, Instituto de Tecnología de Alimentos y Procesos Químicos (ITAPROQ)Universidad de Buenos AiresCiudad Autónoma de Buenos AiresArgentina

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