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A new thermostable peroxidase from garlic Allium sativum

Purification, biochemical properties, immobilization, and use in H2O2 detection in milk

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Abstract

Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40°C and optimum pH was about 5.0. The apparent K m values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50°C for 5h. Moreover POX2 was stable over a pH range of 3.5–11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives.

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Authors and Affiliations

  1. Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080, Tunis Cedex, Tunisia

    Saida Medjeldi Marzouki, M. Issam Smaali & M. Néjib Marzouki

  2. Laboratoire des Interactions Légumineuses-Microorganismes, Institut National de Recherche Scientifique et Technique (INRST), BP 95, 2050, Hammam Lif, Tunisia

    Farid Limam

  3. Institut für Technische Chemie Callinstrasse, 3 D-30167, Hannover, Germany

    Roland Ulber

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  1. Saida Medjeldi Marzouki
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  2. Farid Limam
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  3. M. Issam Smaali
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  4. Roland Ulber
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  5. M. Néjib Marzouki
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Correspondence to M. Néjib Marzouki.

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Marzouki, S.M., Limam, F., Smaali, M.I. et al. A new thermostable peroxidase from garlic Allium sativum . Appl Biochem Biotechnol 127, 201–214 (2005). https://doi.org/10.1385/ABAB:127:3:201

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  • DOI: https://doi.org/10.1385/ABAB:127:3:201

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