A new thermostable peroxidase from garlic Allium sativum: Purification, biochemical properties, immobilization, and use in H₂O₂ detection in milk

Authors: Marzouki, Saida¹; Limam, Farid²; Smaali, M.¹; Ulber, Roland³; Marzouki, M.⁴

Source: Applied Biochemistry and Biotechnology, Volume 127, Number 3, December 2005 , pp. 201-214(14)

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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.

Keywords: Plant peroxidases; garlic; protein purification; immobilization; kinetic characterization

Document Type: Research article

DOI: http://dx.doi.org/10.1385/ABAB:127:3:201

Affiliations: 1: Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080, Tunis Cedex, Tunisia, 2: Laboratoire des Interactions Légumineuses-Microorganismes, Institut National de Recherche Scientifique et Technique (INRST), BP 95, 2050, Hammam Lif, Tunisia, 3: Institut für Technische Chemie Callinstrasse, 3 D-30167, Hannover, Germany, 4: Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080, Tunis Cedex, Tunisia, Email: mn.marzouki@insat.rnu.tn

Publication date: 2005-12-01