title: A new thermostable peroxidase from garlic Allium sativum: purification, biochemical properties, immobilization, and use in H2O2 detection in milk.
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 degrees C and optimum pH was about 5.0. The apparent Km 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 degrees C for 5 h. 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.
authors.affiliation: Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080 Tunis Cedex, Tunisia.
authors.forename: Saida Medjeldi
authors.initials: SM
authors.lastname: Marzouki
authors.forename: Farid
authors.initials: F
authors.lastname: Limam
authors.forename: M Issam
authors.initials: MI
authors.lastname: Smaali
authors.forename: Roland
authors.initials: R
authors.lastname: Ulber
authors.forename: M Néjib
authors.initials: MN
authors.lastname: Marzouki
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D005737:Garlic / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000032:analysis; D008892:Milk / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry* / Q000302:isolation & purification*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D018517:Plant Roots / Q000201:enzymology*
chemical_list: D010940:Plant Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases