A new thermostable peroxidase from garlic Allium sativum: purification, biochemical properties, immobilization, and use in H2O2 detection in milk.
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.
Marzouki
Saida Medjeldi
SM
Unité de Génie Biologique, Institut National de Sciences Appliquées et de Technologie (INSAT), BP 676, 1080 Tunis Cedex, Tunisia.
Limam
Farid
F
Smaali
M Issam
MI
Ulber
Roland
R
Marzouki
M Néjib
MN
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*
D010940:Plant Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases