Isolation and thermal characterization of an acidic isoperoxidase from turnip roots.
An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.
Duarte-Vázquez
Miguel A
MA
Departamento de Investigación y Posgrado en Alimentos, PROPAC, Facultad de Química, Universidad Autónoma de Querétaro, C.U. Cerro de Las Campanas s/n, Querétaro, Qro. 76010, Mexico.
Whitaker
John R
JR
Rojo-Domínguez
Arturo
A
García-Almendárez
Blanca E
BE
Regalado
Carlos
C
D029688:Brassica napus / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification*; D009195:Peroxidase / Q000737:chemistry* / Q000302:isolation & purification*; D018517:Plant Roots / Q000201:enzymology*; D013053:Spectrophotometry
D002241:Carbohydrates; D007527:Isoenzymes; D009195:Peroxidase