Purification and Characterization of Isoperoxidases Elicited by Aspergillus flavus in Cotton Ovule Cultures

Abstract

Two anionic isoperoxidases were isolated from media of Aspergillus flavus-inoculated cotton (Gossypium hirsutum L.) ovule cultures and purified about 150-fold to apparent homogeneity by treatment with Cell Debris Remover and ion exchange chromatography on Accell QMA medium. These isoperoxidases were present in noninoculated cotton ovule cultures at low levels. The major activity peak (B) represented 90% of the recovered peroxidase activity and was electrophoretically homogeneous. The minor activity peak (A) was about 95% pure. Isoelectric focusing analysis showed that B was greater than 95% pure with respect to other peroxidase isozymes, while the enzyme in A was about 90% isozymically pure. Each isoperoxidase displayed a molecular mass of 56 kilodaltons by interpolation from denaturing gel electrophoresis. The B isozyme displayed a molecular mass of 55 kilodaltons by gel filtration chromatography. The pH optima for the cotton ovule isoperoxidases were similar, 5.0 for isozyme A and 6.0 for isozyme B. The isoelectric points for isozymes A and B were 4.2 and 4.4, respectively. Eugenol, guaiacol, and 3,3',5,5'-tetramethylbenzidine were good electron donor substrates, whereas 4-aminoantipyrine was a poor substrate. The absorption spectrum of the material in B revealed a major peak at 400 nanometers and a minor peak at 280 nanometers. The molar extinction coefficient at 400 nanometers (pH 7.0) was calculated to be 1.07 x 105 per square centimeter per mole.Amino acid analysis of isozyme B confirmed the acidic nature of this protein and identified a number of similarities to the anionic peroxidases from tobacco and potato. This glycoprotein was found to contain 12 to 14% sugar (by weight), mainly in the form of galactose and mannose.

Characterisation of an anionic peroxidase from horseradish cv. Balady

“…Similarly, the thermal treatment of peroxidases from apple (Valderrama & Clemente, 2004), orange (Clemente, 1998), mango (Khan & Robinson, 1993), W. somnifera (Johri et al, 2005) and marula fruit (Mdluli, 2005) proved to be non-linear in relation to temperature. The thermal stability of peroxidases is mainly due to the presence of a large number of cysteine residues in the polypeptide chain (Deepa & Arumughan, 2002) and the presence of sugar in their structure (Mellon, 1991). Horseradish POIII activity was tested in the pH range 3.6-8.5 (Fig.…”

Induction of pathogenesis-related proteins during biocontrol of Rhizoctonia solani with Pseudomonas aureofaciens in soybean (Glycine max L. Merr.) plants

“…POD in pepper plants infected by P. capsici showed elevated levels of isoenzymes of 114, 53 and 45 kDa (Jung et al 2004). Two anion isoperoxidases (55 and 56 kDa) were observed in Aspergillus flavus-inoculated cotton (Gossypium hirsutum L.) ovule cultures (Mellon 1991). Peroxidase (35 kDa) was observed following inoculation of barley (Hordeum vulgare L.) with powdery mildew (Erysiphe graminis) (Kerby and Somerville 1992).…”

Kinetic study of a purified anionic peroxidase isolated from Eupatorium odoratum and its novel application as time temperature indicator for food materials

“…Highly heat resistant peroxidase isoenzymes of horseradish and Brussels sprouts are reported to have activation energies for inactivation of 142.3 kJ mol À1 (Ling & Lund, 1978) and 148.8 kJ mol À1 (Regalado, Arvizu, & Garcia-Almendarez, 1999), respectively. Generally, peroxidases and lipoxygenases have high thermostability which is attributed to the presence of sugars in their structure (Mellon, 1991). However, this thermostability cannot be extended to all peroxidase isoenzymes, due to the existence of isoenzymes with different resistance to temperature (Moulding, Grant, Mc Lellan, & Robinson, 1987).…”