|Stipanovic, Robert - Bob|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/9/2006
Publication Date: 9/9/2006
Citation: Golubenko, Z., Khashimova, N., Beresneva, Y., Ibragimov, F.A., Mustakimova, E.C., Abdurashidova, N.A., Akhunov, A.A., Stipanovic, R.D. 2006. Isolation and characterisation of peroxidase from cotton plant leaves [abstract]. In: Proceedings of Peroxidase 2006, July 7-9, 2006, Aveiro, Portugal. p. 76.
Technical Abstract: Peroxidase is a widespread plant enzyme with unique properties with scientific and pharmaceutical applications. However, the use peroxidase is limited because of their high cost. This serves as stimulus to search for new sources of peroxidase enzymes. We have developed methods for isolation and purification of peroxidase from cotton leaves. Cotton leaves may be harvested when the cotton fiber is near maturity and thus does not adversely affect yields. Isolation of peroxidase begins by extraction with 0.1 M phosphate buffer pH 6.0, followed by step sedimentation by centrifugation from an ammonium sulfate solution. Additional purification is accomplished by chromatographic methods using a gel-filtration on TSK-HW-65 and ion-exchange chromatography on DEAE-cellulose. To develop a purified peroxidase preparation and reduce the steps required for purification, antibodies were taken from an immunized rabbit. The isolated antibodies against of acid peroxidase isoform were linked with CNBr-activated Sepharose. A peroxidase extract was passed through this column with the conjugates. A high-purity acid form of the peroxidase enzyme (300 mg) was obtained from one kg of raw plant material (0.03% yield). The physical, chemical and catalytic properties of the isolated enzyme were investigated. The enzyme molecular weight was 37 kDa; pI 4.7; a temperature optimum 30°C; an optimum pH stability 4.7; Km = 2.3 micromolar. The substrate specificity was determined for benzidine (840 E/mg), and for o-dianisidine (776 E/mg).