The curds of cauliflower (Brassica oleracea var.botrytis L.) which are representative of the European biennials, European annuals and Australian types were used to extract aspartate aminotransferase (EC 188.8.131.52) and acid phosphatase (EC 184.108.40.206). These enzymes were separated into their isoenzymes using polyacrylamide gel electrophoresis and were shown to have different numbers of isoenzymes depending upon which of the three main groups of cauliflower cultivars were used. The enzymes examined showed evolutionary divergence of the cauliflower types. Growth and development, storage and frost damage in cauliflowers were examined in relation to peroxidase activity. Physiological age in different parts of the curds and between different size curds was shown to have very little effect on peroxidase activity. Total peroxidase activity in the stem was about 2 times the value in the curds. A gradual increase of peroxidase was found to occur with duration of storage period at 7°C. Peroxidase activity remained low when cauliflowers were kept at low temperatures, namely both -1 + 1°C and -5 + 1°C, but increased rapidly on warming up at 7°C for 2 days. Isolation and partial purification of the peroxidase were carried out using conventional methods, including ammonium sulphate precipitation, Sephadex G-100 molecular sieve and DEAE Cellulose ion exchange chromatography. Further purification was achieved using Phenyl Sepharose hydrophobic interaction chromatography. Peroxidase activity was localized in the 50-95% ammonium sulphate precipitation. Molecular sieve chromatography showed a broad peak of peroxidase activity and molecular weight was estimated to be about 42,000. Polyacrylamide gel electrophoresis and isoelectric focusing of this fraction showed the existence of two peroxidase isoenzymes with P 6.7 and 7.3 using guaiacol as substrate; one of the isoenzymes also possessed a simultaneous IAA oxidase activity. Optimal pH and temperature with guaiacol as a substrate was found to be pH 5.6 and 50°C. Kinetic studies indicated an apparent Km of 8.3 mM for guaiacol at an optimal hydrogen peroxide concentration and a Km of 6.25 mM for hydrogen peroxide at an optimal guaiacol concentration. By including hydrophobic interaction chromatography in the purification scheme, a homogeneous cauliflower peroxidase isoenzyme was obtained. It showed as a single band in polyacrylamide gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Molecular weight estimation of this peroxidase isoenzyme was 41,000+1,000. The elution profiles of white and green cauliflowers and white sprouting broccoli peroxidases by hydrophobic interaction chromatography were found to be different. Green cauliflowers with high frost resistance were found to have high percentage of peroxidase activity bound very tightly to the hydrophobic resin. Similar peroxidase activity patterns using guaiacol, o-dianisidine and pyrogallol were obtained. Antiserum specific for the homogeneous peroxidase from cauliflower (cv. Milreef) was used to examine homology among cauliflower peroxidases and among peroxidases from green and white cauliflowers, white sprouting broccoli and horseradish. The antiserum cross reacted with all cauliflower peroxidases as well as peroxidases from green cauliflower, white sprouting broccoli and horseradish, indicating the antigenic sites were similar.
|Date of Award||1985|