Polyphenol oxidases are important enzymes because of their role in food spoilage, oxidizing monophenols into o-diphenols and/or diphenols into the corresponding o-quinones. The resulting compounds are unstable and can rapidly form brown colored compounds, called melanins. Polyphenol oxidases, (PPOs) have been purified from several sources, particularly from fruits and vegetables. However, successful purification of PPO to homogeneity from plant sources has always been difficult. / The purification procedure of PPOs from cassava tuber consisted of (1) the preparation of cassava acetone powder; (2) the buffer extraction of the acetone powder to obtain a crude extract, followed by one of two possible purification procedures. The first consisted of ammonium sulfate fractionation, ion exchange chromatography on Mono-Q and gel filtration on Superdex G75 to yield two isoenzymes, PPO1 and PP02 having molecular weights of 71.8 +/- 6.0 and 69.6 +/- 1.5 kDa, respectively. The second purification procedure involved hydrophobic interaction chromatography (HIC) on phenyl-sepharose CL-4B followed by gel filtration on Superdex G75 to yield a single active PPO fraction of 68.3 +/- 2.8 kDa molecular weight. / The two isoenzymes obtained by ion exchange chromatography exhibited pH optima of 6.5 (PPO1) and 6.8 (PPO2) and were stable in the pH range of 7.5 to 10.0. These two isoenzymes had a temperature optimum of 30--40°C. PPO2 retained 65% of its original activity after heating at 50°C for 10 min whereas PPO1 was completely inactivated by the same treatment. The PPO fraction obtained by HIC purification exhibited a pH optimum of 7.5 with catechol and D,L-dopa as substrates and was stable in the pH range 4 to 8. Its temperature optima, were 20 and 30°C respectively with D,L-dopa and catechol as substrates and this PPO fraction was able to retain 80% of its original activity after heating at 50°C for 10 min. Unstable enzymes were obtained by the ion exchange chromatography purification procedure suggesting that artifacts were created. / Kinetic studies performed with the PPO fraction obtained by the HIC purification showed that catechol had the highest catalytic efficiency ratio. The Km values were 28.1, 5.27 and 3.72 mM for catechol, catechin and D,L-dopa, respectively. The PPO from the HIC purification procedure was inhibited by benzoic acid and p-coumaric acid and inactivated by diethyldithiocarbamate but not by EDTA. L-Cysteine, ascorbic acid and its derivatives (erythorbic acid and sodium erythorbate) were also inhibitors of the oxidation of catechol, catechin and D,L-dopa.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.34697 |
| Date | January 1997 |
| Creators | Barthet, Véronique J. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001615745, proquestno: NQ44358, Theses scanned by UMI/ProQuest. |
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