A crude enzyme extract (FI) of polyphenol esterase (PPE), obtained from the microbial culture of Aspergillus niger, was partially purified by ammonium sulfate precipitation. The partially purified fraction (FII) was subjected to further purification by ion-exchange chromatography, which resulted in five separated fractions, FIIIa, FIIIb, FIIIc, FIIId and FIIIe), where FIIIa showed the highest PPE activity towards chlorogenic acid, as substrate. The biocatalysis of the PPE with a wide range of mono- and diphenols, as substrates, was shown to inhibit mushroom tyrosinase (PPO) activity. Fraction FIIIa exhibited an inhibitory effect, measured spectrophotometrically, on PPO activity with the monophenols, including 4-hydroxyphenylpyruvic acid and m- and p-cresols and the diphenols, including chlorogenic acid, catechin, 3,4-dihydroxyphenylacetic acid (DHPAA), L-3,4-dihydroxyphenylalanine (L-DOPA), 4-methylcatechol, catechol and caffeic acid; however, using the polarographic method, the inhibition of PPO activity by PPE biocatalysis occurred with the diphenols but not with the monophenols. The selected enzymatic fraction FIIIa was further purified, using size-exclusion chromatography, which resulted in three fractions FIVa, FIVb and FIVc. Although fraction FIVc contained the highest PPE activity, it showed a lack of enzyme stability. Fraction FIIIa was therefore, subjected to further purification by hydrophobic interaction chromatography thereby yielding fractions FVa, FVb, FVc, FVd, FVe, FVf and FVg, where fraction FVc showed the highest PPE activity. The denatured electrophoretic analysis of fraction FVc showed the presence of one major band, with a molecular weight of 60 kDa. The successive purification of PPE resulted in a marked increase in the inactivation of PPO activity with diphenols, as demonstrated by both the lower I50 and inhibition dissociation constant (Ki) values. The purified fraction FVc was shown to exhibit, spectrophotometrically, a competitive and un
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36647 |
| Date | January 2000 |
| Creators | Madani, Wigdan. |
| Contributors | Kenmasha, S. (advisor) |
| 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: 001764696, proquestno: NQ64612, Theses scanned by UMI/ProQuest. |
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