Purification and Characterization of a Polyphenol Oxidase from the Seeds of Field Bean (Dolichos lablab)
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- 作者：Beena Paul and Lalitha R. Gowda
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2000
- 出版时间：September 2000
- 年：2000
- 卷：48
- 期：9
- 页码：3839 - 3846
- 全文大小：99K
- 年卷期：v.48,no.9(September 2000)
- ISSN：1520-5118
文摘
The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparenthomogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography,phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has amolecular weight of 120 ± 3 kDa and is a tetramer of 30 ± 1.5 kDa. Native polyacrylamide gelelectrophoresis of the purified enzyme revealed the presence of a single isoform with an observedpH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. Noactivity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols.Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitiveinhibitor with an apparent Ki of 5.8 × 10-7 M. Ascorbic acid, metabisulfite, and cysteine were alsocompetitive inhibitors.Keywords: Polyphenol oxidase; field bean seed; Dolichos lablab; catecholase; tetramer; substrateinhibition; tropolone