The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.)

Abstract

1. Polyphenol oxidase (EC 1. 10. 3.-) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mp (E'%., 13.5) and 611 m,u (Em%., 0 84) with a shoulder at 330m,. The enzymewasbleached bysubstrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000 + 16000. The copper content was 0-32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 300. 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation ofmost substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation ofthe results to the mechanism oftea fermentation is discussed.

Spinach Thylakoid Polyphenol Oxidase

“…These data may support a mechanism whereby DL-DOPA and 02 sequentially bind to the enzyme (in either an ordered or random mechanism) followed by a release of product and subsequent binding of the second molecule of DL-DOPA to the enzyme. A similar mechanism has been proposed for polyphenol oxidase from tea (8) and grape (15), although in the former instance the enzyme was blue and could oxidize p-phenylenediamine; and in the latter case, the enzyme had cresolase activity. If we assume the first segment of the reaction to be rate-limiting, the kinetic constants can be determined by analyzing the data according to a two-substrate reaction mechanism.…”

Purification and Characterization of a Polyphenol Oxidase from the Seeds of Field Bean (Dolichos lablab)

“…Although the maximum activity with catechol and 4-methyl catechol (Figure 8 a and b) occurred at pH 4.0, for DOPA maximum activity was measured at pH 5.0 (Figure 8b). Differences in pH optima have been reported for partially purified strawberry PPO (Wesche-Ebeling and Montgomery, 1990) and tea-leaf PPO (Gregory and Bendall, 1966). The single pH optimum exhibited by these three substrates further evidences the presence of a single isoform as demonstrated by native PAGE (Figure 4).…”

Polyphenoloxidase in Higher Plants

“…Less information is available on the biosynthesis of this chloroplastlocalized enzyme and on the immunological relationships among the isoenzyme forms found in many plant species. Isoenzyme forms have been suggested to arise from conformational changes (17), association-dissociation phenomena (13), covalent attachment of phenolic material (11), and possible attachment of carbohydrate (4,7,21). Many of the isoenzyme forms differ in mol wt but in recent years the subunit mol wt of PPO has been shown to be approximately 40 to 45 kD in spinach (10), pears (20), olives (3), Neurospora (16), Mucuna pruriens (27), and broad beans (9).…”