Fig 1.
L-tyrosine Hydroxylation by Native Tyrosinase in a Batch Reactor.
(A) Reaction without aeration of the reaction mixture. (B) Reaction with constant aeration. Symbols: oxygen (dashed line); A475 (solid line); L-tyrosine (○); L-DOPA (●). Reaction conditions: 1 mM L-tyrosine in 0.1 M phosphate buffer, pH 7; 30°C; and 20 rpm.
Fig 2.
Effect of the Agitation Rate and Temperature on the Initial Reaction Rates (r).
(A) Effect of the agitation rate using immobilized tyrosinase. (B) Arrhenius plots with native (black circles) and immobilized (empty circles) tyrosinase.
Fig 3.
Simplified Schematic Representation of L-tyrosine Hydroxylation by Tyrosinase.
Reactions without ascorbic acid—black arrows and a reaction system with ascorbic acid (AH2) supplementation—gray arrow. More detailed information about the actions of tyrosinase on L-tyrosine and L-DOPA is summarized in [2–4].
Fig 4.
Stability of Tyrosinase in Reaction Mixture Components and Examples of Oxygen Consumption in Reaction Systems.
(A) Relative activity of native (black) and immobilized tyrosinase (gray) after a 1 h incubation at 30°C in 0.1 M phosphate buffer, pH 7 (Control) containing 1 mM L-tyrosine (L-tyr), 1 mM L-DOPA, or 2 mM ascorbic acid (AH2). (B) An example of the effect of 1 mM L-tyrosine hydroxylation (gray lines) or 2 mM ascorbic acid oxidation (black lines) by native tyrosinase over time on the oxygen concentration in non-aerated (solid lines) and aerated (dashed or dotted lines) reaction mixtures. Red lines represents control experiments with 2 mM ascorbic acid whereas blue lines with 1 mM L-tyrosine, both without tyrosinase. Reaction conditions: 0.1 M phosphate buffer, pH 7; 30°C; and 20 rpm. (C) Relative initial reaction rates of immobilized tyrosinase in three consecutive 1 h processes in the batch reactor. Bars: black–measured from the increase in the L-DOPA concentration or white–measured from the decrease in the L-tyrosine concentration. Reaction conditions: 1 mM L-tyrosine and 2 mM ascorbic acid in 0.1 M phosphate buffer, pH 7; 0.133–0.235 O2; 30°C; and 120 rpm.
Fig 5.
L-tyrosine Hydroxylation by Native Tyrosinase in a Batch Reactor.
Reaction mixtures without (A) and with (B) aeration. Symbols: oxygen (dashed line); A475 (solid line); L-tyrosine (○); L-DOPA (●). Reaction conditions: 1 mM L-tyrosine and 2 mM ascorbic acid in 0.1 M phosphate buffer, pH 7; 30°C; and 20 rpm.
Table 1.
Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Phosphate Buffer (0.1 M, pH 7) in the Presence of Ascorbic Acid (AH2; 2 mM).
Table 2.
Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Reaction Systems with Borate Buffer (BB; 0.5 M), and/or Ascorbic Acid (AH2; 2 mM), and/or Hydroxylamine (HA; 6.7 mM).
Fig 6.
Simplified Schematic Representation of L-tyrosine Hydroxylation to L-DOPA by Tyrosinase in the Reaction System with Boron Buffer.
Boron buffer was applied to create complexes with L-DOPA and ascorbic acid (AH2) that minimize suicide inactivation caused by both compounds; AH2 was added to reduce DOPA-quinone back to L-DOPA; hydroxylamine (HA) was added to reduce met-Tyr to deoxy-Tyr in reduced accessibility of L-DOPA and AH2. The scheme was designed according to that described by Marin-Zamora et al. [28]. The black arrows denote the reaction scheme without additives.
Fig 7.
Stability of Tyrosinase in Presence of Reaction Mixture Components with Borate Buffer.
Relative activity of native (black) and immobilized enzyme (gray) after 1 h incubation at 30°C in 0.5 M borate buffer, pH 9 (Control) containing 1 mM L-tyrosine (L-tyr), 1 mM L-DOPA, 2 mM ascorbic acid (AH2), or 6.7 mM hydroxylamine (HA).
Fig 8.
Operational Stability of Immobilized Tyrosinase.
(A) Relative initial reaction rates of immobilized tyrosinase in three consecutive 1 h processes in the batch reactor. The black, gray, and white bars indicate the first, second, and third runs, respectively. (x2) indicates a two-fold higher enzyme load. (B) L-tyrosine hydroxylation by immobilized tyrosinase in three consecutive processes in the batch reactor. Symbols: L-tyrosine (○); L-DOPA (●). Reaction conditions: 1 mM L-tyrosine, 2 mM ascorbic acid, 6.7 mM hydroxylamine in 0.1 M boron buffer, pH 7; 0.130–0.239 O2; 30°C; and 120 rpm.