Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

<div><p>Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH₂), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH₂. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH₂ complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.</p></div

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).

<p>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).</p

Operational Stability of Immobilized Tyrosinase.

<p>(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.</p

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).

<p>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).</p

Simplified Schematic Representation of L-tyrosine Hydroxylation to L-DOPA by Tyrosinase in the Reaction System with Boron Buffer.

<p>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 <i>met</i>-Tyr to <i>deoxy</i>-Tyr in reduced accessibility of L-DOPA and AH₂. The scheme was designed according to that described by Marin-Zamora et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164213#pone.0164213.ref028" target="_blank">28</a>]. The black arrows denote the reaction scheme without additives.</p

Stability of Tyrosinase in Reaction Mixture Components and Examples of Oxygen Consumption in Reaction Systems.

<p>(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.</p

L-tyrosine Hydroxylation by Native Tyrosinase in a Batch Reactor.

<p>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.</p

Simplified Schematic Representation of L-tyrosine Hydroxylation by Tyrosinase.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164213#pone.0164213.ref002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164213#pone.0164213.ref004" target="_blank">4</a>].</p

Stability of Tyrosinase in Presence of Reaction Mixture Components with Borate Buffer.

<p>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).</p

L-tyrosine Hydroxylation by Native Tyrosinase in a Batch Reactor.

<p>(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.</p