The roles of Tyrosinase and Laccase in enzymic oxidation

Wine oxidizes when exposed to air via two primary mechanisms: enzymic and non-enzymic oxidation. Enzymic oxidation primarily afflicts wine must and requires the presence of the enzyme Tyrosinase (or Laccase, in the case of botrytized must), phenolic compounds (flavonols, anthocyanins, tannins, etc.), oxygen, and metallic co-factors (iron, copper, etc.). The effects of oxidation on wine are browning, loss of fruity aromas, and aldehydic aromas. Because of these characteristics, oxidization is widely viewed as a wine fault. In this post I will focus on enzymatic oxidation.

Polyphenols

According to Jackson (Wine Science), "In contrast to red wines, the limited antioxidant character of white wines (ed: tannins and anthocyanins provide substantive antioxidant capability in red wines with red wine polyphenol content ranging between 300 and 5000 mg/L while white phenolics range between 60 and 200 mg/L) make them more susceptible to oxidative browning." Hydroxycinnamic acid is the most important of the non-flavonoids, comprising 80% of non-skin-contact-white-wine phenolics. It is the first white wine polyphenol to be oxidized.

Further, grape varieties differ markedly in the amount of phenolics released during crushing or extracted during maceration (an extremely important consideration given that phenolics are the main substrate for oxidation activity). The table below shows the levels of flavonoid accumulation during crushing or maceration of selected white varieties.

Table 1. Phenolics released/extracted during crushing/maceration

Variety	Flavonoid Accumulation
Palomino	Low
Sauvignon Blanc	Low
Riesling	Moderate
Semillon	Moderate
Chardonnay	Moderate
Muscat Gordo	Extensive
Colombard	Extensive
Trebbiano	Extensive
Pedro Ximinez	Extensive

Tyrosinase

Polyphenol oxidases (PPOs) are a widespread group of enzymes found in plants, fungi, bacteria, and animals. In plants, they are located in the plastid of the chloroplast and in the mitochondrion, separate from the unsuspecting polyphenols (which are located in the vacuole). 

It is thought that these PPOs contribute to the defense of the plant against predators such as herbivores and insects. 

Enzymic oxidation (which primarily afflicts wine must) requires the presence of the PPO enzyme Tyrosinase (or Laccase, in the case of botrytized must), phenolic compounds (hydroxycinnamic acids, with the main player being caftaric acid but others — including coumaroyl, tartaric acid, and catechin — as alternates) to perform the role of substrate, oxygen, and metallic co-factors (iron, copper, etc.). 

Once the grape berry integrity has been compromised, the enzyme and phenols are exposed to each other and to atmospheric oxygen. Juice or wine that is saturated with oxygen contains about 7 - 8 mg/L (depending on the temperature).

As shown in the figure above, the enzyme has an active site where its interaction with the substrate will eventually occur. The copper contained in the enzyme is located at this active site.

In the presence of oxygen, this copper-containing enzyme oxidizes the phenolic groups to reactivate oxygen molecules known as quinones. The active site of tyrosinase undergoes transitions among mel-, oxy-, and deoxy-forms in a cyclic manner. In each cycle, two molecules of catechol are oxidized and one molecule of oxygen is reduced to water, resulting in the formation of two quinone products.

These quinones, in turn, continue reacting with each other, and other cellular factors, to form brown spots known as melanin.

Because tyrosinase is associated with grape solids, its enzymic activity is significantly diminished once the solids have been removed from the equation. 

Glutathione

These enzymes interact with the substrates to form caftaric acid quinone which, in turn, reacts with glutathione (normally a powerful anti-oxidant) in the must to form Grape Reduction Product (GRP). While my intent was to discuss anti-oxidants in a totally separate post, tight integration of the naturally occurring glutathione into the enzymatic oxidation process dictates that it be an integral part of this discussion. 

Glutathione is a grape- and yeast-produced tripeptide which contains three amino acids: glutamate, cystine, and glycone. It is generally found in must, yeast and wine in its reduced (GSH) or oxidized (GSST) forms, the latter of which contains two molecules of glutathione linked by a sulfide bridge.

Glutathione is important in the wine space, firstly, because of its ability to scavenge ortho-quinones, the main culprit in oxidative browning. The compound plays a critical role in preventing the oxidation of phenols in must by reacting with caftaric ortho-quinones to generate Grape Reduction Product, a stable, colorless compound. The conversion of the oxidized quinone to GRP limits the browning of the juice to some extent (duToit and Kritzinger). 

Once the glutathione is depleted, the remaining caftaric acid quinone reacts with other must constituents to form caftaric acid and begins the oxidation process anew. Browning occurs when the flavanols oxidized by caftaric acid quinones polymerize and precipitate out. Unlike the case of wines, these brown pigments are insoluble in must. The process is illustrated graphically in the figure above.

The GSH form of glutathione can also compete with several aromatic compounds for ortho-quinones, thus protecting and preserving certain wine aromas.

Minimizing the loss of glutathione is one of the keys in white winemaking as there is a strong correlation between the concentration of glutathione in the must and the concentration in the young wine and a positive correlation between glutathione in the wine and the wines freshness and longevity.

The glutathione-to-caftaric-acid ratio can give an indication of the oxidation sensitivity of certain cultivars.

Laccase

Laccase is produced by the phytopathogenic fungus Botrytis cinerea and enters the must with contaminated grape berries. Laccase resides in the glycan sheath surrounding the hyphae of Botrytis. High levels of Botrytis is often correlated with high levels of laccase. 

As in the case with tyrosinase, laccase oxidizes phenolic compounds into quinones which polymerize in the presence of oxygen. Polymerized quinones form pigmented compounds "associated with laccase-induced browning and discoloration."

Both tyrosinase and laccase use catechin, anthocyanin, flavanols, and flavanone as substrates but laccase acts on a far wider range of substrates than does tyrosinase. UCDavis pegs the added scope of laccase as encompassing anthocyanin pigments and ascorbic acid, the latter of which is itself used as an antioxidant. Laccase can oxidize the GRP to the corresponding quinone which can, in turn react with glutathione to form GRP2, GRP3, etc.

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The activity of these enzymes will be impacted by (Boulton, et al, Principles and Practices of Winemaking):

• The concentration of major phenols
• Competition between substrates for binding and reaction
• The caftaric and glutathione content of cultivar (the state at which glutathione is depleted will determine the level of potential browning)
• The ascorbic acid content
• Temperature
•  Wine pH.

Allowing the juice to brown prior to fermentation may be beneficial to some non-aromatic varieties as oxidized phenolics will not contribute to post-fermentation astringency.

In my next post I will discuss how anti-oxidants can be deployed to counteract the effects of oxidation.

©Wine -- Mise en abyme

Walking the tightrope between oxidation and reduction in white wine production: Oxidation

In this series I will be examining the winemaker's challenge in navigating between the twin evils of reduction and oxidation, both faults but both having potentially desirable characteristics close to the center of the continuum. I begin with this post on oxidation.

According to Lukacs' research (Inventing Wine), modern wine did not arise until the advent of the relevant scientific and technological advances of the Enlightenment. Prior to that period, wine drinkers consumed oxidized, sour wines which were "fortified" with all manner of additives designed to either slow its decay or make it more "palatable." Lukacs points out that winemaking in the first half of the 20th century was a reprise of thousands of years past -- "a process of letting nature run its course."

When Emile Peynaud (famed Bourdeaux enologist) began his work in the early 1950s, growers were harvesting early and, as a result, the wines were "excessively green or vegetal." He observed that there was a further striking uniformity about the wines: they were all oxidized.

Wine oxidizes when exposed to air via two primary mechanisms: enzymic and non-enzymic oxidation.  The effects of oxidation on white wine are browning, loss of fruity aromas, and aldehydic aromas. Because of these characteristics, oxidization is widely viewed as a wine fault.

Enzymic Oxidation

Enzymic oxidation (which primarily afflicts wine must) requires the presence of the enzyme Tyrosinase* (or Laccase**, in the case of botrytized must), phenolic compounds (hydroxycinnamic acids, with the main player being caftaric acid but others — including coumaroyl, tartaric acid, and catechin — as alternates) to perform the role of substrate, oxygen, and metallic co-factors (iron, copper, etc.). These enzymes interact with the substrates to form caftaric acid quinone which, in turn, reacts with glutathione (normally a powerful anti-oxidant) in the must to form Grape Reduction Product (GRP). The conversion of the oxidized quinone to GRP limits the browning of the juice to some extent (duToit and Kritzinger). Once the glutathione is depleted, the remaining caftaric acid quinone reacts with other must constituents to form caftaric acid and begins the oxidation process anew. Browning occurs when the flavanols oxidized by caftaric acid quinones polymerize and precipitate out. Unlike the case of wines, these brown pigments are insoluble in must.

Because tyrosinase is associated with grape solids, its enzymic activity is significantly diminished once the solids have been removed from the equation. Laccase is difficult to eliminate from grape juice.

The activity of these enzymes will be impacted by (Boulton, et al, Principles and Practices of Winemaking):

• The concentration of major phenols
• Competition between substrates for binding and reaction
• The caftaric and glutathione content of cultivar (the state at which glutathione is depleted will determine the level of potential browning)
• The ascorbic acid content
• Temperature
•  Wine pH.

Both tyrosinase and laccase use catechin, anthocyanin, flavanols, and flavanone as substrates but, as indicated in Table 2.1 of Boulton et al., laccase acts on a far wider range of substrates than does tyrosinase. UCDavis pegs the added scope of laccase as encompassing anthocyanin pigments and ascorbic acid, the latter of which is itself used as an antioxidant

Non-Enzymic Oxidation

Non-enzymic oxidation, also known as chemical oxidation, occurs in fermented wine. In this case, oxygen does not react directly with phenolic compounds. Rather, it functions through a reaction catalyzed by Cu+ or Fe+ that converts oxygen into a highly reactive radical capable of oxidizing organic compounds.

Non-enzymic oxidation in white wines can result in premature aging, browning, and pinking, all resulting in wine deterioration and loss of quality. Strategies for combating this fault include removing metals -- oxidation catalysts -- and reducing the concentration of phenolic compounds -- oxidation precursors --in wines.

©Wine -- Mise en abyme

Enzymic oxidation of wine must

In my most recent post, I initiated a series on wine faults with a discourse on sulfur taint in wine production. I continue in that vein with a discussion of must oxidation.

Wine oxidizes when exposed to air via two primary mechanisms: enzymic and non-enzymic oxidation. Enzymic oxidation primarily afflicts wine must and requires the presence of the enzyme Tyrosinase (or Lacasse, in the case of botrytized must), phenolic compounds (flavonols, anthocyanins, tannins, etc.), oxygen, and metallic co-factors (iron, copper, etc.). Non-enzymic oxidation, also known as chemical oxidation, occurs in two steps: (i) Oxygen in the air reacts with wine phenols to create hydrogen peroxide and (ii) hydrogen peroxide reacts with ethanol to form acetaldehyde. The effects of oxidation on wine are browning, loss of fruity aromas, and aldehydic aromas. Because of these characteristics, oxidization is widely viewed as a wine fault.

Enzymic oxidation (which primarily afflicts wine must) requires the presence of the enzyme Tyrosinase* (or Laccase**, in the case of botrytized must), phenolic compounds (hydroxycinnamic acids, with the main player being caftaric acid but others — including coumaroyl, tartaric acid, and catechin — as alternates) to perform the role of substrate, oxygen, and metallic co-factors (iron, copper, etc.). These enzymes interact with the substrates to form caftaric acid quinone which, in turn, reacts with glutathione (normally a powerful anti-oxidant) in the must to form Grape Reduction Product (GRP). The conversion of the oxidized quinone to GRP limits the browning of the juice to some extent (duToit and Kritzinger). Once the glutathione is depleted, the remaining caftaric acid quinone reacts with other must constituents to form caftaric acid and begins the oxidation process anew. Browning occurs when the flavanols oxidized by caftaric acid quinones polymerize and precipitate out. Unlike the case of wines, these brown pigments are insoluble in must.

Because tyrosinase is associated with grape solids, its enzymic activity is significantly diminished once the solids have been removed from the equation. Laccase is difficult to eliminate from grape juice.

The activity of these enzymes will be impacted by (Boulton, et al, Principles and Practices of Winemaking):

• The concentration of major phenols
• Competition between substrates for binding and reaction
• The caftaric and glutathione content of cultivar (the state at which glutathione is depleted will determine the level of potential browning)
• The ascorbic acid content
• Temperature
•  Wine pH.

Both tyrosinase and laccase use catechin, anthocyanin, flavanols, and flavanone as substrates but, as indicated in Table 2.1 of Boulton et al., laccase acts on a far wider range of substrates than does tyrosinase. UCDavis pegs the added scope of laccase as encompassing anthocyanin pigments and ascorbic acid, the latter of which is itself used as an antioxidant

A number of suggestions have been advanced to impede/control the oxidative activities of the enzymes:

• Boulton has reported that the addition of sulfur dioxide at levels between 25 and 75 mg/L will reduce tyrosinase activity between 75 and 97%. This inhibition may be the result of a binding of a sulfhydril group on the enzyme or a bisulfite inhibition resulting from the interaction of sulfites and intermediate quinones. (Sulfur dioxide does not significantly impact the operation of laccase (addition of 150 mg/L sulphur dioxide yields only a 20% reduction in oxidative activity); as a matter of fact, it can serve as a substrate. The only way to completely stop laccase is to use HTST to denature it.) Sulfur dioxide can reverse the effects of oxidized quinones to colorless phenols. Sulfites react slowly with oxygen, but react with important intermediates (hydrogen peroxide, acetaldehyde, quinones)
• Judicious harvesting, crushing, pressing to minimize enzyme reaction subtrates and limit oxygen exposure
• Bentonite fining to remove the enzyme
• Hyperoxidation to reduce phenols
• Use healthy grapes
• Keep wine containers full
• Use of inert gas to limit oxygen contact

According to Boulton et al., ascorbic acid can be used as an antioxidant either alone or in cooperation with sulfur dioxide. It does not have any direct effect on the enzyme but it reduces the potential for browning by consuming the available oxygen. It can also delay the onset of browning by converting quinones back to the original phenol. The recommended levels of ascorbic acid addition ranges between 50 and 200 mg/L. Ascorbic acid will react with oxygen to form dehydrascorbic acid and, upon the consumption of the former, browning may be initiated. Conversely, unused ascorbic in finished wines can act like a phenol, leading to the production of acetaldehyde where oxygen is introduced.

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*Tyrosinase is located in the chloroplasts of the grape berry cell membrane and, in the must, is predominantly attached to grape particles. Its concentration in the must is dependent on the variety and the maturity of the fruit. Warmer temperature and higher pH increases the activity of this enzyme.

**Laccase is generally associated with Botrytis and can cause bunch rot and grey mold in grapes. Moisture on the berry surface can cause infection. Farming and harvesting practices can impact the levels of rotten fruit brought into the cellar and, potentially, included in the must. This enzyme is considered more dangerous than tyrosinase because it oxidizes a wider range of phenolic compounds and is resistant to sulphur dioxide. As is the case for tyrosinase, this enzyme is more active at higher temperatures and pH.

©Wine -- Mise en abyme