Polyphenol oxidase phenolic substrates

The phenol oxidases probably play no important role in the elimination of phenolic pressor amines, in spite of the importance that has been attached to the oxidation of the catechol nucleus in the past. The names phenolase and cresolase, polyphenol oxidase, and catechol oxidase serve to identify the enzyme with its mono- or diphenolic substrate, but they usually occur together and are difficultly separated. The enzymes have been purified and their characteristics have been described (56, 104, 106, 156). Beyer (21), Alles (5), and Randall and Hitchings (129) have described the relationship of structure of the phenolic pressor amines to the rate of oxidation of their nucleus in the presence of these enzymes. 

The substrates of the polyphenol oxidase enzymes are phenolic compounds present in plant tissues, mainly flavonoids. These include catechins, anthocyanidins, leucoantho-cyanidins, flavonols, and cinnamic acid derivatives. Polyphenol oxidases from different sources show distinct differences in their activity for different substrates. Some specific examples of polyphenolase substrates are chlorogenic acid, caffeic acid, dicatechol, protocatechuic acid, tyrosine, catechol, di-hydroxyphenylalanine, pyrogallol, and catechins. 

Coche-Guerente, L., Labbe, P., Mengeaud, V. (2001). Amplification of amperometric biosensor responses by electrochemical substrate recycling. 3. Theoretical and experimental study of the phenol-polyphenol oxidase system immobilized in Laponite hydrogels and layer-by-layer self-assembled structures. Anal. Chem. 73 3206-18. 

A portable disposable bioprobe for detection and semiquantitative determination of phenols consists of a mushroom polyphenol oxidase immobilized on a nylon membrane, acting in the presence of 3-methyl-2-benzothiazolinone hydrazone. Maroon to orange colored dyes of (138) are developed, as illustrated for phenol (equation 6), of intensity proportional to the concentration of the substrate, down to 0.05 mgL. Enzyme activity remained unscathed in the pH range 4 to 10, in the presence of various concentrations of salt and metal ions and at temperatures from 5 to 25... 

All these reactions occur in the cell, but on the whole, the cell finds the second and third easier to perform, and, what is more, can perform either of them without the direct intervention of oxygen itself. Reaction (1) does have some importance though. There are enzymes, called oxidases, which catalyse the direct oxidation of their substrates by atmospheric oxygen. They are found in most cells, but are especially common in plants. The effects of one of them, polyphenol oxidase, are familiar to those who peel their apples before eating them. Apples contain traces of a compound called catechol which is oxidized, under the influence of poly phenol oxidase, to a complex, dark brown substance. When an apple is cut its surface is exposed to the air and polyphenol oxidase can begin to work, turning the surface of the apple gradually brown. 

An alternative or complimentary theory for the mode of action of simple phenolic compounds is that they are converted to much more toxic quinones. Pillinger et al. [69] found that various phenolic decomposition products of barley straw were most toxic under conditions favorable for oxidation of the compounds to quinones, and that quinones were up to one thousand-fold more toxic to algae than the parent compounds. The most likely route to conversion to a quinone is enzymatic. Peroxidases and polyphenol oxidases can perform such a reaction, depending on the substrate. However, polyphenol oxidase cannot be detected in most green algae [139] and has not been reported in cyanobacteria. 

Some microbial pathogens can circumvent the defensive response of plants by biotransforming the antimicrobial stilbenoids in a multi-step oxidative detoxification process [106], Research has shown that the pathogenicity of B. cinerea strains is positively correlated with these fungi s production of blue-copper oxidases known as stilbene oxidases or laccases [127,128]. These enzymes are polyphenol oxidases capable of catalyzing the oxidation and polymerization of numerous phenolic substrates [129,130,131,132]. It has been shown that 1 is readily transformed in the presence of B. cinerea culture medium filtrates that contain laccases [107]. Recently, six resveratrol dimers (restrytisols A-C... 

In spite of their frequently brilliant color, it is not so much this property which is important in foods as their tendency to undergo dis-coloration. This is due to their general phenolic character which allows them to serve as effective substrates for oxidase action. They are, in fact, of all classes of phenolic substances, those most universally present in the plant kingdom. Seldom does the analysis of a plant extract fiul to reveal one or more substances of flavonoid character, and if these are absent, chlorogenic acid or one of the closely related coumarins is almost certain to be present. Thus the flavonoid compounds and coumarins are the most commonly available substrates, actual or potential, for polyphenol-oxidase or peroxidase activity. 

Oxidations now known to be catalyzed by copper-containing enzymes were noticed over a century ago, when Schoenbein observed that oxidation of natural substrates resulted in pigment formation in mushrooms. Individual enzymes were gradually identified laccase by Yoshida in 1883 and tyrosinase by Bertrand in 1896. However, it was not imtil potato polyphenol oxidase was isolated in 1937 by Kubowitz that the role of copper was defined. The family of copper oxidases includes a number of enzymes of both plant and animal origin that may very probably be found to react through similar mechanisms, but which exhibit a number of individual characteristics. The enzymes to be described in this section include potato phenol oxidase, mushroom polyphenol oxidase (tyrosinase), laccase, mammalian and insect tyrosinase, and ascorbic acid oxidase. Each of these differs in certain respects from the others, and undoubtedly other related enzymes will be described from other sources that resemble these, but also display individualities. In these cases, identities in nomenclature must not be extended to imply identities in enzyme structure or activity. 

Laccase. A polyphenol oxidase has been purified from the sap of the lac tree by Keilin and Mann. Laccase differs from the potato and mushroom enzyme in several respects. With regard to substrate specificity, it oxidizes p-phenylenediamine more rapidly than catechol. p-Phenylene-diamine is not a substrate for the other polyphenol oxidases described. Laccase apparently is inert with p-cresol. It is not inhibited by carbon monoxide. Unlike the other phenol oxidases, this enzyme is not a pale yellow, but is blue, as is ascorbic acid oxidase (see below). This enzyme, however, is not an ascorbic acid oxidase. 

Laccases are copper containing oxidases that catalyze die one electron oxidation of phenolic substrates (Figure IB) with the concurrent reduction of molecular oxygen to water. Substrates include relatively easily oxidized aromatic conqiounds such as polyphenols, aromatic amines, and medioxy-substituted monophenols (22). Additionally, indirect oxidations are thou to occur in the presence of a suitable redox mediator such as 3-hydroxyandiTaniIate (3-HAA) (23). 

Phenolic compounds are substrates for polyphenol oxidases. These enzymes hydroxylate monophenols to o-diphenols and also oxidize o-diphenols to o-quinones (cf. 2.3.3.2). o-Quinones can enter into a number of other reactions, thus giving the undesired brown discoloration of fruits and fruit products. Protective measures against discoloration include inactivation of enzymes by heat treatment, use of reductive agents such as SO2 or ascorbic acid, or removal of available oxygen. 

The earliest reported demonstration of enzymatic activity in a supercritical fluid was for the reaction of disodium p-nitrophenyl phosphate to p-nitro-phenol, catalysed by alkaline phosphatase. Randolph et aL [26] detected the product in yields of up to 71% in carbon dioxide at 35°C and 100 atm, in the presence of 0.1% v/v water. Hammond et al. [33] found tyrosinase, a polyphenol oxidase, to be catalytically active for the oxidation of 4-methyl phenol in both supercritical carbon dioxide at (36 2)°C and supercritical trifluoro-methane at (34 2)°C, with oxygen, at a total pressure of 345 bar. Use of a flow reactor permitted isolation of greater quantities of the catecholic product (1,2-dihydroxy, 4-methylbenzene). Oxidative activity for 4-chlorophenol substrate was appreciably lower. 

The tendency of plant material towards browning varies within plant species, but the enzyme activity also depends on the type and amount of substrates available. The substrates of polyphenol oxidases may be a large number of different phenolic compounds, but usually only a few are of practical importance (Table 9.16). The individual compounds are given in the section dealing with sensorially active substances. 

Phenolic compounds are present separately from the substrates, predominantly located in the cell vacuoles. Particularly prominent substrates are caffeic acid and its esters, as well as some flavonoid substances, of which monomeric flavan-3-ols (catechins) are the most important. Other groups of phenolic compounds, such as condensed forms of flavan-3-ols and flavan-3,4-diols (tannins), flavonols, flavones, flavans, chalcones, dihydrochalcones and anthocyanins, are only partly oxidised. One of the reasons for this is probably the steric hindrance caused by the corresponding glycosides and substrate specificity of polyphenol oxidases. The content of phenohc compounds depends on genetic factors (on plant species and varieties), the degree of maturity and external (environmental) factors (hght, temperature, nutrients, use of pesticides and so on). The only substrate in animal tissues is the amino acid tyrosine. 

The main substrates of polyphenol oxidases in apples are hydrox-ycinnamic acid derivatives, especially caffeic add depsides, such as chlorogenic acid, and some groups of flavonoid compounds, such as flavan-3-ols, flavonols, dihydrochalcones and (in red-skinned apple varieties) also anthocyanins. In the flesh of apples, the main phenohcs are chlorogenic (3-caffeoylquinic) add, flavan-3-ol epicatechm, and procyanidin B2 representing condensed tannins. These three compounds represent more than 90% of the phenolic compounds present. The main substrates in apple peel are flavan-3-ols and flavonols (quercetin glycosides) and to a lesser extent derivatives of hydroxydnnamic acids are present (chlorogenic add and some other depsides). 

It is generally considered that polyphenol oxidase, which has at least three isoforms, is the key enzyme in the fermentation processes that produce black teas, but there is evidence also for an important contribution from peroxidases with the essential hydrogen peroxide being generated by polyphenol oxidase (Subramanian et al. 1999). The primary substrates for polyphenol oxidase are the flavan-3-ols which are converted to quinones. These quinones react further, and may be reduced back to phenols by oxidizing other phenols, such as gallic acid, flavonol glycosides and theaflavins, that are not direct substrates for polyphenol oxidase (Opie etal. 1993,1995). 

Moreover, there is considerable evidence that PPO is not active as a phenol oxidase in chloroplasts, but is limited as a phenol oxidase by latency or lack of substrate [18]. The latent form of the enzyme can be activated by a wide variety of treatments, including detergents [19], fatty acids [20] trypsin [21] and Ca2+ [22]. The results of Tolbert [21] indicated that light could activate latent PPO because polyphenols can be oxidized photochemically by chloroplast membranes in the absence of other... 

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