Tyrosinase phenol oxidase

Enzymatic electrochemical biosensors are based on immobilized enzymes, whose products can be electrochemically measured after degradation of the substrate at the surface of the biosensor. Many different types of enzymatic biosensors have been developed for environmental monitoring of pesticides, phenols, heavy metals, nitrate, formaldehyde and sulfur oxide, etc. Commonly used enzymes include but are not limited to organophosphorus hydrolase (OPH), AChE, butyrylcholi-nesterase (BChE), horseradish peroxidase (HRP), tyrosinase (phenol oxidases), nitrate reductase, nitrite reductase, formaldehyde dehydrogenase (FDH), and sulfite oxidase (SO). 

In spite of the fact that phenol oxidase probably plays no important role in the inactivation of pressor amines in the body, it has been reported that the injection of the enzyme into hypertensive rats led to a reduction in their blood pressure (141,143)- It is difficult to assess the value of these experiments because of the nonspecific depressor effects of crude protein preparations on blood pressure. For example, Prinzmetal et al. (126) found that their tyrosinase preparations inactivated by boiling decreased the blood pressure of hypertensive patients as well as did their enzymically active preparations. 

Tyrosinase (see Copper Proteins with Dinuclear Active Sites), a copper metalloenzyme with a very broad phylogenetic distribution, is responsible for the browning of fruits and mushrooms.Tyrosinase is a bifimctional phenol oxidase that is able to both hydroxylate monophenols like tyrosine (monooxygenase reaction, (equations)) and snbseqnently oxidize the diphenol product to the corresponding quinone (oxidase reaction, (equation 6)) at a single Type 3 binuclear copper active site. 

Tyrosinase (polyphenol oxidase, a copper-containing monooxygenation enzyme) was used as catalyst for the modification of natural polymers. Phenol moiety-incorporated chitosan derivatives were subjected to tyrosinase-catalyzed cross-linking, yielding stable and self-sustaining gels.90 Tyrosinase also catalyzed the hybrid production between the modified chitosan and proteins. 

A crnde extract of sweet potato Ipomoea-batatas (L.) Lam.) was nsed as a source of phenol oxidases (polyphenoloxidase, tyrosinase, catecholoxidase, EC 1.14.18.1). The extract was directly placed in the carrier of a FIA system with UVD, to promote oxidation of phenolic compounds to o-quinones that condense to form melanin-like pigments with a strong absorption at 410 nm. The determination of phenols in industrial wastewaters showed good agreement with conventional methods (correlation coefficient 0.9954) LOD was 10 p,M, with RSD <2.7% (w = 6). Under optimal storage conditions the enzymatic activity did not vary for at least five months . 

The importance of the functional layer on an electrode to the interaction of an enzyme with the surface is brought out by Kudelski s studies with Cu-containing tyrosinase (a phenol oxidase) [305] and laccase [306]. Using w-functionalized thiols he showed that electron transfer was not prevented between the electrode and the enzyme. That the local environment of the enzyme is important has also been demonstrated in a recent study of human sulfite oxidase using SERRS and cychc... 

Tyrosinases (synonyms phenol oxidases, poly-phenolases or polyphenol oxidases) are copper-containing monooxygenases, which catalyze two consecutive reactions with molecular oxygen as cosubstrate, namely the ortho-hydroxylation of phenols and the oxidation of the resulting catechols to ortho-quinones (Fig. 16.3-4). 

To obtain more information on the nature of the degradation products of the estrogens,. Jellinck (1959) incubated human and rat liver slices as well as mushroom tyrosinase with estrone-/ff-C. The results showed that liver and phenol oxidase of mushroom have a marked ability for converting estrone into water-soluble, ether-insoluble metabolites. The formation of these products was almost completely abolished by cyanide. From an investigation of the estrone degradation products, Jellinck (1959) reached... 

In living cells, various oxidoreductases play an important role in maintaining the metabolism of living systems. So far, peroxidase containing Fe-active site, laccase containing Cu-active site, tyrosinase (polyphenol oxidase, Cu-active site), bilirubin oxidase (Cu-active site), etc, have been reported to act as catalyst for oxidative polymerization of phenol and aniline derivatives and for polymer modification via oxidative coupling. 

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. 

The main difference between the two types of sensors is that the first type (based on phenol oxidases or peroxidase) uses a low applied potential (-100 - 0 mV vs. Ag/AgCl) (3), whereas the second type (based on CDH or GDH) needs a higher potential (300 - 400 mV V5. Ag/AgCl) (9,11,13) to be able to oxidize the phenols and therefore the risk of electrooxidizing interfering compounds in the sample is higher. Tyrosinase, laccase and peroxidase can all be used for both phenolic and diphenolic compounds, however, for laccase and tyrosinase the sensitivity is much higher for catecholic compounds (3). CDH and GDH require a quinone, therefore a diphenol or aminophenol is needed. 

A widely distributed group of enzymes known as the tyrosinases or polyphenol oxidases catalyzes the oxidation of phenolic substances by oxygen. Where these have been isolated, they have been shown to be copper proteins. These enzymes are particularly abundant in plant tissues where they may function as terminal oxidases in place of the cytochrome system the relative importance of the phenol oxidases in plant cell respiration, however, has not yet been determined.The oxidation of ascorbic acid in plant tissues is also due to the presence of a copper enzyme. 

Phenolase Complex. Phenolase (= tyrosinase, or phenol oxidase) converts tyrosine to dopa (= dihydroxyphenylalanine) and oxidizes the dihydroxy derivative further to the quinone stage. Through a series of subsequent reactions, some of which occur spontaneously and wathout enzymic catalysis, the black or brownish black melanin is finally formed (for a schematic representation of the reactions see Chapt. VIII-11). The phenolase is an oxidase with mixed functions, where the product of oxygenation, the hydroquinone derivative, simultaneously acts as... 

Among the carbon-based electrodes, used to replace the mercury electrode, modification of the electrode surface to improve its performance was made by the addition of carbon nanotubes, magnetic or metallic nanoparticles, thin films, etc. The literature reports the use of biosensors incorporating tyrosinase (polyphenol oxidase) for the detection of phenols in aqueous and in organic medium. 

Polyphenol oxidase occurs within certain mammalian tissues as well as both lower (46,47) and higher (48-55) plants. In mammalian systems, the enzyme as tyrosinase (56) plays a significant role in melanin synthesis. The PPO complex of higher plants consists of a cresolase, a cate-cholase and a laccase. These copper metalloproteins catalyze the one and two electron oxidations of phenols to quinones at the expense of 02. Polyphenol oxidase also occurs in certain fungi where it is involved in the metabolism of certain tree-synthesized phenolic compounds that have been implicated in disease resistance, wound healing, and anti-nutrative modification of plant proteins to discourage herbivory (53,55). This protocol presents the Triton X-114-mediated solubilization of Vida faba chloroplast polyphenol oxidase as performed by Hutcheson and Buchanan (57). 

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

While only tyrosinase catalyzes the ortho-hydroxylation of phenol moieties, both tyrosinase and catechol oxidase mediate the subsequent oxidation of the resulting catechols to the corresponding quinones. Various mono- and dinu-clear copper coordination compounds have been investigated as biomimetic catalysts for catechol oxidation [21,194], in most cases using 3,5-di-tert-butylcatechol (DTBC) as the substrate (Eq. 16). The low redox potential of DTBC makes it easy to oxidize, and its bulky tert-butyl groups prevent un-... 

Hypertensin is soluble in alcohol, glacial acetic acid, phenol, and water, and insoluble in ether (61). Because it is inactivated by tyrosinase it probably contains a catechol or phenol group, and by amine oxidase, an amine group on an a-carbon atom (Figure 2). Hypertensin is inactivated by certain phenolic, catecholic, and amine oxidases, by pepsin, trypsin, chymotrypsin, and carboxypeptidase, and by hypertensinase found in plasma. The nature of hypertensinase is unknown, but it is probably not an oxidative enzyme. Because it is heat-labile, hypertensinase can be removed from blood and renin preparations by heating hypertensin itself is heat-stable. Lack of pure preparations of hypertensin has delayed its further chemical identification. 

The interest in catechol oxidase, as well as in other copper proteins with the type 3 active site, is to a large extent due to their ability to process dioxygen from air at ambient conditions. While hemocyanin is an oxygen carrier in the hemolymph of some arthropods and mollusks, catechol oxidase and tyrosinase utilize it to perform the selective oxidation of organic substrates, for example, phenols and catechols. Therefore, establishment of structure-activity relationships for these enzymes and a complete elucidation of the mechanisms of enzymatic conversions through the development of synthetic models are expected to contribute greatly to the design of oxidation catalysts for potential industrial applications. 

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