Tyrosinase-ascorbate system

The rising need for new separation processes for the biotechnology industry and the increasing attention towards development of new industrial eruyme processes demonstrate a potential for the use of liquid membranes (LMs). This technique is particularly appropriate for multiple enzyme / cofactor systems since any number of enzymes as well as other molecules can be coencapsulated. This paper focuses on the application of LMs for enzyme encapsulation. The formulation and properties of LMs are first introduced for those unfamiliar with the technique. Special attention is paid to carrier-facilitated transport of amino acids in LMs, since this is a central feature involved in the operation of many LM encapsulated enzyme bioreactor systems. Current work in this laboratory with a tyrosinase/ ascorbate system for isolation of reactive intermediate oxidation products related to L-DOPA is discussed. A brief review of previous LM enzyme systems and reactor configurations is included for reference. 

Figure 7. Schematic representation of the complete tyrosinase / ascorbate system in a Liquid Membrane showing diffusion of oxygen, carrier-facilitated transport of substrate and product through the LM, and the reactions occurring in the internal aqueous phase.

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

In addition to binding to cytochrome c oxidase, cyanide inhibits catalase, peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, and succinic dehydrogenase activities. These reactions may make contributions to the signs of cyanide toxicity (Ardelt et al. 1989 Rieders 1971). Signs of cyanide intoxication include an initial hyperpnea followed by dyspnea and then convulsions (Rieders 1971 Way 1984). These effects are due to initial stimulation of carotid and aortic bodies and effects on the central nervous system. Death is caused by respiratory collapse resulting from central nervous system toxicity. 

Amplihcation factors of 8 to 12 were claimed for the determination of phenol in a FLA system by a cyclic process depicted in equations 5 (Section VI.A. 1). Phenol is converted to o-benzoquinone in contact with immobilized tyrosinase held in a fixed bed reactor the quinone reacts with ascorbic acid (91) to yield catechol and dehydroascorbic acid (136) catechol can be enzymatically oxidized again to o-benzoquinone and so forth. The accumulated dehydroascorbic acid forms with o-phenylenediamine (137) a highly fluorescent product (kex 345 nm, kfl 410 nm). LOD was ca 0.02 (xM for phenol and catechol the linear range for phenol was 0.1 to 2 p,M and for catechol 0.02 to 2... 

In an alternative approach to mimic tyrosinase activity a copper(I)-copper(n) redox couple and a hydroquinone-quinone redox couple were incorporated in one complex (scheme 17). The hydroquinone moiety should act as an electron shunt between an external reducing agent, i.e. ascorbic acid, zinc or electrochemical reduction, and the copper ions. Catalytic oxygenation by monooxygenases is usually accompanied by the formation of water, with the aid of an external electron and proton source.35 46 Activation of O2 by dinuclear copper(I) complex 58 results in superoxo- or p-peroxo-dicopper(II) complex 59, which oxygenates an external substrate molecule. Internal electron transfer to quinone dicopper(II) complex 60 is followed by quinone to hydroquinone reduction. The electron transfer system shown here is reminiscent of the quinone based systems found in the primary photochemical step of bacterial photosynthesis, and in (metallo)porph3nin-quinone electron transfer systems.In contrast to expectation, the hydroquinone dinuclear copper(II) complex 60 (L = (2-pyridylethyl)formidoyl, scheme 17), designed to mimic step c in this cycle, is a stable system in which the hydroquinone moiety is not oxidized to a quinone structure 61. 

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. 

Polyphenol oxidases and ascorbic acid oxidase, which occur in food, are known to have a Cu /Cu redox system as a prosthetic group. Polyphenol oxidases play an important role in the quality of food of plant origin because they cause the enzymatic browning for example in potatoes, apples and mushrooms. Tyrosinases, catecholases, phenolases or cresolases are enzymes that react with oxygen and a large range of mono and diphenols. 

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