Horseradish phenolic polymerization

Nicell JA, Saadi KW, Buchanan ID. Phenol polymerization and precipitation by horseradish peroxidase enzyme and an additive. Bioresour Technol 1995 54 5-16. 

Horseradish peroxidase Trypsin (protease) Subtilisin (protease) Phenol polymerization Transpeptidation Ester hydrolysis Ethylacetate Butan-l,4-diol Dioxane, chloroform, etc. 

Horseradish peroxidazed polymerization has been extensively studied in recent years. For instance, enzymatic polymerization of tyrosine derivatives was examined. The reaction mechanism is known to involve free radical processes. Oligomerization of brfunctional phenols in the presence of p-CD was studied. Recendy, the role of synthetic polymers used as templates in this process has been studied. ... 

However, an important problem arises during the peroxidative removal of phenols from aqueous solutions PX is inactivated by free radicals, as well as by oligomeric and polymeric products formed in the reaction, which attach themselves to the enzyme (Nazari and others 2007). This suicide peroxide inactivation has been shown to reduce the sensitivity and efficiency of PX. Several techniques have been introduced to reduce the extent of suicide inactivation and to improve the lifetime of the active enzyme, such as immobilization. Moreover, Nazari and others (2007) reported a mechanism to prevent and control the suicide peroxide inactivation of horseradish PX by means of the activation and stabilization effects of Ni2+ ion, which was found to be useful in processes such as phenol removal and peroxidative conversion of reducing substrates, in which a high concentration of hydrogen peroxide may lead to irreversible enzyme inactivation. 

Among other in vitro enzymatic polymerizations that have been studied are the oxidative polymerizations of 2,6-disubstituted phenols to poly(p-phenylene oxide)s (Sec. 2-14b) catalyzed by horseradish peroxidase [Higashimura et al., 2000b] and the polymerization of P-cellobiosyl fluoride to cellulose catalyzed by cellulase [Kobayashi, 1999 Kobayashi et al., 2001],... 

Oxidative polymerization of phenols has been carried out extensively by using horseradish peroxidase and other enzymes. However, these interesting topics lie far beyond the scope of this chapter. 

So far, several oxidoreductases, peroxidase, laccase, polyphenol oxidase (tyrosinase), and so on have been reported to catalyze oxidative polymerization of phenol derivatives among which peroxidase is most often used [165,166]. Peroxidase is an enzyme that catalyzes the oxidation of a donor to an oxidized donor by the action of hydrogen peroxide, liberating two water molecules. Horseradish peroxidase (HRP) is a single-chain p-type hemoprotein that catalyzes the decomposition of hydrogen peroxide at the expense of aromatic proton donors. 

Horseradish peroxidase (HRP, EC 1.11.1.7) catalyzes the ( -dependent oxidation of phenols and amines forming colored polymeric products via radical intermediates. This reaction has been used to detect phenol, bilirubin and aminopyrine (Renneberg et al., 1982). The hydrogen peroxide required was either injected into the measuring cell or generated in the enzyme membrane itself. For the latter reaction, GOD was coimmobilized with HRP. 

Many efforts have been made to base polymers on furfural made from pentoses.159 The polymers may be useful, but tend to have lower thermal stability than the usual synthetic polymers. Polyesters based on furfural were mentioned earlier. The acid-catalyzed polymerization of furfuryl alcohol is used in foundry cores.160 Furfural has been condensed with cardanol (m-pentadecadienylphenol) from cashew nut shell oil in the presence of other phenols to produce polymeric resins.161 Cardanol and hydrogenated cardanol have been polymerized with horseradish peroxidase to soluble polymers in up to 85% yield.162 Plasticizers that are effective in polyvinyl chloride, such as (12.31), have been made from furfural.163... 

Enzymic reactions in SC-CO2 cover oxidations and solvolyses. Good yields (75 % at a residence time of only 13 s) were reported for the enzymic oxidation (immobilized cholesterol oxidase from G. chrysocreas) of cholesterol in supercritical CO2/O2 (9 1) (Scheme 8). Cosolvents, like tert-butyl alcohol, that increase the solubility and, to an even larger extent, those that assist aggregate formation, increase the rate of the reaction (fourfold in this case). [31] However, it appears that this line has not been pursued any further. Horseradish peroxidase was used in the oxidative polymerization of / -cresol by H2O2 in SC-CO2. Cosolvents were useful. The method was evaluated for manufacture of phenolic resins without incorporating formaldehyde. [47]... 

It is now well-established that some enzyme families, including various peroxidases and laccases, catalyze the polymerization of vinyl monomers and other redox active species such as phenol-type structures. Vinyl polymerization by these redox catalysts has recently been reviewed 93). These catalysts have been used to prepare polyanilines 94) and polyphenols 95,96). A few examples of related research are included in this book. For example. Smith et al (57) described a novel reaction catalyzed by horseradish peroxidase (HRP). In the presence of HRP and oxygen, D-glucuronic acid was polymerized to a high molecular weight (60,000) polyether. However, the authors have not yet illucidated the polyether structure. Two other oxidative biotransformations were discussed above i) the sono-enzymatic polymerization of catechol via laccase 31), and ii) the oxidation of aryl silanes via aromatic dioxygenases 30). 

Enzyme-Catalyzed Polymerization. Horseradish peroxidase (HRP) has been used for the oxidation of a wide variety of compounds, eg, phenol derivatives in the presence of hydroperoxide (44). HRP is a kind of heme glycoprotein that catalyzes the oxidation of phenol to phenoxy radicals. Subsequently, the resulting phenoxy radicals couple each other to oligo or polyphenol derivatives step by step (see Oxidative Polymerization). 

The principle of the polymerization of phenols catalyzed by a peroxidase is explained in the following text for the enzyme horseradish peroxidase (HRP). The mechanism of the HRP catalysis is fairly well understood and has been the subject of many investigations [24,51-54]. HRP catalyzes the one-electron oxidation of phenols by a peroxide to form the corresponding phenoxy radicals. Usually, hydrogen peroxide is used as oxidizing reagent. During this process, two water molecules are formed [55] (Eq. 1) ... 

Scheme 1 Polymerization of phenols catalyzed by horseradish peroxidase (HRP)...

So far, most peroxidase-catalyzed oxidative polymerizations have been carried out using the enzyme horseradish peroxidase (HRP). Another useful peroxidase that catalyzes the oxidative polymerization of phenols is soybean peroxidase (SBP). While the use of either HRP or SBP may often lead to similar products and results [77], the enzyme activity, yield, and molecular weight of the resulting polymers can also sometimes depend strongly on the type of enzyme used for the polymerization process. For example, SBP was foimd to be superior to HRP for the efficient polymerization of bisphenol A [140], but the polymerization of phenol with SBP afforded... 

Fig.1 Chemoselective polymerization of phenol derivative by horseradish peroxidase...

A molar equivalent of hydrogen peroxide to monomer and horseradish peroxidase is a well-known redox system that catalyzes the free radical polymerization of phenol, anilines, and their derivatives [6-14]. Horseradish peroxidase-mediated polymerization of styrene and methyl methacrylate, with a monomer (styrene or methyl methacrylate) to hydrogen peroxide ratio of 40 1, did not occur in the absence of 2,4-pentanedione. Therefore, it is likely that this compound is involved in the initiation of free radical formation. A reasonable hypothesis for the horseradish peroxidase-catalyzed polymerization of vinyl monomers is that the enzyme is oxidized by hydrogen peroxide and passes from its native state through two catalytically active forms (Ez and Ezz). Each of these active forms oxidizes the initiator (b-diketone, 2,4-pentanedione) while the enzyme returns to the native form. The Ezz state of enzyme is oxidized by hydrogen peroxide to produce inactive enzyme, Ezzz, which spontaneously reverts to the native form of enzyme. The free radicals produced from the initiator generate radicals in the vinyl monomer to form polymer (Fig. 2). 

In the polymerization in the absence of template, control of the coupling selectivity (regiosdectivity) is often very difficirlt. The authors pay attention to the role of PEG in the polymerization. Phenol was polymerized using horseradish peroxidase (HRP) as a catalyst in phosphate buffer in the presence of PEG at room temperature tmder air. The PEG amormt greatly affected the reaction behaviors. 

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