Oxidation of veratryl alcohol

Although compound I formation is not influenced by pH, reactions of compounds I and II are significantly affected by pH. These reactions are acid-catalyzed 16,17). The rate constant for the oxidation of veratryl alcohol or fenocyanide by lignin peroxidase compound I is 10 times greater at pH 3.5 than at pH 6.0. The enhancement in rate is of the same magnitude for compound II reacting with veratryl alcohol. Therefore, the observed pH dependency for Vmax in catalysis is due to the pH-dependent reactions between the compounds I and n and the reducing substrates. 

It has now been found that the quinones 13, 14 and 15 from the aerobic oxidation of veratryl alcohol by lignin peroxidase were also reduced by fungal mycelium to yield the corresponding hydroquinones. For quinone 14 this reduction had already been reported by Buswell et al. (17) in a study of vanillic acid metabolism. 

Metabolism of Monomeric Lignin Models. Leisola and coworkers (31,37) have reported on the products of the lignin peroxidase catalyzed oxidation of veratryl alcohol. Veratraldehyde was the major product (> 70% yield), together with a number of minor products, the quinones 13 and 14 and the ringopened lactones 16 and 17. In addition, Shimada et al. (38,39) showed that the 6-lactone 18 was also formed. Recently we obtained evidence that the ortho-quinone 15 and the 6-lactone 19 were also products of the lignin peroxidase catalyzed oxidation of veratryl alcohol (Schmidt ef al, Biochemistry, in press). Mechanisms for the formation of those compounds... 

Lignin peroxidase activity, (i.e., peroxide-dependent oxidation of veratryl alcohol at pH 3) was not detected over the 30 days tested, while laccase appeared at day 7. Culture medium from day 7 onwards could also oxidize veratryl alcohol to aldehyde with concomitant conversion of oxygen to hydrogen peroxide. This activity, which was optimal at pH 5.0, was named veratryl alcohol oxidase (VAO). The extracellular oxidative enzyme activities (laccase and veratryl alcohol oxidase) could be separated by ion-exchange chromatography (Figure 2). Further chromatography of the coincident laccase and veratryl alcohol oxidase (peak 2), as described elsewhere (25) resulted in the separation of two veratryl alcohol oxidases from the laccase. 

The oxidation of veratryl alcohol was carried out under air at room temperature. The reaction mixture contained 30 //moles of veratryl alcohol, 0.05 //moles of TDCSPPFeCl, 30 //moles of m-chloroperbenzoic acid (mCPBA) made up to 6 ml using phosphate buffer or as otherwise indicated in Table... 

I. 4-methoxyacetophenone (30 //moles) was added as an internal standard. The reaction was stopped after 2 hours by partitioning the mixture between methylene chloride and saturated sodium bicarbonate solution. The aqueous layer was twice extracted with methylene chloride and the extracts combined. The products were analyzed by GC after acetylation with excess 1 1 acetic anhydride/pyridine for 24 hours at room temperature. The oxidations of anisyl alcohol, in the presence of veratryl alcohol or 1,4-dimethoxybenzene, were performed as indicated in Table III and IV in 6 ml of phosphate buffer (pH 3.0). Other conditions were the same as for the oxidation of veratryl alcohol described above. TDCSPPFeCl remaining after the reaction was estimated from its Soret band absorption before and after the reaction. For the decolorization of Poly B-411 (IV) by TDCSPPFeCl and mCPBA, 25 //moles of mCPBA were added to 25 ml 0.05% Poly B-411 containing 0.01 //moles TDCSPPFeCl, 25 //moles of manganese sulfate and 1.5 mmoles of lactic acid buffered at pH 4.5. The decolorization of Poly B-411 was followed by the decrease in absorption at 596 nm. For the electrochemical decolorization of Poly B-411 in the presence of veratryl alcohol, a two-compartment cell was used. A glassy carbon plate was used as the anode, a platinum plate as the auxiliary electrode, and a silver wire as the reference electrode. The potential was controlled at 0.900 V. Poly B-411 (50 ml, 0.005%) in pH 3 buffer was added to the anode compartment and pH 3 buffer was added to the cathode compartment to the same level. The decolorization of Poly B-411 was followed by the change in absorbance at 596 nm and the simultaneous oxidation of veratryl alcohol was followed at 310 nm. The same electrochemical apparatus was used for the decolorization of Poly B-411 adsorbed onto filter paper. Tetrabutylammonium perchlorate (TBAP) was used as supporting electrolyte when methylene chloride was the solvent. 

Table I. Oxidation of veratryl alcohol by TDCSPPFeCl and mCPBA in various solvents...

Furthermore, oxidation of veratryl alcohol by sulfonated metalloporphyr-ins yields the same two major products observed when LiP is used [63a]. 

In some enzymes, the protein radical appears to participate in substrate oxidation. Evidence exists for the involvement of a surface tryptophan in the oxidation of veratryl alcohol by the ligninase from Phanerochaete chrysosporium [33]. Similarly, tryptophan radicals on the surface of the versatile peroxidases from Pleurotus eryngii and Bjerkandera adjusta [34—36], and a tyrosine in the LiP from Trametes cervina [33], are thought to be involved in substrate oxidation. 

Fig. 5.5 Oxidation of veratryl alcohol to a radical cation that can oxidize other substrates (RX) or can be further oxidized to give veratryl aldehyde...

As already mentioned, in some enzymes radicals generated on surface tryptophan and tyrosine radicals by electron transfer to the ferryl species are involved in abstraction of electrons from substrates [33-36, 40]. Mutation of Trpl71 on the surface of P. chrysosporium LiP to a phenylalanine or serine completely suppresses the veratryl alcohol oxidizing activity of the enzyme [40]. A similar depression in the oxidation of veratryl alcohol occurs on mutation of Trpl64 in the versatile peroxidase from P. eryngii [34, 35]. 

The electrical potential and/or current required for electroenzymatic treatment have been shown to be lower than those needed in electrochemical treatment, which are not economically viable for large-scale. Electroenzymatic oxidation by peroxidases was proposed for the oxidation of veratryl alcohol by LiP [40], Then, electroenzymatic reactors have been used for the treatment of petrochemical wastewater [91], dyes, and textile wastewater [90, 92, 118] and phenol streams [93] utilizing peroxidase immobilized onto inorganic porous Celite beads or directly onto the electrode. The integration of a second electrochemical reactor, which generated hypochlorite in the presence of sodium chloride, has been used for indirect oxidation of the reaction products of the electroenzymatic treatment [91]. 

Lee K, Moon SH (2003) Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase. J Biotechnol 102 261-268... 

Estimated in the oxidation of veratryl alcohol. LiP purified from [10]... 

Kervinen et al. (2005) investigated a homogeneous catalytic reaction, namely, the oxidation of veratryl alcohol to its aldehyde in the liquid phase. In this case, UV-vis spectroscopy, performed by immersing a fiber probe in the reacting medium, allowed detection of changes in the Co (salen) catalyst as well as monitoring of product formation. 

Kumar A, Jain N, Chauhan SMS (2007) Biomimetic oxidation of veratryl alcohol with H Oj catalyzed by iron (III) porphyrins and horseradish peroxidase in ionic liquid. Synlett 411-414... 

MY Balakshin, CL Chen, JS Gratzl, AG Kirkman, H Jakob. Kinetic studies on oxidation of veratryl alcohol by laccase-mediator system. Part 1. Effects of mediator concentration. Holzforschung 54(2) 165-170, 2000. 

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