Veratryl alcohol oxidases

Enzyme Production and Isolation. The production and isolation of veratryl alcohol oxidase (VAO) was described earlier (25). Laccase produced from the same 12-day culture (8 litres) was isolated from the supernatant by precipitation at 0°C with ammonium sulfate (80% saturation). The precipitate was suspended in 0.05 M Na acetate buffer, pH 5.0 and dialysed overnight against 4 litres of buffer. The soluble material was concentrated by ultrafiltration (Amicon PM10) to about 60 mL and applied to a DEAE-Bio-gel A column (2.5 cm x 35 cm). The column was washed with 20 mL of the same buffer, then eluted with a linear gradient from 0 to 0.6 M NaCl (total volume 550 mL). Fractions were monitored for VAO and laccase activity as described below. 

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. 

Enzyme Properties. The two isolated veratryl alcohol oxidases had very similar properties (Table I). The difference in isoelectric points might be accounted for by aspartate content all other amino acid contents except glycine were the same within experimental error (5%). The specific activities (veratryl alcohol as substrate) were significantly different, but both enzymes contained a flavin prosthetic group (25) and converted one molecule of oxygen to one molecule of hydrogen peroxide during alcohol oxidation. 

A possible reductive role for veratryl alcohol oxidase is proposed in Figure 5. Laccases from C. versicolor can produce both polymerization and depolymerization of lignin (29). In phenolic lignin model dimers, laccase can perform the same electron abstraction and subsequent bond cleavage as found for lignin peroxidase (30). The phenolic radical is however likely to polymerize unless the quinoid-type intermediates can be removed, for example by reduction back to the phenol. Veratryl alcohol oxidase, in... 

Ander, P., and Marzullo, L. (1997). Sugar oxidoreductases and veratryl alcohol oxidase as related to lignin degradation./. Biotechnol. 53,115-131. 

Smith AT, Doyle WA (2006) Engineered peroxidases with veratryl alcohol oxidase activity. Patent (International) WO/2006-114616... 

Finally, several enzymes from white-rot fnngi are able to generate hydrogen peroxide, which is required for peroxidatic activity. These include glucose-2-oxidase [123] and veratryl alcohol oxidase [124], Hydrogen peroxide can also be generated nonenzymically by several rontes. 

R Bourbonnais, MG Paice. Veratryl alcohol oxidases from the lignin-degrading basidi-omycete Pleurotus sajor-caju. Biochem J 255 445-450, 1988. 

INHIBITION OF THE VERATRYL ALCOHOL OXIDASE ACTIVITY OF LIGNIN PEROXIDASE H8 BY HYDROXYLAMINODINITROTOLUENE... 

Figure 9. Effect of increasing amounts of hydroxylaminodinitrotoluenes on the veratryl alcohol oxidase activity of lignin peroxidase H8 (32). The reaction mixture contained in a final volume of 600 pi 0.05 pM lignin peroxidase H8, 40 mM sodium tartrate buffer, pH 3.5, 130 pM H2O2, 5.6 mM veratryl alcohol, and various concentrations of hydroxylaminodinitrotoluene (both isomers) [pM] 0, 5,A 10, 16, O 21, A 24, .

Veratryl alcohol plays an important role as a radical cation mediator in lignin biodegradation (18). The oxidative decarboxylation of oxalate (1) or EDTA (42) by the lignin peroxidase is mediated by the veratryl alcohol cation radical (VA ) leading to an apparent inhibition of the veratryl alcohol oxidase activity of the lignin peroxidase. 

An unexpected result was the effect of H202on the lag phase and the lignin peroxidase activity at constant levels of veratryl alcohol and inhibitor (Fig. 12). An increase of the concentration of H2O2 from 64 pM to 512 pM in the assay mixture led to an extension of the lag period. At lower H202-concentrations, down to 13 pM, the veratryl alcohol oxidase... 

---