Veratryl alcohol

Another iron porphyrin complex with 5,10,15,20-tetrakis(2, 6 -dichloro-3 -sulfonatophenyl)porphyrin was applied in ionic liquids and oxidized veratryl alcohol (3,4-dimethoxybenzyl alcohol) with hydrogen peroxide in yields up to 83% to the aldehyde as the major product [145]. In addition, TEMPO was incorporated via... 

VP (E.C. 1.11.1.46) was first described in liquid cultures of Pleurotus eryngii growing on peptone as nitrogen source [82, 83] and Bjerkandera sp. [84], VP is a heme containing structural hybrid between MnP and LiP, as it is able to oxidize Mn2+, veratryl alcohol, simple amines, phenolic, nonphenolic and high molecular... 

V. Christian, R. Shrivastava, D. Shukla, H. Modi and B.R. M. Vyas, Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidati decolorization of Remazol Brilliant Blue R. Enz. Microbiol. Technol., 36 (2005) 327-332. 

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. 

Reactivity of the Oxycomplex. The oxycomplex of lignin peroxidase does not react with veratryl alcohol, one of the lignin peroxidase substrates. Furthermore, the stability of the oxycomplex is not affected by veratryl... 

Unfractionated Preparatioii. Each harvest of the enzyme was concentrated to 401 and diafiltered with 0.01 M sodium acetate, pH 6.0. Enzyme concentrate was bound to Q-Sepharose and eluted with 0.4 M sodium acetate, pH 6.0. Fractions with activity were pooled, sterile filtered and stored in the cold in 10 mM veratryl alcohol. Before further purification, the solution was dialyzed. 

Activity Assays. The standard activity assay mixture of 3 ml contained about 0.1 U/ml lignin peroxidase, 0.4 mM veratryl alcohol (Fluka, purum >97%) and 0.1M sodium tartrate, pH 3.0. The reaction was started by adding 15 fil of 54 mM H2O2 to make a final concentration of 0.28 mM in the reaction. The production of veratraldehyde was followed by recording the change of absorbance for 12 seconds at 310 nm in a cuvette which was thermostated to 37°C. The reaction was started 24 seconds before the recording. One unit of lignin peroxidase is defined as the amount of enzyme required to oxidize one imol of veratryl alcohol to veratraldehyde in one minute. 

For veratryl alcohol treated samples, the concentration of veratryl alcohol in the activity assay mixture was 1-11 mM, the concentration of tartrate was 0.01,0.02 or 0.3 M and the enzyme activity at the beginning was 0.2 U/ml. The samples were at 30°C during the reaction. 

For determination of the Michaelis constant, the activity of purified lignin peroxidase was measured by using the standard activity assay method except that the concentration of veratryl alcohol was varied between 7 fiM and 2.67 mM. 

To study the effect of veratryl alcohol, purified lignin peroxidase or unfractionated enzyme preparation was incubated with buffer, pH 3.0 or 5.0. The concentration of veratryl alcohol in the incubation mixture was 0, 10 or 100 mM. Incubation times were 38 days at 20°C and 40 days at 4°C. The protein concentration of purified enzyme was 80/tg/ml and of unfractionated preparation 180 tg/ml. The incubation mixtures were sterile filtered to prevent microbial growth. 

Purification. One main peak was observed with detection at 405 nm, when purified lignin peroxidase was analyzed by Mono Q chromatography. The retention time of the main peak was 11.8 min and its area was 98.9% of the total peak area. Possibly the enzyme solution contains, however, two isoenzymes which have very similar properties. The Kj of the purified lignin peroxidase for veratryl alcohol was 139 on the basis of Eadie-Hofstee plot. 

Effect of Veratiyl Alcohol. At pH 5.0 the purified lignin peroxidase was not inactivated under the conditions tested. At pH 3.0 the enzyme lost its activity when incubated at 20°C for 38 days, and the presence of veratryl alcohol could not stabilize it. 100 mM veratryl alcohol even inactivated the enzyme to some extent. Ionic strength did not significantly affect the activities. The effect of veratryl alcohol was the same when unfractionated enzyme was used. This time the ionic strength in the activity assay mixture affected the activities, probably because one enzyme in the unfractionated preparation is sensitive to high ionic strength 12),... 

Aitken and Irvine 17) have recently reported on the stability characteristics of lignin peroxidases. They also have shown that the enzyme was most stable at pH 4.5, although higher pH values were not tested. The stability was dependent on protein concentration and veratryl alcohol had a stabilizing effect. The latter result was contrary to our experience. 

These considerations have prompted the formulation of a provisional rationale for the ligninolytic processes elicited by P. chrysosporium. The scheme itself is an extension of a suggestion devised to account for the fate of veratryl alcohol in P. chrysosporium cultures (34). 

These findings led to the proposition that the veratryl alcohol is degraded via the quinone intermediates (Figure 5) to CO2 through a series of transformations involving lignin peroxidase, perhydroxy radicals and the NADP-dependent aryl alcohol oxidoreductase. Veratraldehyde, the major product of lignin peroxidase catalyzed veratryl alcohol oxidation, is rapidly reduced back to veratryl alcohol it is the further metabolism of the side products of the oxidative process, viz. the quinones and lactones, that drives the overall transformation towards completion (34). 

Figure 3. Polymerization of 1 mg mL alkali-isolated straw lignin (dotted line) by 0.8 units mL lignin peroxidase (as concentrated extracellular P, chrysosporium culture fluid) at pH 4.0 in the absence (broken line) and presence (solid line) of 1.2 /imole mL veratryl alcohol 0.54 /imole H2O2 added at 2 hourly intervals for 24 h. Sephadex G75/aqueous 0.5% (w/v) NaOH elution profiles adapted and redrawn from reference 32.

Figure 4. Biodegradation of HCl/dioxane-isolated C-labeled straw lignin by 50 mL washed P. chrysosporium mycelial pellets ( ) in the presence of 2.5 units glucose oxidase accelerative effect engendered by 2 units lignin peroxidase in absence (Q) and presence (H) of 75 /imole veratryl alcohol. Adapted and redrawn from reference 34.

Figure 5. Plausible pathway for P, chrysosporium mediated veratryl alcohol degradation. Adapted and redrawn from reference 34.

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