Characterization and Application of Alcohol Dehydrogenase in the Biotransformation of Veratryl Alcohol

Veratryl Alcohol-Mediated Indirect Oxidation of Phenol by Lignin Peroxidase

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K(m app) value in the steady state and the apparent dissociation constant (K(D)) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.

Aryl-alcohol oxidase and lignin peroxidase from the white-rot fungus Bjerkandera adusta

IntroductionLignin is a three dimensional hydrophobic plant polymer derived from the random coupling of phenylpropanoid precursors. The chemical and physical characteristics of lignin require a nonspecific, extracellular oxidative process for biodegradation. White rot basidiomycetes are the only group of organisms having an efficient extracellular ligninolytic system. These fungi produce peroxidases and laccases that are involved in the initial attack of lignin. The peroxidases work with H 2 O 2 which is also enzymatically produced by the fungi by different H 2 O 2 -producing oxidases. For their proper operation, peroxidases require appropriate cofactors which are the best substrates of the enzymes. Lignin peroxidase (LiP) uses the de novo produced secondary metabolite, veratryl alcohol (3,4-dimethoxybenzyl alcohol) as cofactor; whereas, the cofactor of manganese peroxidase (MnP) is the metal Mn(II) which occurs naturally in wood. The in vitro oxidation of lignin preparations by LiP or MnP is only feasible in the presence of veratryl alcohol or manganese, respectively. Additionally, simple aliphatic organic acid metabolites such as oxalate are involved in lignin degradation. On the one hand, oxalate is a good chelator of Mn(III) generated by MnP. This complex is relatively stable forming a low molecular weight diffusible oxidant capable of oxidizing phenolic lignin. Oxalate is also oxidized by ligninolytic enzymes in the presence of their cofactors generating reactive oxygen species that possibly participate in the oxidation of lignin.The low molecular weight cofactors also influence the physiological regulation of white rot fungi. Veratryl alcohol is known to increase LiP activities in white rot fungal cultures, although, veratryl alcohol itself does not induce lip gene transcription. Manganese is essential for the induction of mnp gene expression and MnP activity in various white rot fungi. In contrast, manganese lowers LiP production so that the highest LiP activities are measured under manganese deficiency. Nonetheless manganese has no direct repressive effect on lip gene transcription.Despite the great research efforts conducted so far on various white rot fungi, the true mechanism of lignin degradation is still not fully understood. However, it is becoming clear that low molecular weight cofactors are important catalytic and physiological regulating components of the ligninolytic system.This thesis was dedicated to study the interrelationship between low molecular weight cofactors and ligninolytic enzymes. Bjerkandera sp. strain BOS55 was used as the fungus of study, since it was found to be an outstanding producer of ligninolytic enzymes and a great variety of secondary aryl metabolites. Moreover, this strain was observed to be a good degrader of polycyclic aromatic hydrocarbons (PAH) and biobleacher of kraft pulp in various screenings. The main objective of the thesis was to gain insight into the role of veratryl alcohol and manganese in regulating the physiology and participating in the ligninolytic system of Bjerkandera sp. strain BOS55.Chapter 2In Chapter 2, it was demonstrated for the first time that manganese inhibits the biosynthesis of veratryl alcohol in white rot fungi. This explains at least in part the general observation that LiP production is lowered in the presence of manganese although manganese itself does not repress the transcription of lip genes. The ten-fold increase in veratryl alcohol biosynthesis caused by manganese deficiency stimulated the LiP titres by protecting the enzyme from inactivation by physiological levels of H 2 O 2 . Adding veratryl alcohol to manganese containing cultures of the fungus sustained high LiP titres similar to that found under manganese deficient conditions. Moreover, a good correlation was observed between the LiP titres and veratryl alcohol concentrations irrespective of whether veratryl alcohol was produced by the fungus or added exogenously.Chapter 3In Chapter 3, the mechanism resulting in manganese inhibition of veratryl alcohol biosynthesis was studied. Potential biosynthetic precursors of veratryl alcohol were added to manganese deficient and sufficient cultures of Bjerkandera sp. strain BOS55 in order to bypass the inhibited step. The addition of fully methylated precursors (veratrate, veratraldehyde) equally increased the production of veratryl alcohol irrespective of the manganese concentration. This observation indicated that the reduction of the benzylic acid to benzyl alcohol group is not inhibited by manganese. All the other known precursors such as phenylalanine, cinnamate, benzoate/benzaldehyde as well as the partially hydroxylated benzylic compounds (e.g. 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate, vanillate, isovanillate) did increase the veratryl alcohol production in the presence of manganese but never as much as that under manganese deficiency. From these observations it was concluded that no single step along the biosynthetic pathway was inhibited by the presence of manganese. Instead, the availability of phenolic precursors is limited when manganese was added. From this study, we also learned that there are several alternative precursors resulting in increased veratryl alcohol production which can potentially originate from lignin degradation. In addition, it was demonstrated that many of the veratryl alcohol precursors (phenylalanine, cinnamate, benzoate, 4-hydroxybenzoate) enhanced the production of anisyl and chloroanisyl metabolites indicating the existence of common precursors in these biosynthetic pathways. Deuterium labelled benzoate and 4-hydroxybenzoate were converted to a broad spectrum of labelled aryl metabolites.Chapter 4In Chapter 4, the interrelationship between cofactors and peroxidases in cultures grown on natural substrates (beech wood and hemp stem wood sawdust) was studied. Beech wood and hemp stem wood substrates, which contain 6 and 25 mg kg -1dry wood of soluble manganese, respectively were favourable for MnP production. Many studies in the past have failed to demonstrate the presence of LiP on natural substrates even in fungi having lip genes. Surprisingly, Bjerkandera sp. strain BOS55 produced very high LiP titres on the wood substrates. The high LiP activity observed suggested that this enzyme may have an important role during wood decay. The significant LiP production in spite of the presence of manganese can be explained by the very high production of veratryl alcohol throughout the incubation. The fact that veratryl alcohol was produced in high amounts although soluble manganese was always present apparently contradicts the findings presented in Chapter 2 . However, the high veratryl alcohol production can be explained by the presence of lignin degradation products such as 4-hydroxybenzoate, protocatechuate, vanillate, isovanillate entering the biosynthetic pathway of veratryl alcohol as was suggested in Chapter 3.Chapter 5Bjerkandera sp. strain BOS55 is a good MnP producer. In Chapter 5, the conditions for optimal MnP production were examined. The highest production was observed in nitrogen rich medium with 0.2 to 1 mM manganese at a pH value of 5.2 and at a temperature of 30 oC. Two interesting phenomena were discovered while studying the physiology of MnP production. Firstly, significant MnP production was also observed in the absence of manganese which previously only has been shown to be the case for Pleurotus spp. However, unlike Pleurotus spp., MnP production in cultures of Bjerkandera sp. strain BOS55 was enhanced in response to increasing Mn levels. Secondly, it was demonstrated that the addition of various organic acid metabolites significantly increased the MnP titres under manganese sufficient conditions. The best results were obtained with glycolate.Chapter 6In Chapter 6, the study on the MnP production was continued in order to better understand why MnP is produced in the absence of manganese and to elucidate the induction mechanism under manganese deficient conditions. In the absence of manganese, oxalate and related organic acids, such as glycolate or glyoxylate were found to induce MnP production. The stimulatory effect of organic acids on MnP production was demonstrated to be due to the increased production of MnP proteins. Additionally, it was shown that the acids induced mnp gene transcription (unpublished data).The major MnP isozyme produced in the absence of Mn and in the presence of glycolate was purified and characterized. Like other MnP isozymes, this enzyme was able to efficiently oxidize Mn. However, unlike other MnP isozymes, it was also able to directly oxidize veratryl alcohol and 1,4-dimethoxybenzene with a very high affinity in the absence of manganese. These nonphenolic substrates are typical substrates of LiP. Methoxyphenols and aromatic amines could also be oxidized in the absence of manganese. The optimal pH for the manganese independent oxidation of all the substrates tested was 3.0 similar to that observed for LiP isozymes. On the other hand, the oxidation of Mn(II) and consequently the manganese dependent oxidation of phenolic substrate reached the highest rate at pH 4.5 as described for many MnP isozymes. The kinetic values in terms of turnover number and affinity for Mn(II) and veratryl alcohol oxidation were similar to those found for other MnP and LiP isozymes. Therefore, the Bjerkandera MnP could be best described as a hybrid enzyme between MnP and LiP, having a binding site for Mn(II) as well as for methoxy aromatics/phenols. This conclusion is supported by the finding that Mn(II) at concentrations greater than 0.1 mM severely inhibited veratryl alcohol oxidation by the enzyme; whereas Mn(II) has no effect on LiP. The fact that this enzyme can oxidize Mn(II) as well as directly oxidize veratryl alcohol and other aromatic amines and phenols clarifies the physiological relevance of the occurrence of this MnP under Mn deficient and sufficient conditions.ConclusionsIn conclusion, this thesis has resulted in new insights into the key regulatory role of manganese in lignin degradation such as the repressive effect of manganese on the production of aryl metabolites. The addition of manganese at concentrations as low as 33μM completely changed the pattern of peroxidases and aryl metabolites production compared to that under manganese deficiency. Additionally, it was observed that MnP is purposefully produced and regulated by organic acids in the total absence of manganese. Under these conditions, a novel type of MnP-LiP hybrid isozyme was isolated which was functional under any manganese regime using either veratryl alcohol or Mn(II) as a cofactor. Under natural conditions, such an enzyme would have considerable physiological significance since soluble manganese is known to be leached out or become oxidized to insoluble MnO 2 during fungal attack, possibly resulting in manganese deficient areas in wood. In manganese deficient areas, veratryl alcohol biosynthesis is stimulated and the enzyme can use this secondary metabolite as an alternative to manganese.Future research should elucidate the newly discovered role of organic acids in regulating MnP in white rot fungi. The physiological significance of this regulation may be due to the role of organic acids as an important source of reduced oxygen radicals. The oxidative stress resulting from radicals may be the signal for MnP gene expression as has been shown to be the case with H 2 O 2 . The radicals are also required for extensive degradation of lignin. Additionally, the presence of organic acids may serve as an early warning for the upcoming presence of bioavailable manganese. The fungal organic acid metabolites are well known for their ability to solubilize insoluble MnO 2 deposits in fungal attacked wood.

Effect of Superoxide and Superoxide Dismutase on Lignin Peroxidase-Catalyzed Veratryl Alcohol Oxidation

Lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA), a secondary metabolite of the fungus, to veratryl aldehyde (VAD). The oxidation of VA does not seem to be simply one-electron oxidation by LiP compound I (LiPI) to its cation radical (VA.+) and the second by LiP compound II (LiPII) to VAD. Moreover, the rate constant for LiPI reduction by VA (3 x 10(5) M-1 s-1) is certainly sufficient, but the rate constant for LiPII reduction by VA (5.0 +/- 0.2 s-1) is insufficient to account for the turnover rate of LiP (8 +/- 0.4 s-1) at pH 4.5. Oxalate was found to decrease the turnover rate of LiP to 5 s-1, but it had no effect on the rate constants for LiP with H2O2 or LiPI and LiPII, the latter formed by reduction of LiPI with ferrocyanide, with VA. However, when LiPII was formed by reduction of LiPI with VA, an oxalate-sensitive burst phase was observed during its reduction with VA. This was explained by the presence of LiPII, formed by reduction of LiPI with VA, in two different states, one that reacted faster with VA than the other. Activity during the burst was sensitive to preincubation of LiPI with VA, decaying with a half-life of 0.54 s, and was possibly due to an unstable intermediate complex of VA.+ and LiPII. This was supported by an anomalous, oxalate-sensitive, LiPII visible absorption spectrum observed during steady state oxidation of VA. The first order rate constant for the burst phase was 8.3 +/- 0.2 s-1, fast enough to account for the steady state turnover rate of LiP at pH 4.5. Thus, it was concluded that oxalate decreased the turnover of LiP by reacting with VA.+ bound to LiPII. The VA.+ concentration measured by electron spin resonance spectroscopy (ESR) was 2.2 microM at steady state (10 microM LiP, 250 microM H2O2, and 2 mM VA) and increased to 8.9 microM when measured after the reaction was acid quenched. Therefore, we assumed the presence of two states of VA.+ bound to LiPII, one ESR-active and one ESR-silent. The ESR-silent species, which could be detected after acid quenching, would be responsible for the burst phase. Both of the VA.+ species disappeared in the presence of 5 mM oxalate. The ESR-active species reached a maximum (3.5 microM) at 0.5 mM VA under steady state. From these studies, a mechanism for VA oxidation by LiP is proposed in which a complex of LiPII and VA.+ reacts with an additional molecule of VA, leading to veratryl aldehyde formation.

Seven natural phenols and two synthetic compounds were evaluated by means of cyclic voltammetry as enhancers for the oxidation of the lignin model compound veratryl alcohol (VAl) and a sulfonated lignin (SL). Their electrochemical behaviors and catalytic efficiencies (CEs) against both substrates were assessed as a function of pH. A general increase in CE of the phenols was for the first time observed in the oxidation of VAl at pH 7 and 8. Methyl syringate (MS), syringic acid (SRC), and syringaldehyde (SRD) exhibited the highest CEs against VAl among the studied phenolic compounds despite the reduced stabilities of their phenoxy radicals. This was a result of favorable stability−reactivity balances, which were apparently influenced by both the chemical structures of the enhancers and the experimental conditions. Violuric acid (VAc) proved the most efficient compound in oxidizing lignin, followed by SRD and MS, which showed regeneration in the interval of pHs studied

Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol brilliant blue R

Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol Brilliant Blue R

Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA) to veratryl aldehyde, with the enzyme-bound veratryl alcohol cation radical (VA.+) as an intermediate [Khindaria et al. (1995) Biochemistry 34, 16860-16869]. The decay constant we observed for the enzyme generated cation radical did not agree with the decay constant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 270, 16745-16748] for the chemically generated radical. Moreover, we have found that the chemically generated VA.+ formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxygen-saturated solutions, with a decay constant of 1.2 x 10(3) s-1, and with a second-order mechanism in argon-saturated solution. The first-order decay constant was pH- independent suggesting that the rate-limiting step in the decay was deprotonation. When VA.+ was generated by oxidation with LiP the decay also occurred with a first-order mechanism but was much slower, 1.85 s-1, and was the same in both oxygen- and argon-saturated reaction mixtures. However, when the enzymatic reaction mixture was acid-quenched the decay constant of VA.+ was close to the one obtained in the Ce(IV) oxidation system, 9.7 x 10(2) s-1. This strongly suggested that the LiP-bound VA.+ was stabilized and decayed more slowly than free VA.+. We propose that the stabilization of VA.+ may be due to the acidic microenvironment in the enzyme active site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potential of VA.+/VA couple using cyclic voltammetery. Due to the instability of VA.+ in aqueous solution the reversible redox potential was measured in acetone, and was 1.36 V vs normal hydrogen electrode. Our data allow us to propose that enzymatically generated VA.+ can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lignin or pollutant degradation.

Application of a biocatalyst at an industrial scale primarily depends on its intrinsic properties, the nature of the support materials, and the scalability of the catalyst. Support materials play an important role in the biocatalytic performance with their mechanical and thermal properties, accessibility, nontoxicity, and ease of derivatization for immobilizion of enzyme. Chicken feather, a readily available poultry waste material, was processed and modified for enzyme immobilization. Free Trametes maxima laccase (TML) was immobilized on the amino-functionalized chicken feather particles (TML@ACFP), and an immobilization yield of 74.24% was achieved. Immobilization improved the pH optimum from 3.0 (TML) to 4.1 (TML@ACFP) and temperature optimum by 5 °C. The kinetics and thermodynamics of thermal inactivation of free TML and immobilized TML@ACFP were studied over the temperature range from 55 to 65 °C. The apparent half-life (t1/2) and decimal reduction time (D-value) for TML was found to be 154.9 and 514.8 min and 256.8 and 853.3 min for TML@ACFP, respectively, at 55 °C. The activation energy for deactivation (Ed) was found to be 117.48 and 137.85 kJ/mol for TML and TML@ACFP, respectively. Gibbs free energy (ΔG) and change in enthalpy (ΔH) were increased from 106.58 and 114.75 kJ/mol for TML to 107.96 and 135.12 kJ/mol for TML@ACFP, respectively, demonstrating its higher stability. The biocatalytic transformation was performed with TML@ACFP for the oxidation of lignin model compound veratryl alcohol. So far, this is the first strategy that uses chicken feather waste derived novel support material for immobilization of enzyme and its application in the biotransformation.

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).

Four air-stable one-dimensional copper(II) coordination polymers (CP1-CP4) with azide linkers were synthesized using tridentate NNS and NNN ligands. Single-crystal X-ray diffraction (XRD) analysis confirmed the molecular structures of CP1, CP3, and CP4. In the presence of TEMPO, all four coordination polymers demonstrated effective catalytic activity for the selective aerobic oxidation of veratryl alcohol, a biomass model compound, under base-free conditions. CP4 exhibited the best catalytic efficiency. Oxidations were conducted at ambient temperature (40 °C) utilizing air as a sustainable oxidant. Selective oxidation of veratryl alcohol to veratraldehyde was also conducted in the presence of a catalytic amount of base (5 mol %), and enhanced reactivity was observed. The green solvents, acetone, and water, were used to maximize sustainability. The optimized reaction conditions were applied to broaden the substrate scope of various lignin model alcohols and substituted benzylic alcohols with wide electronic variability. CP4 exhibited high recyclability, consistently providing quantitative yields even after ten consecutive runs. The catalytic protocol demonstrated sustainability and environmental compatibility, as evidenced by a low E-factor (4.29) and a high Eco-scale score (90). Based on experimental evidence and theoretical calculations, a plausible catalytic cycle was proposed. Finally, the sustainability credentials of the different optimized reaction protocols were evaluated using the CHEM21 green metrics toolkit.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.

On a revised mechanism of side product formation in the lignin peroxidase catalyzed oxidation of veratryl alcohol