Kinetics of Veratryl Alcohol Oxidation by Lignin Peroxidase and in situ Generated Hydrogen Peroxide in an Electrochemical Reactor

The cathodic reduction of oxygen to hydrogen peroxide, the current efficiency for the production of H₂O₂ and the oxidation of veratryl alcohol with an in situ generated hydrogen peroxide‐lignin peroxidase complex were studied in this paper. The complex was prepared by utilizing a novel preparation technique in an electrochemical reactor. The oxidation of veratryl alcohol (VA; 3,4‐dimethoxybenzyl alcohol) was carried out with or without lignin peroxidase under an electric field. The redox properties of veratryl alcohol on a carbon electrode in the presence of lignin peroxidase have been investigated using cyclic voltammetry. The kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to the oxidation when hydrogen peroxide was supplied externally. Further, the oxidation of veratryl alcohol by lignin peroxidase was optimized in terms of enzyme dosage, pH, and electrical potential. The novel electroenzymatic method was found to be effective using in situ generated hydrogen peroxide for the oxidation of veratryl alcohol by lignin peroxidase.     

Lignin and veratryl alcohol are not inducers of the ligninolytic system of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. In this work, we investigated the roles of veratryl alcohol and lignin in the ligninolytic system of P. chrysosporium BKM-F-1767 cultures grown under nitrogen-limited conditions. Cultures supplemented with 0.4 to 2 mM veratryl alcohol showed increased lignin peroxidase activity. Addition of veratryl alcohol had no effect on Mn-dependent peroxidase activity and inhibited glyoxal oxidase activity. Azure-casein analysis of acidic proteases in the extracellular fluid showed that protease activity decreased during the early stages of secondary metabolism while lignin peroxidase activity was at its peak, suggesting that proteolysis was not involved in the regulation of lignin peroxidase activity during early secondary metabolism. In cultures supplemented with lignin or veratryl alcohol, no induction of mRNA coding for lignin peroxidase H2 or H8 was observed. Veratryl alcohol protected lignin peroxidase isozymes H2 and H8 from inactivation by H2O2. We conclude that veratryl alcohol acts as a stabilizer of lignin peroxidase activity and not as an inducer of lignin peroxidase synthesis.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.     

Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production byPhanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.     

Comparison of two assay procedures for lignin peroxidase

The most widely accepted assay for detecting lignin peroxidase, based on the oxidation of veratryl alcohol to veratraldehyde, suffers from some drawbacks. At 310 nm, the wavelength at which the assay is performed, some other materials like lignins, quinonic compounds and aromatics also exhibit strong absorbance thus interfering with the estimation when present in the media. The present study reports the lignin peroxidase production by some white rot fungi under different nutritional conditions. The veratryl alcohol oxidation assay procedure for lignin peroxidase has been compared with another method based on the oxidation of the dye azure B involving absorbance measurements in the visible range. The latter method proved to be much more advantageous over the veratryl alcohol oxidation method, in media supplemented with malt extract, lignin preparations and agricultural residues. The enzyme production by veratryl alcohol assay could be detected only in mineral salts broth. By the azure B assay the enzyme activity was detected in all the media tested. The supplements gave varied response in different media. Veratryl alcohol enhanced the enzyme production in malt extract broth and mineral salts malt extract broth. Among the lignin preparations Indulin AT increased the lignin peroxidase titres from 2 to 20 fold in different fungi. Similarly, wheat straw supplemented in mineral salts broth and malt extract broth, separately, strongly stimulated the lignin peroxidase production. The above studies revealed that azure B assay may act as a substitute or equivalent method.     

A critical study of lignin peroxidase activity assay by veratryl alcohol oxidation

Summary The effects of various parameters on Phanerochaete chrysosporium lignin peroxidase activity as obtained in ligninase assay based on the oxidation of veratryl alcohol were investigated. Marked differences in the ligninase activity were observed when the temperature and pH were varied within the ranges of 23 to 37°C and 2.5 to 4.0, respectively, reported to have been used by various research groups. Further, both veratryl alcohol, and hydrogen peroxide concentration had a significant effect on ligninase activity.     

Lignin and manganese peroxidase activity in extracts from straw solid substrate fermentations

Manganese and lignin peroxidase (MnP, LiP) activities were measured in straw extracts from cultures of Phanerochaete chrysosporium. Out of six MnP substrates, the MBTH/DMAB (3-methyl-2-benzothiazolinone hydrazone/3-(dimethylamino)benzoic acid), gave the highest MnP activity. Detection of LiP activity as veratryl alcohol oxidation was inhibited by phenols in the straw culture extracts. Appropriate levels of veratryl alcohol and peroxide (4 mM and 0.4 mM, respectively), and a restricted sample volume (not larger than 10%) were necessary to detect activity.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase

This paper reports the formation of veratraldehyde by electroenzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase. The novel electroenzymatic method was found to be effective for replacement of hydrogen peroxide by an electrochemical reactor, which is essential for enzyme activity of lignin peroxidase. The effects of operating parameters such as enzyme dosage, pH, and electric potential were investigated. Further, the kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to oxidation when hydrogen peroxide was supplied externally.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn²⁺, veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn²⁺ and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

Central composite experimental design in the optimization of lignin peroxidase production in shake cultures by free and immobilized Phanerochaete chrysosporium

An orthogonal 2³-factorial experimental design was employed in the multivariate optimization of lignin peroxidase production by Phanerochaete chrysosporium in shake cultures both as free pellets and as immobilized on nylon-web, and to provide knowledge on the process for scale-up and control. It was observed that a short starving period after the growth of the mycelium and the depletion of the initial carbon source, followed by the addition of glucose to about 1 g/dm³ level together with the activator markedly enhanced lignin peroxidase production. The optimum concentration of veratryl alcohol as an activator, 2.5 mM with the immobilized fungus system was about double of that with free pellets, and about 6 to 10 times that most often previously employed. Benzyl alcohol could also be used as an activator at an optimal level of about 5.2 mM, although the lignin peroxidase activities obtained were somewhat lower than those with veratryl alcohol. The immobilization appeared to stabilize P. chrysosporium against shear effects, and in the presence of the surfactant Tween 80 in particular high lignin peroxidase activities were obtained already one to two days after the activation.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Comparison of ligninolytic activities of selected white-rot fungi

Summary Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occasion of his 60th birthday     

Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

A Mn(2+)-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn(2+) oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn(2+) and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The K(m) for Mn(2+) was determined to be 1.57 mM and the k(cat) = 5.45 s(-1). These results indicate that the mutated lignin peroxidase is less effective in Mn(2+) oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.     

Amperometric detection of lignin-degrading peroxidase activities from Phanerochaete chrysosporium

An amperometric enzyme sensor for rapid and simultaneous detection of the lignin-degrading peroxidase activities secreted by Phanerochaete chrysosporium was developed, using H₂O₂, hydroquinone and veratryl alcohol as substrates. In the amperometric measurement, samples of culture filtrate with different lignin-degrading peroxidase activities measured by spectrophotometry were placed into electrochemical cells. The slope of the current increase (Δ_current/Δ_time) upon the addition of H₂O₂ into the culture filtrate solution containing hydroquinone was used as the index for total activity of lignin peroxidase and manganese peroxidase. Then a specific detection of lignin peroxidase was achieved by the addition of veratryl alcohol, which led to current decrease due to the redox competition between veratryl alcohol and hydroquinone. A good linear correlation was found between the electrochemical response and lignin peroxidase activity, manganese peroxidase activity in the range of 8.14–29.79 U l⁻¹ and 0.085–1.37 U l⁻¹, respectively. A regression model was established describing the relationship. The amperometric sensor described here is more rapid, sensitive and precise than conventional spectrophotometric assays, free from interference of turbidity and UV–vis-light-absorbing substances. In this paper, it was also applied in the detection of lignin-degrading peroxidases in compost bioremediation using P. chrysosporium, showing considerable advantages.