Mechanism of versatile peroxidase inactivation by Ca(2+) depletion

Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca(2+) depletion. In this work, the spectroscopic characterizations of Ca(2+)-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca(2+) content. At pH 7.5, UV-Vis spectra indicate a heme-Fe(3+) transition from 5-coordinated high-spin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca(2+)-depleted form. This Fe(3+) hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca(2+)-depleted enzyme has a high spin Fe(3+). EPR measurements on VP confirm the differences in the Fe(3+) spin states at pH 4.5 and at 7.5 for both, native and Ca(2+)-depleted enzymes. In addition, EPR spectra recorded after the addition of H(2)O(2) to Ca(2+)-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca(2+)-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca(2+)-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.     

Gene cloning,heterologous expression,in vitro reconstitution and catalytic properties of a versatile peroxidase

The gene of a peroxidase described as being involved in carotenoid degradation was cloned from a strain that was conserved as Lepista irina (CBS 458.79). Gene sequencing revealed high nucleotide and amino-acid identity with Pleurotus eryngii gene vpl, which encodes a versatile peroxidase with unique catalytic properties, and only reported in Pleurotus and Bjerkandera species. Re-identification of the supposed L. irina strain revealed that, in fact, it is a P. eryngii strain. The new P. eryngii peroxidase was expressed in Escherichia coli, and the recombinant protein folded in the presence of cofactor to obtain the active form. The purified enzyme was able to oxidize Mn²⁺, veratryl alcohol, substituted phenols, and both low and high redox-potential dyes, demonstrating that it belongs to the versatile peroxidase family (named VPL3). These catalytic properties agreed with the presence of both Mn²⁺ and aromatic-substrate oxidation sites in its molecular structure.     

Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium

Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pK_a values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pK_a value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.     

Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: production, partial characterization and application for the elimination of endocrine disruptors

Versatile peroxidase (VP) from Bjerkandera adusta was insolubilized in the form of cross-linked enzyme aggregates (CLEA®s). Of the initially applied activity 67% was recovered as CLEA®s. Co-aggregation of VP with glucose oxidase from Aspergillus niger led to an increased activity recovery of 89%. The combined CLEA®s showed higher stability against H₂O₂ and exerted VP activity upon glucose addition. The elimination of the endocrine disrupting chemicals bisphenol A, nonylphenol, triclosan, 17α-ethinylestradiol and the hormone 17β-estradiol (10 mg L⁻¹ each) and the removal of their estrogenic activity by combined CLEA®s were tested in batch experiments. Within 10 min, the combined CLEA®s were able to remove all the endocrine disruptors except triclosan (residual concentration 74%). The removal of the estrogenic activity was higher than 55% for all compounds, except triclosan. A membrane reactor continuously operated with combined CLEA®s could almost completely remove bisphenol A (10 mg L⁻¹) for 43 h.     

Depolymerisation of lignosulfonate by peroxidase of the white-rot basidiomycete, Pleurotus ostreatus

In the presence of a H₂O₂-generating system, the peroxidase of Pleurotus ostreatus that decolorise Remazol Brilliant Blue R catalysed the partial depolymerisation of lignosulfonate. The UV spectrum of the lignosulfonate changed with time with the absorbance at 280 nm increasing gradually. Gel permeation profile showed that the pattern of molecular weight distribution was changed to a lower molecular weight region. Oxidation products were identified as 2,6-dimethoxy-1,4-benzoquinone (I), benzoic acid (II), butyl phthalate (III), and bis (2-ethylhexyl) phthalate (IV) by GC/MS procedures.     

Biodecolorization of azo,anthraquinonic and triphenylmethane dyes by white-rot fungi and a laccase-secreting engineered strain

One laccase-secreting engineered strain and four white-rot fungi were tested for their capacity to decolorize nine dyes that could be classified as azo, anthraquinonic and triphenylmethane dyes. Trametes versicolor was the most efficient of the tested strains under these experimental conditions. Anthraquinonic dyes were decolorized more easily than the other two types. Small structural differences among the dyes could significantly affect decolorization. None of the strains showed lignin peroxidase or veratryl alcohol oxidase activity. None of the dyes were decolorized completely by laccase alone. It is likely that other phenoloxidases, such as Mn-dependent and versatile peroxidase, were also involved in decolorization of the dyes.     

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca²⁺ and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.     

Hericenols A-D and a chromanone from submerged cultures of a Stereum species

Extracts of submerged cultures of a Stereum species afforded four new pentasubstituted phenolic compounds, named hericenols A, B, C, and D (1-4), 6-hydroxymethyl-2,2-dimethylchroman-4-one (5) and the known erinapyrone C. Hericenol A (1) showed weak antimicrobial activity while hericenol C (3) was weakly cytotoxic. The structures of the metabolites were determined by spectroscopic techniques.     

Free-radical polymerization of acrylamide by manganese peroxidase produced by the white-rot basidiomycete Bjerkandera adusta

Acrylamide was polymerized to give polyacrylamide using manganese peroxidase (MnP) produced by the basidiomycete Bjerkandera adusta. The molecular weight of the polymer synthesized by MnP was 155000, higher than those obtained with other reaction systems using horseradish peroxidase and a redox initiator. The ¹³C-NMR spectrum showed that polyacrylamide was atactic. Electron spin resonance analysis revealed that 2,4-pentanedione added as an initiator was first oxidized to generate a carbon-centered radical, which initiated radical additive polymerization of acrylamide.     

白囊耙齿菌产锰过氧化物酶条件优化及其对染料的脱色

锰过氧化物酶（manganese peroxidase,MnP）是白腐真菌降解多种异生物质的主要降解酶之一。本研究对白囊耙齿菌Irpex lacteus产MnP的酶活曲线进行监测,利用单因素和正交试验对I. lacteus产MnP的发酵条件进行优化,同时检测了I. lacteus的MnP粗酶液对5种染料的脱色效果。结果显示,I. lacteus在培养5d时MnP活性较大;I. lacteus产MnP较优的条件为：可溶性淀粉20g/L、尿素1g/L、pH 6.3、CaCl₂ 1mmol/L、FeCl₃ 1mmol/L,该条件下MnP活性达29.24U/L,与优化前MnP活性相比提高了1.25倍;I. lacteus的MnP粗酶液对5种染料均可脱色,其中对直接大红和活性红的脱色效果更为明显,脱色5d后的脱色率分别达到82%和81%。     

Effect of culture temperature on the heterologous expression of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> versatile peroxidase in <Emphasis Type="Italic">Aspergillus</Emphasis> hosts

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 °C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L^-1 was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.)

We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids.     

Continuous removal of endocrine disruptors by versatile peroxidase using a two‐stage system

The oxidant Mn³⁺‐malonate, generated by the ligninolytic enzyme versatile peroxidase in a two‐stage system, was used for the continuous removal of endocrine disrupting compounds (EDCs) from synthetic and real wastewaters. One plasticizer (bisphenol‐A), one bactericide (triclosan) and three estrogenic compounds (estrone, 17β‐estradiol, and 17α‐ethinylestradiol) were removed from wastewater at degradation rates in the range of 28–58 µg/L·min, with low enzyme inactivation. First, the optimization of three main parameters affecting the generation of Mn³⁺‐malonate (hydraulic retention time as well as Na‐malonate and H₂O₂ feeding rates) was conducted following a response surface methodology (RSM). Under optimal conditions, the degradation of the EDCs was proven at high (1.3–8.8 mg/L) and environmental (1.2–6.1 µg/L) concentrations. Finally, when the two‐stage system was compared with a conventional enzymatic membrane reactor (EMR) using the same enzyme, a 14‐fold increase of the removal efficiency was observed. At the same time, operational problems found during EDCs removal in the EMR system (e.g., clogging of the membrane and enzyme inactivation) were avoided by physically separating the stages of complex formation and pollutant oxidation, allowing the system to be operated for a longer period (～8 h). This study demonstrates the feasibility of the two‐stage enzymatic system for removing EDCs both at high and environmental concentrations. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:908–916, 2015     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

Production of versatile peroxidase from Pleurotus eryngii by solid-state fermentation using agricultural residues and evaluation of its catalytic properties

Interest in production of ligninolytic enzymes has been growing over recent years for their use in various applications such as recalcitrant pollutants bioremediation; specifically, versatile peroxidase (VP) presents a great potential due to its catalytic versatility. The proper selection of the fermentation mode and the culture medium should be an imperative to ensure a successful production by an economic and available medium that favors the process viability. VP was produced by solid-state fermentation (SSF) of Pleurotus eryngii, using the agricultural residue banana peel as growth medium; an enzymatic activity of 10,800 U L^?1 (36 U g^?1 of substrate) was detected after 18 days, whereas only 1800 U L^?1 was reached by conventional submerged fermentation (SF) with glucose-based medium. The kinetic parameters were determined by evaluating the H₂O₂ and Mn²⁺ concentration effects on the Mn³⁺-tartrate complex formation. The results indicated that although the H₂O₂ inhibitory effect was observed for the enzyme produced by both media, the reaction rates for VP obtained by SSF were less impacted. This outcome suggests the presence of substances released from banana peel during the fermentation, which might exhibit a protective effect resulting in an improved kinetic behavior of the enzyme.