Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus.

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k(cat). While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

Isolation and selection of novel basidiomycetes for decolorization of recalcitrant dyes.

Thirty wood-rotting basidiomycetes, most of them causing white rot in wood, were isolated from fruiting bodies growing on decaying wood from the Sierra de Ayllón (Spain). The fungi were identified on the basis of their morphological characteristics and compared for their ability to decolorize Reactive Black 5 and Reactive Blue 38 (as model of azo and phthalocyanine type dyes, respectively) at 75 and 150 mg/L. Only eighteen fungal strains were able to grow on agar plates in the presence of the dyes and only three species (Calocera cornea, Lopharia spadicea, Polyporus alveolaris) decolorized efficiently both dyes at both concentrations. The ligninolytic activities, involved in decolorization dyes (laccases, lignin peroxidases, Mn-oxidizing peroxidases), were followed in glucose basal medium in the presence of enzyme inducers. The results indicate a high variability of the ligninolytic system within white-rot basidiomycetes. These fungal species and their enzymes can represent new alternatives for the study of new biological systems to degrade aromatic compounds causing environmental problems.

Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus 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 degrees 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.

Main lipophilic extractives in different paper pulp types can be removed using the laccase-mediator system.

Lipophilic extractives in wood and other lignocellulosic materials exert a negative impact in pulp and paper manufacturing causing the so-called pitch problems. In this work, the appropriateness of an enzymatic treatment using the laccase-mediator system for pitch biocontrol is evaluated. With this purpose, three pulp types representative for different raw materials and pulping processes-eucalypt kraft pulping, spruce thermomechanical pulping, and flax soda-anthraquinone pulping-were treated with a high-redox-potential laccase from the basidiomycete Pycnoporus cinnabarinus in the presence of 1-hydroxybenzotriazole as a redox mediator. The gas chromatography and gas chromatography/mass spectrometry analyses of the lipophilic extractives from the enzymatically treated pulps revealed that the laccase-mediator treatment completely or greatly removed most of the pitch-causing lipophilic compounds present in the different pulps including: (1) free and conjugated sitosterol in eucalypt paper pulp; (2) resin acids, sterol esters, and triglycerides in spruce pulp; and (3) sterols and fatty alcohols in the flax pulp. Different amounts of free and conjugated 7-oxosterols were found as intermediate products in the oxidation of pulp sterols. Therefore, the laccase-mediator treatment is reported as an efficient method for removing pitch-causing lipophilic compounds from paper pulps obtained from hardwood, softwood, and nonwoody plants.

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 Ca(2+) 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.

Pycnoporus cinnabarinus laccases: an interesting tool for food or non-food applications.

The effects of the addition of ferulic acid and ethanol in P. cinnabarinus ss3 culture medium in fermentor were compared in 15-L fermentor. In the presence of 30 g l(-1) ethanol, laccase activity (270,000 U/L1) was 3-fold higher as compared with ferulic acid-induced cultures, and 150-fold higher as compared with non-induced cultures, respectively. High-quality flax pulp was bleached in a totally-chlorine free (TCF) sequence using a laccase-mediator system constituted by laccase from Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as mediator. Up to 90% delignification and strong brightness increase were attained after the laccase-mediator treatment followed by H2O2 bleaching. This TCF sequence was further improved by applying H2O2 under pressurized O2. In this way, up to 82% ISO brightness was obtained (compared with 37% in the initial pulp and 60% in the peroxide-bleached control) as well as very low kappa number. A positive evaluation of the laccase has been also performed in a food application. The colour of a tea-based beverage was significantly improved by incubating an infusion of green tea with the Pycnoporus laccase.

Fungal bioturbation paths in a compact disk.

We report here on bioturbation traces, with micro-dendrite textures, composed of a mixture of altered aluminum and polycarbonate, which have been developed in a common compact disk (CD), destroying information pits. Fungal hyphae proliferated in these deteriorated zones, and Geotrichum-type fungus was isolated from surface-sterilized CD fragments. The severe biodeterioration described is attributed to the slow growth of this arthroconidial fungus on the CD material in the tropical indoor environment of Belize, Central America (approximately 30 degrees C, approximately 90% humidity).

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.

The biotechnological control of pitch in paper pulp manufacturing.

At present, microbial and enzymatic preparations for the control of triglyceride-containing pitch deposits during the manufacture of mechanical and sulfite paper is commercially available. However, biotechnological products for pitch control in other pulping processes, such as alkaline pulping, are under development. These products include new fungi for the removal of steroids involved in pitch deposit formation in chlorine-free pulps, to be used as a biological pretreatment of wood before pulping. Simultaneously, tailor-made enzymes are being produced using protein-engineering techniques, enabling the specific removal of pitch contaminant compounds from paper pulp.

A new versatile peroxidase from Pleurotus.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) have been investigated in Phanerochaete chrysosporium. A third ligninolytic peroxidase has been described in Pleurotus and Bjerkandera. Two of these versatile peroxidases (VPs) have been cloned, sequenced and characterized. They have high affinity for Mn(2+), hydroquinones and dyes, and also oxidize veratryl alcohol, dimethoxybenzene and lignin dimers. The deduced sequences show higher identity with Ph. chrysosporium LiP than MnP, but the molecular models obtained include a Mn(2+)-binding site. Concerning aromatic substrate oxidation, Pl. eryngii VP shows a putative long-range electron transfer pathway from an exposed trytophan to haem. Mutagenesis and chemical modification of this tryptophan and the acidic residues forming the Mn(2+)-binding site confirmed their role in catalysis. The existence of several substrate oxidation sites is supported further by biochemical evidence. Residue conservation in other fungal peroxidases is discussed.

Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: purification and characterization of the recombinant enzyme.

Aryl-alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin biodegradation by some white-rot fungi. The enzyme catalyzes the extracellular oxidation of aromatic alcohols to the corresponding aldehydes. The electron acceptor is molecular oxygen yielding H(2)O(2) as the product. Herein we describe, for the first time, the expression of AAO from Pleurotus eryngii in the ascomycete Aspergillus nidulans. The activity of the recombinant enzyme in A. nidulans cultures is much higher than found in the extracellular fluid of P. eryngii. The recombinant enzyme showed the same molecular mass, pI and catalytic properties as that of the mature protein secreted by P. eryngii. The enzymic properties are also similar to those reported from other Pleurotus and Bjerkandera species.

Time course of fungal removal of lipophilic extractives from Eucalyptus globulus wood.

Free and esterified sitosterol, the main lipophilic constituents of eucalypt wood extractives, have been associated with the formation of pitch deposits during manufacturing of environmentally-sound paper pulp from Eucalyptus globulus wood. These, and other lipophilic compounds, were analyzed by gas chromatography-mass spectrometry in the course of wood treatments (up to 7 weeks) with four extractive-degrading fungi in order to optimize biotechnological control of pitch deposition in eucalypt pulp (with moderate loss of wood weight). In contrast to commercialized fungi used in pitch control, which are not able to degrade sitosterol, the fungi investigated in this paper produced a rapid decline of both free and esterified sterols in wood. The degradation rate of steroid hydrocarbons and squalene was moderate, and the amount of steroid ketones (probably formed during oxidative degradation of steroids) and triglycerides increased at different stages of wood treatment. Up to 95% removal of total steroids (including free and esterified sterols, steroid ketones and steroid hydrocarbons) by fungi was obtained at the end of wood treatment under the solid-state fermentation conditions used. The most promising results from the point of view of industrial applicability, however, were obtained after 1-2 weeks of treatment with either Phlebia radiata or Poria subvermispora, which enabled 70% steroid removal with a moderate wood weight loss of 1-4%.

Production of hydroxyl radical by the synergistic action of fungal laccase and aryl alcohol oxidase.

A mechanism for the production of hydroxyl radical (*OH) during the oxidation of hydroquinones by laccase, the ligninolytic enzyme most widely distributed among white-rot fungi, has been demonstrated. Production of Fenton reagent (H2O2 and ferrous ion), leading to *OH formation, was found in reaction mixtures containing Pleurotus eryngii laccase, lignin-derived hydroquinones, and chelated ferric ion. The semiquinones produced by laccase reduced both ferric to ferrous ion and oxygen to superoxide anion radical (O2*-). Dismutation of the latter provided the H2O2 for *OH generation. Although O2*- could also contribute to ferric ion reduction, semiquinone radicals were the main agents accomplishing the reaction. Due to the low extent of semiquinone autoxidation, H2O2 was the limiting reagent in Fenton reaction. The addition of aryl alcohol oxidase and 4-methoxybenzyl alcohol (the natural H2O2-producing system of P. eryngii) to the laccase reaction greatly increased *OH generation, demonstrating the synergistic action of both enzymes in the process.

Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species.

Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes tersicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases.

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.

Aryl-alcohol oxidase protein sequence: a comparison with glucose oxidase and other FAD oxidoreductases.

Aryl-alcohol oxidase (AAO), an FAD-dependent enzyme involved in lignin degradation, has been cloned from Pleurotus eryngii. The AAO protein is composed of 593 amino acids, 27 of which form a signal peptide. It shows 33% sequence identity with glucose oxidase from Aspergillus niger and lower homology with other oxidoreductases. The predicted secondary structures of both enzymes are very similar. For AAO, it is predicted to contain 13 putative alpha-helices and two major beta-sheets, each of the putative beta-sheets formed by six beta-strands. The ADP binding site and the signature-2 consensus sequence of the glucose-methanol-choline (GMC) oxidoreductases were also present. Moreover, residues potentially involved in catalysis and substrate binding were identified in the vicinity of the flavin ring. They include two histidines (H502 and H546) and several aromatic residues (Y78, Y92 and F501), as reported in other FAD oxidoreductases.

Different fungal manganese-oxidizing peroxidases: a comparison between Bjerkandera sp. and Phanerochaete chrysosporium.

Two manganese-oxidizing peroxidases differing in glycosylation degree were purified from fermenter cultures of Bjerkandera sp. They were characterized and compared with the three manganese-oxidizing peroxidase isoenzymes obtained from the well-known ligninolytic fungus Phanerochaete chrysosporium. All the enzymes showed similar molecular masses but those from P. chrysosporium had less acidic isoelectric point. Moreover, the latter strictly required Mn2+ to oxidize phenolic substrates whereas the Bjerkandera peroxidases had both Mn-mediated and Mn-independent activity on phenolic and non-phenolic aromatic substrates. Taking into account these results, and those reported for Bjerkandera adusta and different Pleurotus species, we concluded that two different types of Mn(2+)-oxidizing peroxidases are secreted by ligninolytic fungi.

Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii.

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH(2)) and 2, 6-dimethoxy-1,4-benzohydroquinone (DBQH(2)) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(*-) + O(2) <--> Q + O(2)(*-)). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O(2)(*-) on quinone formation rates. Then, the production of H(2)O(2) in laccase reactions, as a consequence of O(2)(*-) dismutation, confirmed that semiquinones autoxidized. The highest H(2)O(2) levels were obtained with DBQH(2), indicating that DBQ(*-) autoxidized to a greater extent than did MBQ(*-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O(2)(*-) consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O(2)(*-), besides undergoing dismutation, reacts with Mn(2+), Fe(3+), and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 microM concentrations of MBQ(*-) and DBQ(*-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(*-) autoxidation rose to 13.8% and that of DBQ(*-) increased to 39.9%.

Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius.

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N-carbohydrate content and an estimated molecular mass of 70.5 kDa and pI of 3.95. The kinetic studies showed the highest enzyme affinity against p-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.

Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii.

A versatile peroxidase able to oxidize Mn(2+) as well as phenolic and nonphenolic aromatic compounds is produced in peptone-containing liquid cultures of Pleurotus eryngii encoded by the gene mnpl. The regulation of its transcript levels was investigated by Northern blotting of total RNA. High-peroxidase transcripts and activity were found in cultures grown in glucose-peptone medium, whereas only basal levels were detected in glucose-ammonium medium. The addition of more than 25 microM Mn(2+) to the former medium did not result in detectable peroxidase transcripts or activity. Potential regulators were also added to isolated mycelium. In this way, it was shown that high transcript levels (in peroxidase-expressing mycelium) were maintained on peptone, whereas expression was not induced in short-term incubation experiments. Similar results were obtained with Mn(2+) ions. Strong induction of mnpl expression was caused by exogenous H(2)O(2) or by continuous H(2)O(2) generation during redox cycling of menadione. By the use of the latter system in the presence of Fe(3+), which catalyzes the reduction of H(2)O(2) to hydroxyl radical, it was shown for the first time that the presence of this strong oxidant causes a rapid increase of the transcripts of a ligninolytic peroxidase. In conclusion, peptone and Mn(2+) affect the levels of transcripts of this versatile peroxidase in culture, and reduced oxygen species induce short-term expression in isolated mycelium, probably via a stress response mechanism.