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.

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.

Copper trafficking: the solution structure of Bacillus subtilis CopZ.

A sequence with a high homology (39% residue identity) with that of the copper-transport CopZ protein from Enterococcus hirae and with the same MXCXXC metal-binding motif has been identified in the genome of Bacillus subtilis, and the corresponding protein has been expressed. The protein, constituted by 73 amino acids, does bind copper(I) under reducing conditions and fully folded in both copper-bound and copper-free forms under the present experimental conditions. The solution structure of the copper-bound form was determined through NMR spectroscopy on an 15N-labeled sample. A total of 1508 meaningful nuclear Overhauser effects, 38 dihedral phi angles, and 48 dihedral psi angles were used in the structural calculations, which lead to a family of 30 conformers with an average rmsd to the mean structure of 0.32 +/- 0.06 A for the backbone and of 0.85 +/- 0.07 A for the heavy atoms. NMR data on the apoprotein also show that, also in this form, the protein is in a folded state and essentially maintains the complete secondary structure. Some disorder is observed in the loop devoted to copper binding. These results are compared with those reported for CopZ from E. hirae whose structure is well-defined only in the apo form. The different behaviors of copper-loaded E. hirae and B. subtilis are tentatively accounted for on the basis of the presence of dithiothreitol used in the latter case, which would stabilize the monomeric form. The comparison is extended to other similar proteins, with particular attention to the copper-binding loop. The nature and the location of conserved residues around the metal-binding site are discussed with respect to their relevance for the metal-binding process. Proposals for the role of CopZ are also presented.

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.

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.

Heterologous expression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn(2+) and different aromatic substrates.

A versatile ligninolytic peroxidase has been cloned from Pleurotus eryngii and its allelic variant MnPL2 expressed in Aspergillus nidulans, with properties similar to those of the mature enzyme from P. eryngii. These include the ability to oxidize Mn(2+) and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase.

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.

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites.

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii.

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58-60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C alpha-distance 1.2 A). However, this peroxidase includes a Mn2+ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn2+. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.

A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta.

A novel peroxidase, oxidizing Mn2+ and different aromatic compounds, was isolated. Hydroquinones, substituted phenols, dyes, other aromatic compounds and Mn2+ were compared as reducing substrates, and conclusions presented in the light of a molecular model built by homology modeling. The enzymes showed the fastest reaction rates with Mn2+, but the highest affinity corresponded to hydroquinones and dyes. Oxidation of Reactive Black 5 (an azo-dye not oxidized by Mn3+) was non-competitively inhibited by Mn2+. These findings, together with identification of putative Mn-binding site (involving Glu36, Glu40, Asp175 and inner heme propionate) and long-range electron transfer pathways, indicate that different sites are involved in substrate oxidation.

Purification and catalytic properties of two manganese peroxidase isoenzymes from Pleurotus eryngii.

The ligninolytic basidiomycetes Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor-caju did not exhibit detectable levels of manganese peroxidase (MP) when grown in liquid media with ammonium tartrate as N source. However, after examination of cells grown on different organic N-based media, high MP activity was obtained in peptone medium, up to nearly 3 U/ml in cultures of P. eryngii. Moreover, Mn2+ supplementation was not used to produce MP, since all Mn2+ concentrations assayed (1-4000 microM) inhibited production of this enzyme in liquid medium. Two MP isoenzymes were purified to homogeneity from shaken or stationary cultures of P. eryngii grown in peptone medium. The purification process (which included chromatography on Biorad Q-cartridge, Sephacryl S-200 and Mono-Q) attained 56% activity yield with a purification factor of 25. The isoenzymes differed in pI (3.75 and 3.65), N-terminal sequence and some catalytic properties. They were in some aspects (e.g, molecular mass of 43 kDa) similar to Phanerochaete chrysosporium MP but exhibited some distinct characteristics, including Mn(2+)-independent peroxidase activities against 2,6-dimethoxyphenol and veratryl alcohol, and higher resistance to H2O2. Recent studies have shown that MP are ubiquitous enzymes in ligninolytic fungi, but the results obtained suggest that differences in catalytic properties probably exist between different Mn(2+)-oxidizing peroxidases produced by these fungi.