Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of “alpeorujo” (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.     

Coriolopsis rigida,a potential model of white-rot fungi that produce extracellular laccases

In the last two decades, a significant amount of work aimed at studying the ability of the white-rot fungus Coriolopsis rigida strain LPSC no. 232 to degrade lignin, sterols, as well as several hazardous pollutants like dyes and aliphatic and aromatic fractions of crude oil, including polycyclic aromatic hydrocarbons, has been performed. Additionally, C. rigida in association with arbuscular mycorrhizal fungi appears to enhance plant growth, albeit the physiological and molecular bases of this effect remain to be elucidated. C. rigida's ability to degrade lignin and lignin-related compounds and the capacity to transform the aromatic fraction of crude oil in the soil might be partially ascribed to its ligninolytic enzyme system. Two extracellular laccases are the only enzymatic components of its lignin-degrading system. We reviewed the most relevant findings regarding the activity and role of C. rigida LPSC no. 232 and its laccases and discussed the work that remains to be done in order to assess, more precisely, the potential use of this fungus and its extracellular enzymes as a model in several applied processes.     

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.     

Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH₂). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH₂ extended the reactions catalyzed by these enzymes to the production of H₂O₂, the oxidation of Mn²⁺, the reduction of Fe³⁺, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN₃, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ Methoxy-p-hydroquinone - GLC gas liquid chromatography - TMSi trimethylsilane - TLC thin layer chromatography     

Induction of colonial growth and replica plating of the white rot basidiomycete Phanaerochaete chrysosporium.

A medium containing L-sorbose and sodium deoxycholate was devised which induces colonial growth of Phanaerochaete chrysosporium in the presence of a variety of supplements. The restricted size and heavy conidial production of the colonies permits high plating densities and the use of a replica-plating technique for copying the pattern of colony growth.     

Induction of colonial growth and replica plating of the white rot basidiomycete Phanaerochaete chrysosporium.

A medium containing L-sorbose and sodium deoxycholate was devised which induces colonial growth of Phanaerochaete chrysosporium in the presence of a variety of supplements. The restricted size and heavy conidial production of the colonies permits high plating densities and the use of a replica-plating technique for copying the pattern of colony growth.     

Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M(r) of 40,000 and 66, 000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity ( approximately 100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.     

Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium

An H2O2-requiring oxygenase found in the extracellular medium of ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium was purified by DEAE-Sepharose ion-exchange chromatography and gel filtration on Sephadex G-100. Sodium dodecyl sulfate (SDS)-disc gel electrophoresis indicated that the purified protein was homogeneous. The Mr of the enzyme as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 41,000. The absorption spectrum of the enzyme indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme (407 nm) shifted to 435 nm in the reduced enzyme and to 420 nm in the reduced-CO complex. The pyridine hemochrome absorption spectrum indicated that the enzyme contained one molecule of heme as iron protoporphyrin IX. Both CN- and N-3 bound readily to the native enzyme, indicating an available coordination site and that the heme iron was high spin. The purified enzyme generated ethylene from 2-keto-4-thiomethyl butyric acid, and oxidized a variety of lignin model compounds, including the diarylpropane, 1-(3'4'-diethoxyphenyl)1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I); a beta-ether dimer, 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (V); an olefin, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2 propene (III); and a diol, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2-propane diol (IV). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures.     

Transformation of 2-hydroxydibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerization products

Laccase, a ligninolytic enzyme, was secreted by each ofthe white rot fungi Trametes versicolor and Pycnoporus cinnabarinusduring growth in a nitrogen-rich medium under agitated conditions. Afteraddition of 2-hydroxydibenzofuran to cell-freesupernatants of the cultures, yellow precipitates wereformed. These precipitates were poorly soluble in waterand therefore readily separated from the supernatant. Theproducts formed were more hydrophobic than thesubstrate, as indicated by their longer retention times on areverse phase high-performance liquid chromatographycolumn. Mass spectrometric analysis of the purifiedproducts indicated the formation of oligomers. Analysis ofthe mixture of products by gas chromatography and massspectrometry after derivatization with diazomethanesuggested the formation of at least three dimeric and ninetrimeric products. Carbon-carbon and carbon-oxygenbonds were identified in the dimers and trimers,respectively. The nuclear magnetic resonance spectrum ofthe main dimer suggested coupling of the two monomersat the carbon one position.     

Transformation of 2-hydroxydibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerization products

Laccase, a ligninolytic enzyme, was secreted by each of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus during growth in a nitrogen-rich medium under agitated conditions. After addition of 2-hydroxydibenzofuran to cell-free supernatants of the cultures, yellow precipitates were formed. These precipitates were poorly soluble in water and therefore readily separated from the supernatant. The products formed were more hydrophobic than the substrate, as indicated by their longer retention times on a reverse phase high-performance liquid chromatography column. Mass spectrometric analysis of the purified products indicated the formation of oligomers. Analysis of the mixture of products by gas chromatography and mass spectrometry after derivatization with diazomethane suggested the formation of at least three dimeric and nine trimeric products. Carbon-carbon and carbon-oxygen bonds were identified in the dimers and trimers, respectively. The nuclear magnetic resonance spectrum of the main dimer suggested coupling of the two monomers at the carbon one position.     

Studies of the production and characterization of laccase activity in the basidiomycete Coriolopsis gallica, an efficient decolorizer of alkaline effluents

The basidiomycete Coriolopsis gallica decolorizes alkaline paper effluents efficiently. In this work, we found that C. gallica produces laccase during this decolorization process. This enzymatic activity was produced in all media studied; however, the highest enzymatic activity was obtained in a medium containing paper effluent, where laccase was detected on the 2nd day of the experiment. The laccase activity of C. gallica was purified and characterized. The amino-terminal sequence of this protein showed the highest similarity with the laccase I of the basidiomycete PM1 and with Coriolus hirsutus laccase. Received: 20 April 1998 / Accepted: 21 September 1998     

An improved transformation vector for the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium

In this study, a lignin peroxidase-encoding gene (LIP) of Phanerochaete chrysosporium was disrupted by inserting into its coding region the kanamycin-resistance determinant from Tn903. The resulting recombinant plasmid, pUGLG1: kan, was transformed into P. chrysosporium with the expectation that the disrupted gene might replace the homologous LIP gene in the chromosome. However, the results showed that pUGLG1: kan sequences do not integrate into the chromosome; instead, the plasmid is maintained intact in the transformants in an extrachromosomal state. Our data also show that pUGLG1: kan undergoes replication in P. chrysosporium, is maintained as a circular element, is recoverable from meiotic and mitotic progeny, although at a low frequency, and can be recovered intact by Escherichia coli transformation. These results suggest that the GLG1 component of pUGLG1: kan contains as yet unidentified sequences that allow its autonomous replication in P. chrysosporium transformants.     

Use of a shuttle vector for the transformation of the white rot basidiomycete, Phanerochaete chrysosporium

A novel shuttle vector based spheroplast transformation system for the lignin degrading filamentous fungus P. chrysosporium is described. The transformation vector, designated pRR12, consists of the yeast integration plasmid YIp5, a putative autonomous replication sequence (ars) of P. chrysosporium, and a 2.2 kb PvuII fragment carrying kanr determinant from plasmid pNG35, which confers resistance against both kanamycin and the related antibiotic G418. Two different strains of P. chrysosporium (ME446 and BKM-F) were transformed to G418 resistance using vector pRR12. Approximately 20 transformants per micrograms of vector DNA were obtained. The transforming vector pRR12 could be recovered from the total DNA of transformants by E. coli transformation, albeit at a low frequency.     

Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium

In Phanerochaete chrysosporium fruit body formations is subject to strong catabolite repression by glucose in the presence of physiological levels of nitrogen. Walseth cellulose was found to be the best source of carbon for the induction of fruit body and consequent basidiospore synthesis. Ejected basidiospores collected from cultures grown under these conditions for two weeks are contaminated with neither conidia nor mycelial fragments and are therefore suitable for genetic analysis of recombination. Under conditions of nitrogen limitation, the glucose catabolite repression of fruit body synthesis was relieved. Exogenous adenosine 3,5-monophosphate but not other related nucleotides, also relieved glucose catabolite repression of fruit body formation.     

Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.     

Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.     

Microbial scission of sulfide linkages in vulcanized natural rubber by a white rot basidiomycete, Ceriporiopsis subvermispora

A white rot basidiomycete, Ceriporiopsis subvermispora, degraded vulcanized natural rubber (NR) sheets on a wood medium. The fungus decreased the total sulfur content of the rubber by 29% in 200 days, accompanied by the cleavage of sulfide bonds between polyisoprene chains. X-ray photoelectron spectroscopy (XPS) demonstrated that C. subvermispora reduced the frequency of S-C bonds by 69% with a concomitant formation of S-O bonds during the culture period. Dipolar decoupling/magic angle spinning (DD/MAS) solid state 13C NMR revealed that the fungus preferentially decomposed monosulfide bonds linked to a cis- and trans-1,4-isoprene backbone but the cleavage of polysulfide bonds was also observed. In contrast, no decrease in weight or devulcanization of rubber was observed in cultures of a white rot fungus, Dichomitus squalens. The oxidative cleavage of sulfide bonds by C. subvermispora demonstrates that ligninolytic basidiomycetes are potential microbes for the biological devulcanization of rubber products.