Comparative Stability Assessment of Laccases from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus in the Presence of Effectors

Stability characteristics of the laccases of the basidiomycetes Coriolus hirsutus and Coriolus zonatus were measured comparatively at temperatures of 25 and 40°C in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). Stabilization effects of cations on the laccases from C. hirsutus and C. zonatus decreased in the descending series Cu²⁺ > Mg²⁺ > Ca²⁺ and Ca²⁺ > Mg²⁺ > Mn²⁺, respectively. Tween 20 caused insignificant stabilization of the two enzymes. The C. zonatus laccase was also insignificantly stabilized as a result of treatment with bovine serum albumin. The enzymatic activity of the laccase preparations from C. hirsutus and C. zonatus was conserved virtually completely after vacuum drying (84 and 93%, respectively). The most effective stabilizer of the C. hirsutus laccase was found to be dextran (17 kDa). Dry preparations treated with this agent conserved up to 95% of the enzymatic activity. The most effective stabilizer of the C. zonatus laccase was polyacrylic acid (102% of the initial activity).     

Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus.

The paper reports on two fungal laccases from Coriolus hirsutus and Coriolus zonatus and their type-2 copper-depleted derivatives. Temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (EPR) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. The intact enzymes showed highly cooperative thermal unfolding transitions at about 90 degrees C. Type-2 copper depletion led to uncoupling of the domains characterized by a different melting pattern which resolved three subtransitions. Melting curves monitored optically at 290, 340 and 610 nm showed additional transitions below thermal unfolding temperature. EPR spectra of the intact laccases showed the disintegration of the trinuclear copper cluster accompanied by loss of one of the copper ions and disappearance of the strong antiferromagnetic coupling in the type-3 site at 70 degrees C and above 70 degrees C. The copper centers of type-2 copper-depleted laccase showed reduced thermotolerance.     

Certain biochemical and physicochemical properties of the inducible form of extracellular laccase from basidiomycetes Coriolus hirsutus

An inducible form of extracellular laccase (EC 1.14.18.1) was isolated from the basidiomycete Coriolus hirsutus. The induction was performed with 0.11 microM syringaldazine, a substrate of laccase. The inducible form of the enzyme consisted of two isoforms, laccase I1 and laccase I2, whose molecular weights were 69 +/- 2 and 67 +/- 2 kDa, respectively. The isoelectric points of these isoenzymes were found to be 3.5 and 4.2, respectively. The optimum pH range for both laccases was 4.4-4.6, and the optimum temperature was 50 degrees C. The thermal stability of these isoenzymes was examined, and KM values for the substrates syringaldazine and pyrocatechol were determined. Our biochemical and physicochemical studies demonstrated that inducible laccase isoforms differed from constitutive forms in molecular weight, IP, KM, and thermal stability. However, their optimum pH ranges and temperatures were identical.     

Laccases from Basidiomycetes: physicochemical characteristics and substrate specificity towards methoxyphenolic compounds

Laccases from the Basidiomycetes Coriolus hirsutus, Coriolus zonatus, Cerrena maxima, and Coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. The kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. As laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. The stoichiometries of the enzymatic reactions were determined: guaiacol and sinapinic acid are one-electron donors and their oxidation apparently results in the formation of dimers. It was established that k _cat/K _m, which indicates the effectiveness of catalysis, increases in the series guaiacol, ferulic acid, and sinapinic acid. This fact might be connected with the influence of substituents of the phenolic ring of the substrates. This phenomenon was established for fungal laccases with different physicochemical properties, amino acid composition, and carbohydrate content. This suggests that all fungal laccases possess the same mechanism of interaction between organic substrate electron donors and the copper-containing active site of the enzyme and that this interaction determines the observed values of the kinetic parameters.     

Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971).

A new lignin-degrading basidiomycete, strain PM1 (= CECT 2971), was isolated from the wastewater of a paper factory. The major ligninolytic activity detected in the basidiomycete PM1 culture supernatant was a phenoloxidase (laccase). This activity was produced constitutively in defined or complex media and appeared as two protein bands in native gel electrophoresis preparations. No enzyme induction was found after treatment with certain potential laccase inducers. Laccase I was purified to homogeneity by gel filtration chromatography, anion-exchange chromatography, and hydrophobicity chromatography. The enzyme is a monomeric glycoprotein containing 6.5% carbohydrate and having a molecular weight of 64,000. It has an isoelectric point of 3.6, it is stable in a pH range from 3 to 9, and its optimum pH is 4.5. The laccase optimal reaction temperature is 80 degrees C, the laccase is stable for 1 h at 60 degrees C, and its activity increases with temperature. Spectroscopic analysis revealed that the enzyme has four bound copper atoms, a type I copper, a type II copper, and a type III binuclear copper. The amino-terminal sequence of the protein is very similar to the amino-terminal sequences of laccases from Coriolus hirsutus and Phlebia radiata.     

Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus

Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, DEAE Sepharose CL-6B, Sephacryl S-200 HR, Hitrap SP, and Mono S chromatography. The purification was 14.5-fold with an overall yield of 32.3%. The enzyme is a monomeric glycoprotein with 11% carbohydrate content, an isoelectric point of 7.4, and a molecular mass of 73 kDa. The N-terminal amino acid sequence showed low homology to those of the laccases of other white-rot basidiomycetes. Spectroscopic analyses revealed a typical laccase active site in the C. hirsutus enzyme, as all three Cu centers were identified. The absorption spectrum showed a type 1 signal at around 600 nm and a type 3 signal near 330 nm. Type 3 Cu showed fluorescence emission near 418 nm and an excitation maximum at 332 nm. The EPR spectrum yielded parameters for the type 1 and type 2 Cu of gII = 2.191 and AII = 0.0097 cm(-1), and gII = 2.222 and AII = 0.0198 cm(-1), respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for the enzyme was reached at 45 degrees C, and the pH optima of the enzyme varied and was substrate dependent in the range of 2.5 to 4.0. The enzyme oxidized a variety of the usual laccase substrates, including lignin-related phenols and had highest affinity toward guaiacol. Under standard assay conditions, the apparent Km value of the enzyme toward guaiacol was 10.9 microM. The enzyme catalyzed single electron transfer via the phenoxy radical as an intermediate and was completely inhibited by L-cysteine and sodium azide but not by EDTA.     

Optimization of conditions for cultivation of the basidiomycete Coriolus hirsutus--producer of extracellular laccase

The effects of various factors on the biosynthesis of extracellular laccase (EC 1.14.18.1) by the basidiomycete Coriolus hirsutus (Wulf.: Fr.) Quel. no. 072 during submerged cultivation were examined. Optimal parameters for cultivation in a fermenter of 10 l were determined: temperature, 28 degrees C; stirrer rotation speed, 160 rpm; and the inoculum volume, 15% of the working volume of the fermenter. The filtrate contained peroxidase, laccase, and phenol oxidase activities and displayed a high thermal stability.     

Transformation of the white-rot basidiomycete Coriolus hirsutus using the ornithine carbamoyltransferase gene

An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg+ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 10(3)-10(4) transformants per mug DNA per 10(6)-10(7) oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg+ phenotype of the transformants was stably inherited through mitosis.     

Effect of Coriolus hirsutus laccase on atrazine adsorption and desorption by different types of soil

Study of adsorption-desorption behavior of herbicide atrazine in soils of different geographical zones in the presence of Coriolus hirsutus laccase was performed. Laccase was shown to significantly increase adsorption coefficient and to facilitate irreversible adsorption of atrazine to soil. Supposably, laccase catalyzes oxidative binding of atrazine to soil.     

Increase of the detoxification potential of basidiomycetes by induction of laccase biosynthesis

The effect of oxidoreductase inducers guaiacol and syringaldazine on the ability of Coriolus hirsutus, Coriolopsis fulvocinerea, Cerrena maxima, and cocultivated Coriolus hirsutus/Cerrena maxima to degrade atrazine in submerged cultures was studied. All the basidiomycetes reduced atrazine concentration with and without syringaldazine or guaiacol. The degree of atrazine degradation was higher in induced cultures (77–98% vs. 68–94% without induction). Of the four cultures, the highest detoxifying potential was observed in Coriolopsis fulvocinerea with and without an inducer (98% with guaiacol), and the lowest was in Cerrena maxima. Inducers decreased the residual atrazine concentration differently in the different cultures. A long-term increase of laccase production was observed in both induced and uninduced cultures, whereas the activity of Mn-peroxidase decreased. The results indicate that laccase plays a larger role in atrazine biodegradation than Mn-peroxidase.     

Increase of the detoxification potential of basidiomycetes by induction of laccase biosynthesis

The effect of oxidoreductase inducers guaiacol and syringaldazine on the ability of Coriolus hirsutus, Coriolopsis fulvocinerea, Cerrena maxima, and cocultivated Coriolus hirsutus/Cerrena maxima to degrade atrazine in submerged cultures was studied. All the basidiomycetes reduced atrazine concentration with and without syringaldazine or guaiacol. The degree of atrazine degradation was higher in induced cultures (77-98% vs. 68-94% without induction). Of the four cultures, the highest detoxifying potential was observed in Coriolopsis fulvocinerea with and without an inducer (98% with guaiacol), and the lowest was in Cerrena maxima. Inducers decreased the residual atrazine concentration differently in the different cultures. A long-term increase of laccase production was observed in both induced and uninduced cultures, whereas the activity of Mn-peroxidase decreased. The results indicate that laccase plays a larger role in atrazine biodegradation than Mn-peroxidase.     

Purification and characterization of extracellular laccase fromPleurotus ostreatus

Laccase (EC 1.10.3.2) from the culture filtrate of a strain of white rot basidiomycetePleurotus ostreatus was purified using DEAE-Toyopearl 650M and butyl-Toyopearl 650M column chromatographies and Superdex 75 HR 10/30 fast protein liquid chromatography. Molecular weight of the purified laccase was about 55,000, and the isoelectric point was 3.0. The optimum pH for enzyme activity was 6.5, and the optimum temperature was 50°C. This enzyme contained 7.4% sugar and two copper atoms per molecule. The substrate specificity was similar to those of other fungal laccases. Comparison of the N-terminal amino acid sequence of theP. ostreatus laccase with those fromPleurotus ostreatus Florida,Coriolus hirsutus, Phlebia radiata, basidiomycete PM1 (CECT 2971),Trametes villosa, Pycnoporus cinnabarinus, Ceriporiopsis subvermispora, andAgaricus bisporus showed 95, 65, 60, 55, 55, 55, 50, and 35% similarity, respectively, in the first 20 residues. No similarity in this region was detected with laccases fromNeurospora crassa, Aspergillus nidulans, andCryptococcus neoformans.     

毛云芝菌利用糖蜜酒精废水产漆酶培养基优化

为提高毛云芝菌的漆酶产量,降低生产成本,同时使糖蜜废水中的营养成分得到充分利用,变废为宝,以糖蜜废水作为碳源,对毛云芝菌发酵产漆酶进行研究。在单因素试验基础上,以不同浓度糖蜜废水、尿素含量为自变量,漆酶活力为因变量,采用Box-Behnken设计方法得到最佳的培养基配方为糖蜜废水浓度47%,尿素添加量0.5%,漆酶活力可达1 810.0 U/mL,约为优化前的6倍。说明毛云芝菌利用糖蜜废水作为碳源生产漆酶是可行的,为糖蜜废水资源化利用高效生产有价值产品奠定了理论基础。     

Cloning and sequencing of a second laccase gene from the white-rot fungus Coriolus versicolor

A second laccase gene, CVLG1, was isolated from Coriolus versicolor. CVLG1 encodes a precursor protein of 526 amino acids which contains a 23-amino acid signal sequence, and the coding region is interrupted by 11 introns. The number of potential N-glycosylation sites in this product is 12 and the greatest among that of polyporales laccases. Moreover, this protein shares about 70% homology with other polyporales laccases. Genomic Southern analysis showed that C. versicolor laccases are encoded by more than four genes including CVLG1 and a transposed allele of this gene.     

Electroreduction of O(2) to water at 0.6 V (SHE) at pH 7 on the "wired" Pleurotus ostreatus laccase cathode

O(2) was electroreduced to water at 0.6 V (SHE) near neutral pH on the "wired" Pleurotus ostreatus laccase cathode. We previously reported high-current density (5 mA cm(-2)), four-electron electroreduction of O(2) to water on a "wired" Coriolus hirsutus laccase electrode at +0.7 V (SHE) in pH 5 in citrate buffer. Since the enzyme was inhibited by chloride and because its activity declined steeply when the pH was raised to neutral, the rate of O(2) electroreduction in a physiological buffer solution was only approximately 1% of that at pH 5 in absence of chloride. Here we show that substitution of the C. hirsutus laccase by laccase from P. ostreatus allows the upward extension of the pH range of O(2) electroreduction. The current density of the electrode made with laccase from P. ostreatus in pH 7 citrate buffer was approximately 100 microA cm(-2) and at pH 7 and in phosphate buffered NaCl (PBS, 20 mM phosphate, 0.1 M NaCl) it still retained 6% of its maximal (1 mA cm(-2)) current density at pH 5 in citrate buffer. The electrocatalyst consisted of the crosslinked P. ostreatus laccase and the electron conducting redox polymer PVI-Os(dmebpy)(tpy)(2+/3+) [PVI=poly(N-vinyl imidazole) with about 1/5th of the rings complexed with (Os-dmebpy-tpy)(2+/3+); dmebpy=4,4'-dimethyl-2,2'-bipyridine; tpy=2,2',6',2"-terpyridine].     

Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations are expensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)2]PF6 and Ru(bpy)2CO3 under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)2]PF6 and 3 for Ru(bpy)2CO3. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Fungal decomposition of oat straw during liquid and solid state fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Catalysis by Laccase (From Coriolus uersicolor) in Microheterogeneous Media of the Water/Organic Solvent/Surfactant Type

Catalysis by laccase from Coriolus uersicolor solubilized in the ternary systems of surfactant/water/organic solvent type, namely, Aerosol OT/water/octane, Brij 56/water/cyclohexane and egg lecithin/water/octane + pentanol + methanol mixture, has been studied. The laccase activity is found to depend, in principle, not only on the water/surfactant molar ratio, but on the surfactant concentration (with its hydration degree being constant) as well. The following inferences should be emphasized. Firstly, in all the systems under study, the catalytic activity (k_cat) of laccase entrapped into surfactant reversed micelles increases more than 50 times (when the surfactant concentration is extrapolated to zero) compared with the k_cat value in aqueous solution. Secondly, the catalytic activity (k_cat) of laccase entrapped in hydrated Aerosol OT aggregates, having lamellar, reversed cylindrical (hexagonal) and reversed micellar structure, depends greatly on the aggregate type. In other words, the phase transitions, i.e. an alteration in the packing of hydrated Aerosol OT molecules, evokes a sharp reversible change in the enzymatic activity. Thirdly, in the same phase, the catalytic activity of the solubilized enzyme depends on the linear dimensions of water cavities inside the surfactant aggregates (i.e. on the water content in the system under study). All these effects, regulating enzymatic activity, are probably caused by an alteration of the conformational mobility of laccase molecules incorporated into the inner polar cavities inside the surfactant aggregates.     

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