Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETαA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae α-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the α-factor signal peptide was 9.79 U ml⁻¹. The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

Different proportions of laccase isoenzymes produced by submerged cultures of Trametes versicolor grown on lignocellulosic wastes

The white-rot fungus Trametes versicolor grown in submerged culture produced two laccase isoenzymes, LacI and LacII. Addition of insoluble lignocellulosic materials into the culture medium increased the total laccase activity. The proportion of laccase isoenzymes also changed depending on the lignocellulosic material employed, with ratios of activity LacII/LacI from 0.9 (barley straw) to 4.4 (grape stalks). Besides, this proportion played an important role in the dye decolourisation.     

Degradation of styrene by white-rot fungi

Degradation of styrene in the gaseous phase was investigated for white-rot fungi Pleurotus ostreatus (two strains), Trametes versicolor, Bjerkandera adusta and Phanerochaete chrysosporium. Fungi were grown in liquid culture and the gas/mycelium contact surface was enhanced with the help of perlite. The influence of various inducers on styrene degradation was studied. The best inducers for styrene degradation were lignosulphonate for P. ostreatus and T. versicolor and wood meal for B. adusta and P. chrysosoporium. Under these conditions all fungi were able to degrade styrene almost completely in 48 h at a concentration of 44 μmol/250 ml total culture volume; one strain of P. ostreatus was able to remove 88 μmol styrene under these conditions. Three transformation products of [¹⁴C]styrene in cultures of P. ostreatus were identified: phenyl-1,2-ethanediol, 2-phenylethanol and benzoic acid; 4% of the styrene was metabolised to CO₂ in 24 h and no other volatile products were found. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Cloning and characterization of a laccase gene from the white-rot basidiomycete <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 The gene lccK encoding a laccase of the white-rot basidiomycete Pleurotus ostreatus wild-type strain collected in Japan has been cloned, sequenced, and characterized. The isolated gene consists of 2929 bp with the coding region interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA elements were identified. Two putative N-glycosylation sites and four putative copper-binding sites found in other fungal laccase are conserved in lccK. The cDNA contains an open reading frame of 1599 bp and the gene encodes 533 amino acids preceded by a signal peptide of 23 amino acids. The nucleotide sequence of the lccK cDNA showed high homology with those of laccases of other basidiomycetes. Received: August 22, 2002 / Accepted: October 9, 2002 Present address: Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjo-nakano, Akita 010-0195, Japan Correspondence to:K. Okamoto     

Accumulation and degradation of dead-end metabolites during treatment of soil contaminated with polycyclic aromatic hydrocarbons with five strains of white-rot fungi

The white-rot fungi Trametes versicolor PRL 572, Trametes versicolor MUCL 28407, Pleurotus ostreatus MUCL 29527, Pleurotus sajor-caju MUCL 29757 and Phanerochaete chrysosporium DSM 1556 were investigated for their ability to degrade the polycyclic aromatic hydrocarbons (PAH) anthracene, benz[a]anthracene and dibenz[a,h]anthracene in soil. The fungi were grown on wheat straw and mixed with artificially contaminated soil. The results of this study show that, in a heterogeneous soil environment, the fungi have different abilities to degrade PAH, with Trametes showing little or no accumulation of dead-end metabolites and Phanerochaete and Pleurotus showing almost complete conversion of anthracene to 9,10-anthracenedione. In contrast to earlier studies, Phanerochaete showed the ability to degrade the accumulated 9,10-anthracenedione while Pleurotus did not. This proves that, in a heterogeneous soil system, the PAH degradation pattern for white-rot fungi can be quite different from that in a controlled liquid system. Received: 20 March 1996 / Received revision: 2 July 1996 / Accepted: 8 July 1996     

Production of ligninolytic enzymes and synthetic lignin mineralization by the bird's nest fungus Cyathus stercoreus

Production of ligninolytic enzymes and degradation of ¹⁴C-ring labeled synthetic lignin by the white-rot fungus Cyathus stercoreus ATCC 36910 were determined under a variety of conditions. The highest mineralization rate for ¹⁴C dehydrogenative polymerizates (DHP; 38% ¹⁴CO₂ after 30 days) occurred with 1 mM ammonium tartrate as nitrogen source and 1% glucose as additional carbon source, but levels of extracellular laccase and manganese peroxidase (MnP) were low. In contrast, 10 mM ammonium tartrate with 1% glucose gave low mineralization rates (10% ¹⁴CO₂ after 30 days) but higher levels of laccase and manganese peroxidase. Lignin peroxidase was not produced by C. stercoreus under any of the studied conditions. Mn(II) at 11 ppm gave a higher rate of ¹⁴C DHP mineralization than 0.3 or 40 ppm, but the highest manganese peroxidase level was obtained with Mn(II) at 40 ppm. Cultivation in aerated static flasks gave rise to higher levels of both laccase and manganese peroxidase compared to the levels in shake cultures. 3,4-Dimethoxycinnamic acid at 500 μM concentration was the most effective inducer of laccase of those tested. The purified laccase was a monomeric glycoprotein having an apparent molecular mass of 70 kDa, as determined by calibrated gel filtration chromatography. The pH optimum and isoelectric point of the purified laccase were 4.8 and 3.5, respectively. The N-terminal amino acid sequence of C. stercoreus laccase showed close homology to the N-terminal sequences determined from other basidiomycete laccases. Information on C. stercoreus, whose habitat and physiological requirements for lignin degradation differ from many other white-rot fungi, expands the possibilities for industrial application of biological systems for lignin degradation and removal in biopulping and biobleaching processes. Received: 29 January 1999 / Received revision: 5 July 1999 / Accepted: 9 July 1999     

Overproduction of recombinant laccase using a homologous expression system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants g^–1 of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.     

Solid-state fermentation of sugarcane bagasse with Flammulina velutipes and Trametes versicolor

The mushroom Flammulina velutipes and the white-rot fungus Trametes versicolor were cultivated separately on sugarcane bagasse for 40 days. Trametes versicolor produced laccase and manganese-peroxidase activities, showing a simultaneous degradation of lignin and holocellulose. However, only phenoloxidase activity was found with Flammulina velutipes. A preferential degradation of lignin was detected in F. velutipes, which exhibited a greater reduction in the ratio of weight loss to lignin loss than T. versicolor. A decrease in the syringyl/guaiacyl ratio observed with both fungi indicated the preferential degradation of non-condensed (syringyl-type) lignin units. An increase in the relative abundance of aromatic carboxylic acids suggested that the oxidative transformation of lignin unit side-chains was occurring. This was more noticeable with Flammulina velutipes than with T. versicolor.     

Amelioration of the ability to decolorize dyes by laccase: relationship between redox mediators and laccase isoenzymes in Trametes versicolor

The effect of redox mediators in the dye decolorization by two laccase isoenzymes from Trametes versicolor cultures supplemented with barley bran has been investigated. All the redox mediators tested, 1-hydroxybenzotriazole (HBT), promazine (PZ), para-hydroxybenzoic acid (pHBA) and 1-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), led to higher dye decolorization than those obtained without mediator addition. Among the different tested mediators, PZ was the most effective one at a low range of concentration (0.5–50 μM) and the natural mediator employed, pHBA did not improve significantly the degree of decolorization, and was slightly inhibitory.The two laccase isoenzymes, LacI and LacII, showed different decolorization capability depending on the mediator used. No significant differences were detected for NNDS, however LacII was more effective than LacI in the presence of PZ, while in the presence of HBT LacI was the fastest and the most effective isoenzyme.     

An Evaluation of Soil Colonisation Potential of Selected Fungi and their Production of Ligninolytic Enzymes for Use in Soil Bioremediation Applications

Initially sixteen fungi were screened for potential ligninolytic activity using decolourisation of a polymeric dye Poly R-478. From this, four fungi were selected, Trametes versicolor, Pleurotus ostreatus, Collybia sp., and an isolate (identified as Rhizoctonia solani) isolated from a grassland soil. Differences in the ligninolytic enzyme profiles of each of the fungi were observed. All of the four fungi tested produced MnP and laccase while the Collybia sp. and R. solani produced LiP in addition. Enzyme activity levels also varied greatly over the 21 days of testing with T. versicolor producing levels of MnP and laccase three to four times greater than the other fungi. The four fungi were then tested for their ability to colonise sand, peat (forest) and basalt and marl mixed till (field) soils through visual measurement and biomass detection in soil microcosms. Trametes versicolor and the Collybia sp. failed to grow in any of the non-sterilised soils whereas the R. solani and P. ostreatus isolates grew satisfactorily. Primers were␣designed to detect MnP and laccase genes in P.␣ostreatus and RTPCR was used to detect that these genes are expressed in forest and field soils.     

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.     

Degradative ability of 2,4,6-tribromophenol by saprophytic fungi Trametes versicolor and Agaricus augustus isolated from chilean forestry

Trametes versicolor and Agaricus augustus, with a maximum tolerable concentration (MTC) of 80 μg ml⁻¹ tribromophenol (TBP), were selected to evaluate TBP biodegradation capacity. These fungi were capable of decreased TBP concentrations and A. augustus was also capable of biotransforming TBP to tribromoanisole (TBA). Peroxidase and laccase activities were observed in the T. versicolor supernatant but not in that of A. augustus. These tolerance levels could be due to either lignolytic enzymes that degrade TBP or the ability of the fungi to biotransform TBP to tribromoanisole, respectively. The sustained ability of T. versicolor to degrade TBP (total of 40 μg ml⁻¹) in the presence of an additional carbon source suggests that it may have potential applications in the degradation of forestry industry waste.     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.