Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

Enzymatic characterization of Chilean native wood-rotting fungi for potential use in the bioremediation of polluted environments with chlorophenols

This work presents a preliminary report of a series of studies on the ability of several indigenous wood-rotting fungi from Chile to produce hydrolytic and ligninolytic enzymes and the evaluation of these native microorganism to future research on potential applications in bioremediation programs. Wood-rotting Basidiomycete fungi were collected from indigenous hardwood forest in the South of Chile. Twenty-eight strains were identified and qualitative enzymatic tests for peroxidases, laccase, tyrosinase, xylanase and cellulase production were performed in solid medium. Eleven selected strains were evaluated in liquid medium to quantify their ligninolytic enzyme production and their capacity to grow in solid medium supplemented with 2,4-dichlorophenol (2,4-DCF), 2,4,6-trichlorophenol (2,4,6-TCF) and pentachlorophenol (PCP). PCP degradation and ligninolytic enzymes production were also evaluated in liquid medium. Results showed that laccase was present in 28 of the selected strains (≈73%). Peroxidase was present in 40% and cellulase in 37% of the strains. Xilanase and tyrosinase were obtained in a smaller percentage in the strains (28% and 7%, respectively). The 11 selected strains showed high concentrations of lignin peroxidase (Lip) and manganese peroxidase (MnP). Anthracophyllum discolor (Sp4), produced LiP and MnP at 90.3 and MnP 125.5 U L⁻¹ respectively, compared to the control fungus Phanerochaete chrysosporium CECT-2798 that produced 58.1 and 118.4 U L⁻¹ of LiP and MnP. Tolerance test showed that native Chilean fungi did not present high tolerance to 2,4,6-TCF and PCP but were quite tolerant to 25 and 50 mg L⁻¹ of 2,4-DCF. However, pre-acclimatization in 2,4-DCP notably improved the growth in medium with 2,4,6-TCP and PCP. PCP in liquid medium was efficiently degraded by the fungi Anthracophyllum discolor, Lenzites betulina (Ru-30) and Galerina patagónica (Sp3), and the major MnP activity was produced by A. discolor (Sp4) (67 U L⁻¹).     

Partial purification and characterization of the novel halotolerant and alkalophilic laccase produced by a new isolate of Bacillus subtilis LP2

Laccases (benzenediol: oxygen oxidoreductases, [EC1.10.3.2] are mostly known as members of the blue multicopper oxidase family that are used in very different industrial applications: textile, pulp and paper, food, cosmetics industries, bioremediation process, biosensor, biofuel and organic synthesis. Stability against the extreme conditions is an important property and it makes laccase suitable for several industrial processes. Laccase should have salt resistance to be used in textile dye degradation because the textile wastewaters include dyes with high concentrations of salts, especially NaCl. Bacterial laccases are preferable to be used for bioremediation process due to their high stability to extremely salt contaminated and alkalophilic environment. Bacillus subtilis LP2 was identified as a source of alkali-tolerant, salt resistant laccase. Laccase showed activity over a wide pH (4–10) and temperature (30–80?°C) range. Maximum laccase activity was observed as 140.4?U/mg (umol/min*mg) at pH 8 and 50?°C with the substrate guaiacol. Stability of laccase was determined as 60% and 20% after incubation of the enzyme for different time intervals of 20 and 40?min at 50?°C and pH 8. SDS (10?mM) and EDTA (5?mM) decreased laccase activity from 100% to 0% and 56%, respectively. Despite the other inhibitors, NaCI increased the activity of laccase to 167% at 500?mM concentration. Laccase from Bacillus subtilis LP2 barely showed the activity on the substrates vanillin and L-tyrosine. These results clearly show that laccase from Bacillus subtilis LP2 has high potential to be used for several applications in textile industry.     

Isolation and characterization of novel Serratia marcescens (AY927692) for pentachlorophenol degradation from pulp and paper mill waste

Seven aerobic bacterial strains were isolated from pulp paper mill waste and screened for pentachlorophenol (PCP) tolerance on PCP containing mineral salt agar medium (MSM). The organism was characterized by 16S rDNA sequencing which showed 99.7% sequence similarity with Serratia marcescens. PCP degradation was routinely monitored with spectrophotometric analysis and further confirmed by HPLC analysis. Among seven strains, ITRC S₇ was found to degrade up to 90.33% of 1.127 mM (300 mg/l) of PCP and simultaneous release of chloride ion (2.435 mM) emphasized the bacterial dechlorination in the medium in presence of glucose as an additional carbon and energy source under optimized condition within 168 h incubation. In absence of glucose bacterium was unable to utilize PCP indicating the phenomenon of co-metabolism. Bacterium was identified as S. marcescens (AY927692), was a novel and potential aerobic bacterial strain capable of degrading PCP in axenic condition. Further, this strain may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Bioremediation of Polycyclic Aromatic Hydrocarbons in Creosote-Contaminated Soil by Peniophora incarnata KUC8836

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are present in products made from creosote, coal tar, and asphalt. When wood pile treated with creosote is placed in soil, PAHs can contaminate it. Creosote has been used for wood preservation in the past and is composed of approximately 85% PAHs and 15% phenolic compounds. PAHs cause harmful effects to humans and the environment because of their carcinogenic and mutagenic properties. White rot fungi can degrade not only lignin, but also recalcitrant organic compounds such as PAHs. Among numerous white rot fungi used in previous studies, four species were selected to degrade PAHs in a liquid medium. From this evaluation of the degradation of PAHs by the four fungal isolates, two species were ultimately selected for the highest rates of removal. Following 2 weeks of incubation with Peniophora incarnata KUC8836, the degradation rates of phenanthrene, fluoranthene, and pyrene were 86.5%, 77.4%, and 82.6%, respectively. Mycoaciella bispora KUC8201 showed the highest degradation rate for anthracene (61.8%). Hence, bioremediation of creosote-contaminated soil with an initial concentration of 229.49 mg kg^?1 PAHs was carried out using the two selected fungi because they could simultaneously degrade 13 more PAHs than the comparison species. More importantly, isolates of P. incarnata KUC8836 were discovered as powerful degraders of PAHs by producing laccase and manganese-dependent peroxidase (MnP), with 1.7- and 1.1-fold higher than the comparison species, respectively. Therefore, the white rot fungus may be proposed for the removal of PAHs and xenobiotic compounds in contaminated environments.     

Production of Wood Decay Enzymes,Loss of Mass,and Lignin Solubilization in Wood by Diverse Tropical Freshwater Fungi

In vitro production of cellulase and xylanase was common among diverse freshwater ascomycetes and their hyphomycetous anamorphs. Production of enzymes involved in lignin degradation was rare. Most isolates were capable of causing mass loss in angiosperm wood, although values were low, at ~10% during a 24-week period. A few isolates caused higher mass loss of up to 26.5%, and five of these were shown to solubilize significant amounts of lignin. This is the first report of lignin solubilization by freshwater fungi. Torula herbarum (hyphomycete) and Ophioceras dolichostomum (ascomycete) produced indices of lignin solubilization equivalent to those of terrestrial white-rot basidiomycetes. In all cases wood decay was 2.2- to 3-fold higher in exposed rather than submerged conditions.     

Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor

One-electron oxidation activity, as measured by ethylene generation from 2-keto-4-thiomethylbutyric acid, phenol oxidase activity, and the generation of hydroxyl radical were examined in cultures of the lignin-degrading white-rot basidiomycete fungus, Trametes (Coriolus) versicolor. The activity levels of specific lignin-degrading enzymes and cellulases, as well as the rate of wood degradation, also were examined. The fungus secreted a low-molecular-weight substance (M(r) 1000-5000) that catalyzed a redox reaction between molecular oxygen and an electron donor, to produce the hydroxyl radical via hydrogen peroxide. During wood decay, T. versicolor also produced significant amounts of laccase and lignin peroxidase, carboxymethyl cellulase, and Avicelase. The roles of the hydroxyl radical, phenol oxidases, and cellulases in wood degradation by white-rot fungi are discussed. That the hydroxyl radical produced by the low-molecular-weight substance secreted by T. versicolor results in new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase or manganese peroxidase is suggested.     

Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota

Pentachlorophenol (PCP) bioremediation by the fungal strains amongst the cork-colonising community has not yet been analysed. In this paper, the co- and direct metabolism of PCP by each of the 17 fungal species selected from this community were studied. Using hierarchical data analysis, the isolates were ranked by their PCP bioremediation potential. Fifteen isolates were able to degrade PCP under co-metabolic conditions, and surprisingly Chrysonilia sitophila, Trichoderma longibrachiatum, Mucor plumbeus, Penicillium janczewskii and P. glandicola were able to directly metabolise PCP, leading to its complete depletion from media. PCP degradation intermediates are preliminarily discussed. Data emphasise the significance of these fungi to have an interesting potential to be used in PCP bioremediation processes.     

Isolation and Growth Characteristics of Chromium(VI) and Pentachlorophenol Tolerant Bacterial Isolate from Treated Tannery Effluent for its Possible Use in Simultaneous Bioremediation

The bacterial strains resistant to pentachlorophenol (PCP) and hexavalent chromium [Cr(VI)] were isolated from treated tannery effluent of a common effluent treatment plant. Most of the physico-chemical parameters analyzed were above permissible limits. Thirty-eight and four bacterial isolates, respectively were found resistant to >50 μg/ml concentration of [Cr(VI)] and the same level of PCP. Out of the above 42 isolates, only one was found simultaneously tolerant to higher levels of both PCP (500 μg/ml) and Cr(VI) (200 μg/ml), and hence was selected for further studies. To the best of our knowledge, this is the first report in which a native bacterial isolate simultaneously tolerant to such a high concentrations of Cr(VI) and PCP has been reported. The culture growth was best at 0.4% (w/v) glucose as an additional carbon source and 0.2% (w/v) ammonium chloride as a nitrogen source. The growth results with cow urine as a nitrogen source were comparable with the best nitrogen source ammonium chloride. The isolate exhibited resistance to multiple heavy metals (Pb, As, Hg, Zn, Co & Ni) and to antibiotics nalidixic acid and polymixin-B. The efficacy of bacterial isolate for growth, PCP degradation (56.5%) and Cr(VI) bioremediation (74.5%) was best at 48 h incubation. The isolate was identified as Bacillus sp. by morphological and biochemical tests. The 16S rDNA sequence analysis revealed 98% homology with Bacillus cereus. However, further molecular analysis is underway to ascertain its likelyhood of a novel species.     

Bioremediation of a Chilean Andisol contaminated with pentachlorophenol (PCP) by solid substrate cultures of white-rot fungi

This study provides a first attempt investigation of a serie of studies on the ability of Anthracophyllum discolor, a recently isolated white-rot fungus from forest of southern Chile, for the treatment of soil contaminated with pentachlorophenol (PCP) to future research on potential applications in bioremediation process. Bioremediation of soil contaminated with PCP (250 and 350 mg kg⁻¹ soil) was investigated with A. discolor and compared with the reference strain Phanerochaete chrysosporium. Both strains were incorporated as free and immobilized in wheat grains, a lignocellulosic material previously selected among wheat straw, wheat grains and wood chips through the growth and colonization of A. discolor. Wheat grains showed a higher growth and colonization of A. discolor, increasing the production of manganese peroxidase (MnP) activity. Moreover, the application of white-rot fungi immobilized in wheat grains to the contaminated soil favored the fungus spread. In turn, with both fungal strains and at the two PCP concentrations a high PCP removal (70–85%) occurred as respect to that measured with the fungus as free mycelium (30–45%). Additionally, the use of wheat grains in soil allowed the proliferation of microorganisms PCP decomposers, showing a synergistic effect with A. discolor and P. chrysosporium and increasing the PCP removal in the soil.     

Degradation of wood and enzyme production by Ceriporiopsis subvermispora

The degradation of the components of Japanese beech and Japanese cedar wood was measured over time in cultures of the white-rot fungus Ceriporiopsis subvermispora. Although there was no initial degradation of cedar wood, after 12 weeks the mass loss of both cedar and beech wood was 15–20%. The mass losses of filter paper in beech wood-containing cultures and glucose cultures after 12 weeks were 87% and 70%, respectively. The ratio of lignin loss to mass loss of both beech and cedar wood cultures approached 2.0. Although the cellulose loss in cedar wood was very low throughout the 12-week incubation, C. subvermispora degraded the hemicellulose in Japanese cedar much more effectively than that in Japanese beech. These results confirm that C. subvermispora is a selective lignin degrader. During the 12-week incubation with Japanese beech wood, C. subvermispora continuously produced at least one of three phenol oxidases: laccase was produced initially, followed by Mn-independent peroxidase activity peaking at 6 weeks and Mn-dependent peroxidase activity peaking at 10 weeks. Lignin peroxidase and carboxymethylcellulase activities peaked after 3 weeks of incubation. Avicelase activity was present throughout the incubation period, although the activity was very low. The low-molecular-mass fraction of the extracellular medium, which catalyzes a redox reaction between O₂ and electron donors to produce hydroxyl radical, may act synergistically with the enzymes to degrade wood cell walls.     

Biopulping of wood chips with Phlebia brevispora BAFC 633 reduces lignin content and improves pulp quality

The white-rot fungus Phlebia brevispora BAFC 633 produces laccases in large proportions. In this work P. brevispora BAFC 633 was grown on Pinus taeda wood chips in 10-L bioreactors. To select the biopulping experimental conditions, we analyzed the variables affecting enzymatic laccase activity in the culture supernatants, indicating that the suitable incubation temperature was 30 °C in order to promote enzyme stability. Phlebia brevispora BAFC 633 secreted 744 U/g of laccase, selectively removing lignin during biotreatment of wood chips, causing a reduction in Kappa number and 10% weight loss, and creating a more open structure and better access to the pulping liquor, which would require less chemical consumption, thus diminishing the environmental impact of the chemical pulping process.These results support the biotechnological potential of P. brevispora BAFC 633 for biopulping processes and enhance the potential for bioprospecting native isolates of the microflora of our country's natural environment.     

Laccase production and wood degradation by a white-rot fungus Daedalea flavida

A comparative study has been conducted on seven white rot fungi to investigate their abilities to produce laccase and selectively degrade lignin. Laccase was produced constitutively on the different media tested. Of the different lignins, phenolic compounds and sugars involved, the highest laccase yield was obtained on indulin AT. Salicylic acid inhibited enzyme activity. A temperature of 20°C and 0.2% of indulin AT were found to be optimum for enzyme activity. No correlation was found between the amount of enzyme and fungal mass produced. During semisolid degradation of angiospermic wood sawdust, Daedalea flavida caused a total weight loss of 11%, with a lignin loss of 15.77% during two months of decay. Lignin removal was comparatively selective during the first month, during which time laccase production was also higher, indicating its probable role in lignin degradation.     

Degradation of poplar wood by Fomes sclerodermeus: production of ligninolytic enzymes in sawdust of poplar and cedar

Fomes sclerodermeus is a white-rot fungus. Its production of laccase, manganese peroxidase and lignin peroxidase on sawdust-based media was evaluated. No lignin peroxidase activity was measured in any media tested. The higher production of laccase and manganese peroxidase were found on media containing poplar sawdust. F. sclerodermeus was grown on wood blocks of poplar during six months. Dry weight losses of the blocks reached a mean value of 51%. The quantification of cellulose and lignin in the 6-months incubated blocks showed losses of up to 58 and 56% for cellulose and lignin, respectively. The decay examined under microscope revealed mycelium colonizing the lumen of vessel elements, cell wall thinning and entire degradation of the radial parenchyma.     

Remediation of pentachlorophenol-contaminated soil by composting with immobilized Phanerochaete chrysosporium

Summary To reduce and eliminate the hazards of pentachlorophenol (PCP) to the soil, the method of inoculating free and immobilized white rot fungi, Phanerochaete chrysosporium to PCP-polluted soils was investigated. Three parallel beakers A, B, C are adopted with the same components of soil, yard waste, straw and bran for aerated composting to degrade the PCP in soil. A was with no inoculants as control, B was added with the inoculants of immobilized P.␣chrysosporium, C was inoculated with non-immobilized P. chrysosporium, and additionally D contained only PCP-contaminated soils also as control. By contrastive analyses, the feasibility of applying composting to the bioremediation of the PCP-polluted soil was discussed. From the experimental results, it could be seen that the degradation rate of PCP by the immobilized fungi exceeded 50% at day 9, while that of the non-immobilized fungi achieved the same rate at day 16. However, the final degradation rates of PCP for both of them were beyond 90% at day 60 and that the rate of A was much lower than the others. The above data have shown that the degradation effect of inoculating P. chrysosporium was better than that of no inoculation, and that of the immobilized fungi was better than that of non-immobilized ones. Meanwhile, shown by all the indicators the composts of A, B and C were mature and stabilized at the end of the experiment. Therefore, the method of composting with immobilized P.␣chrysosporium is effective for the bioremediation of PCP-contaminated soil.     

Effects of Incubation Time and Temperature on In Vitro Selective Delignification of Silver Leaf Oak by Ganoderma colossum

The effects of incubation time and temperature on the ability of isolates of the chlamydosporic and thermophilic fungus Ganoderma colossum (Fr.) C. F. Baker to cause selective delignification of Quercus hypoleucoides A. Camus were evaluated by standard in vitro agar block tests. Chemical and scanning electron microscopy studies of decayed wood were used to determine the extent of selective delignification or simultaneous decay caused by each fungal isolate. At 35 deg C, the percent weight loss increased from 6.1% after 4 weeks to a maximum of 32.5 to 33.0% after 16 and 20 weeks of incubation. The average percent Klason lignin-chlorite holocellulose ratios (PKL/CHC) decreased from 0.35 in the control wood block to 0.22 in wood blocks incubated for 12 weeks; this indicated selective delignification. The average PKL/CHC increased for the 16- and 20-week incubation periods, indicating greater removal of polysaccharides during longer incubation periods. In temperature studies, the percent weight loss after 12 weeks was 26 to 27% between 30 and 40 deg C and less than 16% for the 25 and 45 deg C treatments. The average PKL/CHC ranged from 0.18 to 0.16 between 35 and 40 deg C, whereas they were 0.23 and 0.31 for the 25 and 45 deg C treatments, respectively. Scanning electron microscopy confirmed an optimum temperature range near 35 to 40 deg C and incubation times of 8 to 12 weeks for selective delignification. Under these conditions, ray parenchyma, fiber tracheids, and vessels were devoid of middle lamella; pit regions of cells were visible with significantly enlarged apertures; and individual cells were separated and clearly delimited. Extensive delignification of wood occurred throughout the wood blocks evaluated. Incubation times longer than 12 weeks resulted in greater degradation of wood cell walls and thus in greater removal of the polysaccharide component of the wood. For incubation times of 4 weeks or a temperature of 25 deg C, limited to no degradation of cells was observed. At 45 deg C, walls of fiber tracheids were eroded and ray parenchymal cells were extensively degraded, indicating that simultaneous degradation of cell walls occurred. Thus, the incubation temperature influenced the type of decay by G. colossum observed on oak wood blocks: extensive selective delignification at 35 to 40 deg C after more than 8 weeks of incubation or simultaneous decay at 45 deg C with 14% weight loss after 12 weeks of incubation. Isolates of G. colossum may prove useful in studies on mechanisms of delignification and biotechnological applications (e.g., biopulping) of lignin-degrading fungi.     

血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解

白腐菌是目前已知的唯一能将木质素彻底降解的微生物,而漆酶在木质素分解过程中起着重要的作用,被广泛应用于农作物秸秆或甘蔗渣等多种类型生物质的生物预处理和生物降解。本研究利用白腐菌产漆酶发酵培养基对30株血红密孔菌Pycnoporus sanguineus菌株进行筛选,得到了多株漆酶高产菌株,并研究了血红密孔菌发酵粗酶液和菌丝对烟梗的生物降解条件。研究结果表明:血红密孔菌及其产生的漆酶表现出了对烟梗木质素较强的生物降解能力。在漆酶浓度为40U/mL、温度30℃、pH4.5的条件下处理24h,烟梗中木质素的降解率可达到50.4%,纤维素和半纤维素的降解率分别为17.5%和17.3%;漆酶浓度为5U/mL、温度30℃、pH4.5的条件下处理48h,木质素降解率可达到65.1%。血红密孔菌菌丝也表现出对烟梗较好的生物降解效果,接种培养7d后烟梗中木质素降解率可达30%以上,21d后木质素的降解率可达79.1%,而纤维素和半纤维素的降解率仅为20%和12%左右。本研究不但为生物质材料的生物预处理和生物降解提供了优质的白腐菌及漆酶资源,还为通过烟梗的生物预处理提高烟草梗丝和卷烟品质提供了重要参数,具有一定的应用前景。     

Characterization of a strong CCA-treated wood degrader,unknown <Emphasis Type="Italic">Crustoderma</Emphasis> species

In this study, basidiomycete isolates that possessed a strong ability to degrade chromated copper arsenate (CCA)-treated wood were characterized. These fungal isolates, which were collected from CCA-treated pine log wastes, showed no recognizable morphological properties on culture media. Nucleotide sequence analysis of the large subunit rDNA of the isolates revealed that they were one species. Based on the high sequence similarity (>95%) and close phylogenetic relationship with several known species of Crustoderma, the fungal isolates characterized in this study were classified as a Crustoderma sp. In a wood degradation test, Crustoderma isolate KUC8611 produced a remarkably higher weight loss in CCA-treated Pinus radiata (68.7%), Pseudotsuga menziesii (39.7%), and Tsuga heterophylla (38.5%) wood than other evaluated basidiomycete species, including Crustoderma flavescens and Crustoderma corneum. In addition, extracellular enzymes for cellulose and protein degradation were detected when the isolates were cultured in chromogenic media, which supports the finding that isolate KUC8611 is a wood degrader. Furthermore, an in vitro test for metal tolerance revealed that isolate KUC8611 showed strong arsenic tolerance, but that it could not tolerate copper. Finally, isolate KUC8611 produced lower amounts of oxalic acid than copper-tolerant fungi such as Fomitopsis palustris and Antrodia vaillantii. To the best of our knowledge, this is the first study to report the degradation of CCA-treated wood by a Crustoderma species.     

Synergistic biodegradation of pentachlorophenol by <Emphasis Type="Italic">Bacillus cereus</Emphasis> (DQ002384), <Emphasis Type="Italic">Serratia marcescens</Emphasis> (AY927692) and <Emphasis Type="Italic">Serratia marcescens</Emphasis> (DQ002385)

The consortium of Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) were used for pentachlorophenol (PCP) degradation. The consortia showed better overall removal efficiencies than single strains by utilization of PCP as a carbon and energy source confirmed by pH dependent dye indicator bromocresol purple (BCP) in mineral salt media (MSM). Mixed culture was found to degrade up to 93% of PCP (300 mg/l) as compared to single strains (62.75–90.33%), at optimized conditions (30 ± 1°C, pH 7 ± 0.2, 120 rpm) at 168 h incubation. PCP degradation was also recorded at 20°C (62.75%) and 37°C (83.33%); pH 6 (70%) and pH 9 (75.16%); 50 rpm (73.33%) and 200 rpm (91.63%). The simultaneous release of chloride ion up to 90.8 mg/l emphasized the bacterial dechlorination in the medium. GC–MS analysis revealed the formation of low molecular weight compound, i.e., 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone, from degraded sample as compared to control.     

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999