红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

Production of ligninolytic exoenzymes and14C-Pyrene mineralization byPleurotus sp. in lignocellulose substrate

Pleurotus sp. was grown in liquid medium and on a solid straw substrate, and activities of laccase and manganese-dependent peroxidase (MnP) were recorded. The activities were the highest in a rich, glucose corn-steep liquid medium. In straw cultures, laccase activity was about ten times lower. Under solid state conditions, MnP production was the highest during days 20–40, when laccase activity already had declined. In straw cultures, mineralization of¹⁴C-pyrene was measured as release of¹⁴CO₂. The highest rates of pyrene mineralization occurred during days 20–45,i.e. the period of high MnP activities, suggesting a role of this enzyme in PAH degradation. Within 60d, 24% of pyrene was mineralized.     

Enhanced production of laccase in <Emphasis Type="Italic">Coriolopsis rigida</Emphasis> grown on barley bran in flask or expanded-bed bioreactor

In this work, the effect on laccase activity of adding xylidine, veratryl alcohol and copper sulphate to cultures of Coriolopsis rigida under submerged cultivation conditions have been investigated. The highest activities (approximately 6 × 10⁵ nkat/l) were obtained when the inducers copper sulphate (2 mM) and xylidine (10 mM) were added simultaneously. In addition, operating in the optimal conditions, it was possible to maintain the sustained production of laccase (around 3 × 10⁵ nkat/l) for successive repeated batch cultures in an expanded-bed laboratory scale bioreactor. On the other hand, in vitro phenol degradation by laccase obtained in the bioreactor was studied with/without an effective mediator 1-hydroxybenzotriazol (HBT). The presence of a radical mediator plays an important role inducing the degradation of phenol, and without mediator the polymerization of phenol was detected.     

Purification of recombinant laccase from Trametes versicolor in Pichia methanolica and its use for the decolorization of anthraquinone dye

A recombinant laccase from Trametes versicolor in Pichia methanolica was produced constitutively in a defined medium. The recombinant laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 64 kDa by SDS-PAGE. The purified recombinant laccase decolorized more than 90% of Remazol Brilliant Blue R (RBBR) initially at 80 mg l⁻¹ after 16 h at 45°C and pH 5 when 25 U laccase ml⁻¹ was used. The purified recombinant laccase could efficiently decolorize RBBR without additional redox mediators.     

An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol

Rhizopus nigricans, isolated from an industrial effluent (paper mill), was resistant to pentachlorophenol (PCP) in Petri dishes and in submerged cultures (100 and 25 mg l^–1 respectively). It was shown that this strain of R. nigricans can remove PCP in submerged culture. When 12.5 mg of PCPl^–1 were added at 48 h, this compound had been completely removed by 144h. Results indicated that the fungus did not produce extracellular lignin peroxidase (LiP) and laccase, but extracellular phenoloxidase production was observed. The synthesis of the latter enzyme was stimulated by the presence of PCP and/or tyrosine. These results indicate that this fungus, and probably other filamentous fungi, have an interesting potential to be used in processes for chlorophenol biodegradation.     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Effect of Cu2+, Mn2+ and aromatic compounds on the production of laccase isoforms by Coprinus comatus

Six different extracellular laccase isoforms were identified in submerged cultures of the commercially important edible mushroom, Coprinus comatus. Although laccase activity (~55 IU/L) was readily detectable in unsupplemented control cultures containing 1.6 μM Cu²⁺ after 22-day incubation, mean enzyme levels (~150–185 IU/L) were 2.7–3.4-fold higher in cultures supplemented with 0.5–3.0 mM Cu²⁺. Laccase production was also stimulated by Mn supplementation over the range 0.05–0.8 mM Mn²⁺, and the peak value of ~225 IU/L recorded after 22 days in cultures containing 0.8 mM added Mn²⁺ was 4.5-fold higher compared with unsupplemented controls. Of 12 aromatic compounds tested for their effect on laccase isozyme production by C. comatus, highest laccase levels (~188 IU/L), equivalent to a 4.4-fold increase compared with unsupplemented controls (~43 IU/L), were recorded after 22 days in cultures supplemented with 3.0 mM caffeic acid. Other aromatic compounds tested all stimulated laccase production, with peak enzyme levels 1.3–3.3-fold higher compared with unsupplemented controls. Extracellular laccase levels in cultures supplemented with optimal concentrations of Mn²⁺ and caffeic acid together were 38% and 15% lower, respectively, compared with cultures containing the separate supplements. Lac1 was the most abundant laccase isoform produced under all the conditions tested, but marked differences were observed in the production patterns of Lac2–Lac6.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam gave maximum activities of 220 U lignin peroxidase ml^–1, 150 U manganese peroxidase ml^–1, 50 U laccase ml^–1 and 6.2 U protease ml^–1 at the retention time of 24 h for 60 days. Protease secretion destabilized the produced lignin peroxidase, manganese peroxidase and laccase.     

Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products

More than 90% of the antibiotics ciprofloxacin (CIPRO) and norfloxacin (NOR) at 2 mg L⁻¹ were degraded by Trametes versicolor after 7 days of incubation in malt extract liquid medium. In in vitro assays with purified laccase (16.7 nkat mL⁻¹), an extracellular enzyme excreted constitutively by this fungus, 16% of CIPRO was removed after 20 h. The addition of the laccase mediator 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt led to 97.7% and 33.7% degradation of CIPRO and NOR, respectively. Inhibition of CIPRO and NOR degradation by the cytochrome P450 inhibitor 1-aminobenzotriazole suggests that the P450 system also plays a role in the degradation of the two antibiotics. Transformation products of CIPRO and NOR were monitored at different incubation times by triple-quadrupole and quadrupole time-of-flight mass spectrometry, and can be assigned to three different reaction pathways: (i) oxidation of the piperazinyl substituent, (ii) monohydroxylation, and (iii) formation of dimeric products.     

Changes in production of lignin degrading enzymes during interactions between mycelia of the tropical decomposer basidiomycetes Marasmiellus troyanus and Marasmius pallescens

During the interaction of two tropical agaric fungi, Marasmius pallescens and Marasmiellus troyanus, on agar media, initial deadlock between the two mycelia was ultimately followed by take-over by M. troyanus. When shaken liquid cultures of these two fungi were mixed, a rapid increase in laccase and manganese peroxidase activity, but no lignin peroxidase, was detected in the culture supernatant. Even more rapid and elevated induction of laccase occurred when filter-sterilized supernatant of Marasmius pallescens was added to Marasmiellus troyanus cultures, but the reciprocal experiment (addition of M. troyanus supernatant to M. pallescens cultures), did not lead to any increase in laccase activity. Addition of autoclaved supernatant of M. pallescens also induced laccase activity from M. troyanus cultures, but over a period of days rather than hours. Although both M. troyanus, and to a lesser extent M. pallescens, are able to produce laccases in shaken liquid culture following addition of the inducer 2,5-dimethylalanine, these experiments suggest that the presence of heat-stable and heat-labile laccase inducers secreted by M. pallescens mycelia lead to induction of laccases by M. troyanus.     

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.     

Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents

In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated. Substrates included wood chips, cereal grain, wheat husk and wheat bran. Suitability for transformation of chlorophenols depended on laccase production by the fungus. The greatest amount of laccase (<25 Units g⁻¹ substrate) was produced on wheat husk and wheat bran over 30 days colonisation. Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75–80% of pentachlorophenol (50 ppm) within 24 h. Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation. Addition of 8–12% yeast extract to the pellets increased laccase production five-fold. Colonised pellets were added to chlorophenol solutions in 200–4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. Received: 10 April 2000 / Received revision: 4 July 2000 / Accepted: 10 July 2000     

Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K _m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN₃, NaF, and HgCl₂.     

Optimizing Production of Extracellular Laccase from Grammothele Subargentea CLPS No. 436 Strain

The production of extracellular laccase by the Grammothele subargentea CLPS no. 436 strain in liquid cultures grown on a carbon-limited basal medium was significantly enhanced when culture conditions, including the addition of CuSO₄·5H₂O or veratryl alcohol, were consecutively optimized. A laccase activity as high as 1954.5 mU ml⁻¹ of liquid medium was obtained under optimum conditions, which corresponded to non-agitated cultures supplemented with 0.6 mM CuSO₄·5H₂O. Veratryl alcohol at 1 mM was less effective than CuSO₄·5H₂O for increasing laccase activity levels; the supplementation of veratryl alcohol resulted only in maximum levels of 44 mU ml⁻¹ in non-agitated cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Extracellular laccase activity in Tetraploa aristata

Tetraploa aristata CLPS 419 produced maximum extracellular laccase activity at over 9 mU ml^–1 in shaking cultures supplemented with glucose and 3.5 mU ml^–1 in sucrose-grown ones. Laccase activity did not exceed 0.7 mU ml^–1 in stationary cultures with glucose and was not detected in similar cultures with sucrose or in ones grown on lignin.