Laccase activity of lignicolous aquatic hyphomycetes isolated from the River Nile in Egypt

Eleven species of aquatic hyphomycetes were isolated from 92 samples of different lignin sources (unidentified wood segments, skeleton and neck of leaves, bark). The most common species were Pyramidospora casuarina (on 3.7% of samples), Triscelophorus monosporus (3.2%) and Flagellospora curvula (3%). Varying levels of laccase activity were present in most of the fungi included in this study. The laccase plate assay was found to be much less reliable than the spectrophotometric assay. Several factors including type of growth medium, the media pH and assay pH had marked effects on laccase activity. A few species produced high levels of laccase in both malt extract (ME) medium and low N medium; however, a majority of the species produced laccase in low nitrogen (N) medium (pH 4.5) but not in the ME medium. When the tested species were grown in low N medium at pH 4.5, six species showed acidic laccase (pH 4.5) activity; of these, four also showed alkaline laccase (pH 8.2) activity. Alatospora acuminata and Tetracladium marchalianum exhibited laccase activity only when grown in the low N medium at pH 8.2. These results indicate that aquatic hyphomycetes may play a role in the decomposition of lignin materials in freshwater environments. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and Characterization of Laccase from Chaetomium thermophilium and Its Role in Humification

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.     

Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.     

Effect of Direct-Current Electric Field on Enzymatic Activity and the Concentration of Laccase

This work investigates the effect of direct-current electric field on the extracellular enzymatic activity, concentration and other experimental parameters of laccase from Trametes versicolor. The results showed that laccase could significantly contribute to the change of pH at the end of graphite electrode. In addition, it increased the electrical conductivity of the water. In the experiment, the optimum pH and catalytic pH range for laccase activity were 3.0 and pH 2.5–4.0. The application of 6 V direct current showed significant effects on the laccase enzyme activity. The activity of laccase was enhanced in the anodic region, but at the same time was strongly inhibited at the cathode. The electric charge characteristics of laccase were changed when exposed to electric field, and some laccases molecules moved to the anode, which produced a slight migration phenomenon. This study is the basis of combination of laccase and electrical technology, at the same time, providing a new direction of enhancing laccase activity. Compared to immobilization, using electric field is simple, no chemical additives, and great potential.     

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.     

灵芝漆酶转化除草剂2,4-D丁酯的初步研究

来自灵芝ST（Ganoderma sp.ST）的漆酶在无氧化还原介质存在时能够直接转化苯氧羧酸类除草剂2,4-D丁酯。对酶转化2,4-D丁酯的条件进行了优化,包括pH值、给酶量和反应温度。灵芝ST漆酶在pH值4.0～8.0和15～30℃的范围内均能够有效转化2,4-D丁酯（初始浓度0.5mg/L）,在最适的pH值7.0、给酶量25U/L和温度30℃条件下,转化率随着酶作用时间的延长而提高,在24h左右达到最高（〉99.5%）。酶活力在作用时间内稳定,并且用自来水代替缓冲液获得的转化率相当。     

Catalytic behavior and detoxifying ability of a laccase from the fungal strain Cerrena unicolor

The kinetics and stability of a laccase isolated and purified from the fungal strain Cerrena unicolor were studied. The enzyme was produced in a great yield without inducers. Kinetic parameters were determined by using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate. At high ABTS concentrations (> 10 mM) a substrate inhibition phenomenon appeared and an inhibition constant K_i of 24 mM was determined. The pH- and temperature-profiles as well as the sensitivity of the enzyme to several deactivation agents were almost similar to those observed with laccase from different origins. Freezing-thawing treatment, high temperature, acidic pH (< 3.0) and acetonitrile strongly affected laccase activity. The laccase showed a good ability to oxidize different phenolic substances; a significant enhancing effect was showed by ABTS acting as co-substrate. These results seem to suggest that this new laccase preparation may be suitable for environmental purposes.     

Production of Laccase by Trametes hirsuta Grown in an Immersion Bioreactor and its Application in the Docolorization of Dyes from a Leather Factory

An alternative system for producing laccase on a bioreactor scale by the white‐rot fungus Trametes hirsuta is proposed. The experiments were performed in an immersion bioreactor (employing cuttings of stainless steel sponges as a support) and the culture medium was supplemented with copper sulfate (1 mM). Operating under these conditions, it was possible to obtain a maximum laccase activity of nearly 5,000 U/L within 9 days. In addition, the ability of the crude laccase produced to decolorize two synthetic acid dyes utilized in the leather industry (Luganil Green and Sella Solid Red) was investigated. The effect of the pH and the enzyme activity on decolorization was analyzed. It was found that a pH of 4.0 and a laccase activity of 300 U/L were optimal for Luganil dye decolorization (16.2 % in 2 hours). Sella Solid Red showed its highest decolorization (around 40 % in 2 hours) when used at pH 5.0 and at a laccase activity of 1,000 U/L.     

Oxygen-reducing enzyme cathodes produced from SLAC, a small laccase from Streptomyces coelicolor

The bacterially-expressed laccase, small laccase (SLAC) of Streptomyces coelicolor, was incorporated into electrodes of both direct electron transfer (DET) and mediated electron transfer (MET) designs for application in biofuel cells. Using the DET design, enzyme redox kinetics were directly observable using cyclic voltammetry, and a redox potential of 0.43 V (SHE) was observed. When mediated by an osmium redox polymer, the oxygen-reducing cathode retained maximum activity at pH 7, producing 1.5 mA/cm2 in a planar configuration at 900 rpm and 40 degrees C, thus outperforming enzyme electrodes produced using laccase from fungal Trametes versicolor (0.2 mA/cm2) under similar conditions. This improvement is directly attributable to differences in the kinetics of SLAC and fungal laccases. Maximum stability of the mediated SLAC electrode was observed at pH above the enzyme's relatively high isoelectric point, where the anionic enzyme molecules could form an electrostatic adduct with the cationic mediator. Porous composite SLAC electrodes with increased surface area produced a current density of 6.25 mA/cm2 at 0.3 V (SHE) under the above conditions.     

Novel laccases of Ganoderma sp. KU-Alk4, regulated by different glucose concentration in alkaline media

Physiological regulation of laccase production from Ganoderma sp. KU-Alk4, isolated in Thailand, was controlled by the initial glucose concentration in liquid culture. Different laccase isozymes were produced using different starting concentrations of glucose. With 1% glucose, two isozymes, KULac 1 and 2 were produced, while with 4% glucose, three different isozymes, KULac 3, 4 and 5, were produced. The KULacs differed in their molecular mass, ranging from 53 to 112 kDa. KULac 2 was a new laccase that had a different N-terminal amino acid sequence from other laccases previously reported. All the isozymes had optimum pH at 3.5 and were stable over the wide range of pH, 3.0–10.0, especially in alkaline pH. It is noteworthy that the activities of the four KULacs with 2,6-dimethoxyphenol were extremely high up to 90°C. They retained 100% of their activities at 60°C for 1 h.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation.

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions.     

A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: Purification, characterization and indigo carmine degradation

γ-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu²⁺-centre. It worked optimally at 55 °C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 even after 60 days at 4 °C. K_m and V_max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 °C) to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT.     

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 degrees C for 3 days, and then at 30 degrees C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.     

一株产漆酶细菌的分离鉴定及酶学性质研究

[目的]分离获得产漆酶的细菌菌株,研究漆酶的酶学性质并应用于染料脱色.[方法]利用含铜的富集培养基筛选产漆酶细菌;通过形态特征、生理生化试验及16SrDNA序列分析等方法进行鉴定;以丁香醛连氮为底物测定漆酶的酶学性质;通过测定染料在最大吸收波长下吸光值的变化评价漆酶对染料的脱色效果.[结果]从森林土壤中筛选到一株漆酶高产菌株LS05,初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);菌株LS05的芽孢漆酶以丁香醛连氮为底物的最适pH为6.6,最适温度为70℃;该酶具有较好的稳定性,经70℃处理10h或在pH 9.0条件下放置10d后可保留活性.对抑制剂SDS和EDTA具有一定的抗性,在碱性条件下可有效脱色不同的工业染料,RB亮蓝、活性黑和靛红1h内的脱色率达93％以上.[结论]Bacillus amyloliquefaciens LS05的芽孢漆酶在高温和碱性条件下稳定性强,相对于真菌漆酶具有更好的工业应用特性,可有效用于工业染料废水的处理.     

Phenol-oxidase (laccase) activity in strains of the hyphomycete Chalara paradoxa isolated from olive mill wastewater disposal ponds

Production of laccase activity by nine strains of Chalara paradoxa isolated from olive mill wastewater disposal ponds were studied. Enzyme extracts obtained from cultured broths by adsorption on hydroxyapatite showed a single band of laccase activity on ABTS after polyacrylamide gel electrophoresis (PAGE). They showed small mobility differences, with molecular masses of 67 to 68 kDa. Enzymes from the different strains oxidized a variety of phenolic and non-phenolic substances, and they could be divided into two groups according to their relative activities on substrates. All laccases showed a dual pH dependence of activity, with a maximum in the range of pH 3.0 to 4.5 for ABTS, o-dianisidine and 2,6-dimethoxyphenol, and pH 6.0 (Group 1) or pH 6.5 (Group 2) for syringaldazine and other substrates. Optimal temperatures were in the range of 10 to 28 degrees C for two strains (maximum at 10 degrees C) and 10 to 37 degrees C for the rest. The different enzymes were partially inactivated by heating at 60 degrees C and totally inactivated at 70 degrees C. Laccases were stable in a pH range of 3.0 to 9.0 (except for strain 36A, which was partially inactivated at pH 3.0), but became inactivated at pH 2.0. Altogether these data suggest that Ch. paradoxa strains produce different laccase isoenzymes.     

Biochemical characterization and potential for textile dye degradation of blue laccase from Aspergillus ochraceus NCIM-1146

In our study, we produced intracellular blue laccase by growing the filamentous fungus Aspergillus ochraceus NCIM-1146 in potato dextrose broth. The enzyme was then purified 22-fold to a specific activity of 4.81 U/mg using anion-exchange and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed maximum substrate specificity toward 2,2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid than any other substrate. The optimum pH and temperature for laccase activity were 4.0 and 60°C, respectively. The purified enzyme was stable up to 50°C, and high laccase activity was maintained at pH 5.0 ∼ 7.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol, and L-cysteine. Purified laccase decolorized various textile dyes within 4 h in the absence of redox mediators. HPLC and FTIR analysis confirmed degradation of methyl orange. The metabolite formed after decolorization of methyl orange was characterized as p-N,N′-dimethylamine phenyldiazine using GCMS.     

Removal of estrogenic activity from endocrine-disrupting chemicals by purified laccase of Phlebia tremellosa

A white-rot basidiomycete, Phlebia tremellosa, produced a laccase that showed increased activity during degradation of phthalates. A laccase was purified through the ion exchange chromatography and preparative gel electrophoresis, and the estimated molecular weight was 75 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 degrees C, respectively. The K(m) value of the enzyme was 55.7 microM, and the V(max) was 0.0541 OD min(-1) U(-1) for o-tolidine. Purified laccase reduced the estrogenic activity of four different endocrine-disrupting chemicals. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was involved in the removal of estrogenic activity.     

Covalent immobilization of laccase on activated carbon for phenolic effluent treatment

Summary Laccase was covalently immobilised to activated carbon using four derivatisation methods. The highest bound activity was obtained using diimide coupling of laccase to carboxyl groups on the carbon. The maximum bound activity was reached at 11.5 mg laccase/g carbon. The carbon-immobilised laccase (CIL) was stable at pH values from 4.0 to 9.0. CIL stored at 4°C lost 38± 5% activity in the first 4 days, then a further 22±5% in 126 days. CIL showed increased stability to low pH although the pH optimum was unchanged. The activation energy of CIL was lower than soluble laccase. Oxidation of 2,6-dimethoxyphenol (DMP) by CIL in a packed-bed system was only 30±10% of that in a fluidised bed system. Of the initial activity 10–30% was retained after oxidation of seven batches of DMP. CIL removed colour from two industrial effluents. Colour was removed from pulp mill bleach plant effluent at 115 colour units per enzyme unit per hour and the removal rate increased with increasing effluent concentration. Correspondence to: R. G. Burns     

Decolorization of Alizarin Red and other synthetic dyes by a recombinant laccase from Pichia pastoris

A cDNA encoding for a laccase was isolated from the white-rot fungus Lenzites gibbosa by RT-PCR and expressed in the Pichia pastoris. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as pH, cultivation temperature, copper concentration and methanol concentration, were optimized. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a MW of ~61.5 kDa. The purified enzyme behaved similarly to the native laccase produced by L. gibbosa and efficiently decolorized Alizarin Red, Neutral Red, Congo Red and Crystal Violet, without the addition of redox mediators. The decolorization capacity of this recombinant enzyme suggests that it could be a useful biocatalyst for the treatment of dye-containing effluents. This study is the first report on the synthetic dye decolorization by a recombinant L. gibbosa laccase.