Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

Summary Laccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.     

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.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.     

Biochemical characterization and molecular evidence of a laccase from the bird's nest fungus Cyathus bulleri

Cyathus bulleri, a bird's nest fungus, known to decolorize polymeric dye Poly R-478, was found to produce 8 U ml(-1) of laccase in malt extract broth. Laccase activity appeared as a single band on non-denaturing gel. Laccase was purified to homogeneity by anion exchange chromatography and gel filtration. The enzyme was a monomer with an apparent molecular mass of 60 kD, pI of 3.7 and was stable in the pH range of 2-6 with an optimum pH of 5.2. The optimal reaction temperature was 45 degrees C and the enzyme lost its activity above 70 degrees C. Enzyme could oxidize a broad range of various phenolic substrates. K(m) values for ABTS, 2,6-dimethoxyphenol, guaiacol, and ferulic acid were found to be 48.6, 56, 22, and 14 mM while K(cat) values were 204, 180, 95.6, and 5.2, respectively. It was completely inhibited by KCN, NaN(3), beta-mercaptoethanol, HgCl(2), and SDS, while EDTA had no effect on enzyme activity. The N-terminal amino acid sequence of C. bulleri laccase showed close homology to N-terminal sequences of laccase from other white-rot fungi. A 150 bp gene sequence encoding copper-binding domains I and II was most similar to the sequence encoding a laccase from Pycnoporus cinnabarinus with 74.8% level of similarity.     

Yellow laccase from the fungus Pleurotus ostreatus D1: purification and characterization

Yellow laccase was isolated from a solid-phase culture of the fungus Pleurotus ostreatus D1 and characterized. It is a copper-containing enzyme with molecular weight 64 kDa. Its absorption spectrum lacks the maximum at 610 nm, characteristic of fungal laccases and corresponding to type I copper atom. The optimum pH values for the enzyme were determined. They proved to be: 7.0 for syringaldazine, 8.0 for pyrocatechol, and 4.0 for 2,2'-azine-bis-(3-ethylbenzothiazoline-6-sulfonate and 2,6-dimethoxyphenol. Kinetic parameters (Km and Vmax) for oxidation of these substrates were determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme was studied. It was shown that yellow laccase from Pleurotus ostreatus D1 oxidized anthracene to anthraquinone by 95% without any mediator.     

Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood

Trametes versicolor was shown to produce extracellular laccase during surface cultivation on glucose, wheat straw and beech wood. Growth on both wheat straw and beech wood led to an increase as high as 3.5-fold in extracellular laccase activity, in comparison with growth on glucose. The corresponding yields in fungal biomass reached only about 20% of the value obtained on glucose. Manganese peroxidase activity␣appeared during growth on wheat straw and beech wood. Mycelia grown on glucose, wheat straw and beech wood also showed intracellular laccase activities, monitored with 2,6-dimethoxyphenol, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) and 3,4-dihydroxyphenylalanine (l-DOPA). Assaying intracellular laccase with 2,6-dimethoxyphenol, syringaldazine and l-DOPA showed the maximum oxidation rates to be at pH values different from those producing maximum oxidation rates with extracellular laccase. In each case most of the total laccase activity was recovered from the culture filtrates. Growth on wheat straw and beech wood led to increased values for both extra- and intracellular laccase activities, based on fungal dry weight, in comparison with growth on glucose. Received: 18 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

一色齿毛菌漆酶的酶学特性及染料脱色研究

染料由于具有复杂的化学结构通常难以降解。本文从白腐菌一色齿毛菌LS0547中纯化出胞外漆酶并用于染料脱色实验。SDS-PAGE结果显示纯化的漆酶分子量大小为63.7kDa。漆酶氧化底物ABTS的最适pH为2.2,最适温度为50℃。叠氮钠可强烈抑制漆酶活性,半胱氨酸和二硫苏糖醇可部分抑制漆酶活性。漆酶氧化ABTS,丁香醛连氮和2,6-二甲氧基苯酚的米氏常数分别为0.217,0.306和0.199mmol/L。粗酶和纯化的漆酶用于不同化学结构的染料的脱色研究,结果表明一色齿毛菌纯化漆酶可快速对RB亮蓝进行脱色,偶氮胭脂红和结晶紫的脱色效果低于RB亮蓝,测试的三种染料均可在没有介体存在的条件下被漆酶脱色,显示出一色齿毛菌漆酶在染料废水处理中的应用前景。     

Isolation and biochemical characterization of laccase and tyrosinase activities in a novel melanogenic soil bacterium

Bacterial polyphenol oxidases (PPOs) with a high oxidizing capacity for a wide range of substrates could make their applications in phenolic biotransformation, food processing, cosmetics, and textile industry. We have isolated a melanogenic soil bacterium by differential screening of a number of strains which were isolated from the Iranian microflora. The taxonomic characterization of this strain indicates that it belongs to the genus Bacillus (HR03), and has the ability to produce all types of PPOs; cresolase (EC 1.14.18.1), cathecolase (EC.1.10.3.1), and laccase (EC 1.10.3.2). We studied the tyrosinase activity using l-tyrosine and l-dopa as substrates and the laccase activity with specific substrates such as syringaldazine and 2,6-dimethoxyphenol. The optimum pH and temperature, obtained for all types of polyphenol oxidases, were at about pH 5.5 and 55 °C, respectively. Tyrosinase-like enzyme of this strain shows a lag period in its tyrosine hydroxylase activity that could be avoided by the addition of small amounts of l-dopa and sodium dodecyl sulfate (SDS). In addition, tyrosinase and laccase were activated by SDS below the critical micelle concentration and were inhibited by 1 mM EDTA. We tested the resistance of melanized-cells against UVA, UVC and H₂O₂. Results show that melanin protects strain HR03 against UV lights and the oxidant.     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.     

Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile

An extracellular laccase-producing ascomycete was isolated from soil and identified as Paraconiothyrium variabile using rDNA sequence analysis. Typical laccase substrates including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol (DMP), and guaiacol were oxidized by the purified enzyme (designated as PvL). The molecular mass of PvL was 84 kDa and it showed a pI value of 4.2. The enzyme acted optimally at pH 4.8 and exhibited an optimum temperature of 50 °C. Using ABTS, PvL represented Km and Vmax of 203 μM and 40 μmol min(-1) mg(-1), respectively. After 24 h incubation at pH 4.8 and 4 °C, 80% of the initial activity of PvL remained. The enzyme was inhibited by Fe2+, Hg2+, and Mn2+, but induced by Cu2+. EDTA (10 mM), 1,4-dithiothreitol (DTT) (0.1 mM), and NaN3 (10 mM) were found to completely inhibit PvL. Sixty-eight percent of Malachite green was decolorized by 4 U/mL of PvL after 15 min incubation at 30 °C.     

Purification,molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete <Emphasis Type="Italic">Trametes</Emphasis> sp. strain AH28-2

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.     

Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds

The extracellular laccase produced by the ascomycete Trichoderma atroviride was purified and characterized and its ability to transform phenolic compounds was determined. The purified laccase had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 °C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 °C. Under standard assay conditions, K_m values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator.     

Homologous cloning,expression, and characterisation of a laccase from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> and enzymatic decolourisation of an indigo dye

The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this enzyme in various industrial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L(-1) of a bacterial laccase with a high purity and without any extensive purification. This is the highest production yield reported in the literature for a bacterial laccase. The secreted enzyme achieved oxidation under a wide pH range depending on the substrate: 4.0 for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and 9.0 for 2,6-dimethoxyphenol. Furthermore, this bacterial laccase was found to be quite resistant under various conditions. It withstands pH from 3.0 to 9.0, shows a great thermostability at 70 degrees C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN(3), the laccase of Streptomyces coelicolor was fully active under the same conditions. To assess application potential of this laccase, we have investigated its ability to decolourise Indigo carmine. This enzyme was able to rapidly decolourise the dye in the presence of syringaldehyde as a redox mediator.     

Extracellular laccase produced by an edible basidiomycetous mushroom, Grifola frondosa: purification and characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K(m) values of Lac 1 toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.     

Effect of Ethanol on Enzymatic Activity of Fungal Laccases

Blue laccase from Coriolus versicolor and blue and yellow laccases from Panus tigrinus were isolated, purified and studied in acetate buffer solutions, with and without addition of various amounts of ethanol, using syringaldazine and 2,6-dimethoxyphenol as substrates. Effect of ethanol on blue laccases could be successfully described using the mixed inhibition model, over the range of 0–2.5 M ethanol concentrations. Yellow laccase from P. tigrinus behaves differently, which may be explained by the presence of some extra molecules in its structure, which possibly stabilize the enzyme and might be exchanged in ethanol solutions.     

Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) > phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 degrees C, pH 4.5, yielded values of 26, 0.43, and 0.45 microm and 18, 660, and 1175 s(-1), respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.     

An alkali-stable enzyme with laccase activity from entophytic fungus and the enzymatic modification of alkali lignin

Mycelia Sterilia YY-5, an entophytic fungus, was isolated from Rhus chinensis Mill and its extracellular enzyme had a higher laccase activity (MS-Lac). After been purified by anion exchange and gel filtration chromatography, MS-Lac, which had a molecular mass of 45 kDa, was found to be an alkali-stable enzyme with an optimum pH of 10.0 and capable of retaining 80% activity after incubation for 72 h with syringaldazine as substrate. It was also found that syringaldazine had a higher affinity than 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as substrate for MS-Lac, which was determined in sodium phosphate buffer (pH 6.0, 0.1 M) at 30 °C. Meanwhile, the lignin modification, catalyzed by MS-Lac, indicated that it could oxidize the phenolic hydroxyl, side chain substituent or carbonyl group of spruce alkali lignin in cetyltrimethylammonium bromide (CTAB) reversed micelles (20 mM, pH 6.0, W/O = 40) and steam-exploded wheat straw alkali lignin in NaOH solution (20 mM, pH 10.0).     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.