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Delignification of plant-based biomass is one of the most important steps to determine the commercial success of bioavailability, and enzymatic delignification mediated via laccases could significantly improve the yield in an eco-friendly and precise manner. In this study, an extracellular laccase is newly identified and characterized from Trichoderma asperellum strain BPLMBT1. The purified laccase with specific activity of 51.0 U/mg, possesses a molecular mass of 65.0 kDa with absorbance peaks of 610–630 and 330 nm in the UV–Vis spectrum indicating the presence of the inclusive types I and III copper catalytic centers. The wide thermal and pH stability of this laccase, with optimal temperature of 80 °C and 3.0, are demonstrated by biochemical characterization and kinetics determination. In addition, the delignification of sweet sorghum stover biomass by the purified laccase resulting in an optimal lignin removal of 76.93% reveals its potential of utilizing delignified biomass for biohydrogen production 402.01 mL of biohydrogen was achieved after 84 h of anaerobic fermentation, which is 3.26-fold higher than the control without enzymatic pretreatment. The overall outcomes demonstrate an efficient way to utilize the thermostable laccase for biofuels conversion from lignocellulosic biomass.

Laccase, an important oxidoreductase, is widely distributed in various organisms. Termites are known to decompose lignocellulose efficiently with the aid of gut microorganisms. However, few laccases have been characterized from termite or its gut microbes. We aimed to screen the strain capable of degrading lignocellulose from fungus-growing termites. In this study, Bacillus stratosphericus BCMC2 with lignocellulolytic activity was firstly isolated from the hindgut of fungus-growing termite Macrotermes barneyi. The laccase gene (BaCotA) was cloned both from the BCMC2 strain and termite intestinal metagenomic DNA. BaCotA was overexpressed in E. coli, and the recombinant BaCotA showed high specific activity (554.1 U/mg). BaCotA was thermostable with an optimum temperature of 70 °C, pH 5.0. Furthermore, BaCotA was resistant to alkali and organic solvents. The enzyme remained more than 70% residual activity at pH 8.0 for 120 min; and the organic solvents such as methanol, ethanol and acetone (10%) had no inhibitory effect on laccase activity. Additionally, BaCotA exhibited efficient decolorization ability towards indigo and crystal violet. The multiple enzymatic properties suggested the presented laccase as a potential candidate for industrial applications. Moreover, this study highlighted that termite intestine is a good resource for either new strains or enzymes.

A laccase producing new bacterial strain (Alcaligenes faecalis XF1) was isolated from green site of Chandigarh (India) by standard screening method. Nutrient broth medium containing 0.2 mM CuSO4 was used for the production of laccase. Maximum production (110 U/ml) was achieved after four days of incubation. The extracellular laccase from the medium was purified by simple salt precipitation and ion exchange technique to get 3.8 fold purified protein with 1637.8 U/mg specific activity. Purified laccase (named as LAC1*) revealed its optimum activity at pH 8.0 and 80 °C temperature, and displayed remarkable stability in the range of 70–90 °C and in the pH range (pH 7.0–9.0). The single bands on SDS-PAGE represents the purity of LAC1* with molecular weight of ∼71 kDa. The kinetic parameters for 2,6-DMP oxidation was Km, Vmax and kcat were 480 μM, 110 U/mL and 1375 s−1. Enzyme activity of the LAC1* was significantly enhanced by Cu2+, Mg2+, Mn2+, SDS and NaCl, and was slightly inhibited in the presence of conventional inhibitors like cysteine, EDTA and sodium azide. Extracellular nature and significant stability of LAC1* under extreme conditions of temperature, pH, heavy metals, halides and detergents confined its suitability for various biotechnological and industrial applications which required these qualities of laccase. So after recognizing all these properties the purified laccase was studied for its application in decolorization of industrial dyes. Keywords: Laccase, Alcaligenes faecalis, Decolorization

Background: Laccase has been considered important for the degradation of lignocellulose by wood rot fungi. The properties and functions of laccase in white rot fungi have been investigated extensively, but those from brown rot fungi remain largely unknown. In this paper, a laccase isoforms Pplcc2 from the brown rot fungus Postia placenta MAD-698-R was expressed heterologously in Pichia pastoris GS115 , purified and the properties of the enzyme were determined. Results: The molecular weight of the protein was determined to be 67 kDa using SDS-PAGE. It cannot oxidize syringaldazine (SGZ), but it can oxidize 2,2'-azino-di-(3-ethylbenzothialozin-6-Sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP). Specific activity for ABTS was 1960 ± 19 Unit/mg. The catalytic constant ( k cat ) was 1213 ± 18.3 s - 1 for ABTS and 293.2 ± 21.9 s - 1 for DMP. K m was 22.08 μM for ABTS and 11.62 μM for DMP. The optimal pH for the oxidation of ABTS and DMP was 3.5 and 5.0 respectively. The optimal temperature for the oxidation of ABTS and DMP was 60°C. Conclusions: This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to the understanding the roles played by laccases in brown rot fungi.

Laccases are multicopper polyphenol oxidases that are able to catalyze the oxidation of a wide range of phenolic compounds with the simultaneous reduction of O2 to H2O. Despite their promising industrial uses, feasible incorporation of laccases in harsh processes requires the bioprospecting and/or engineering of enzymes to be stable and active in acidic or alkaline pHs, high temperatures, oxidative conditions and tolerant to high salinity and/or organic solvents. Here we used a PCR-based screening to clone two novel laccase coding sequences from the white-rot basidiomycete Trametes trogii. Recombinant expression of lcc3 gene in Komagataella (=Pichia) pastoris showed that it encodes a thermo active and thermostable laccase with an optimum temperature of 50°C and with a half-life of 45 minutes at 70°C and a stability higher than 3 h at 60°C. Furthermore, recombinant LCC3 was capable of decolorizing between 50% and 100% of indigoid, triarylmethane, azoic and anthraquinonic synthetic dyes in the presence of the natural redox mediator acetosyringone within 2 h of incubation at pH 6 and 70°C. Fil: Campos, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Levin, Laura Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Wirth, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina

© 2019 Elsevier Ltd As the most abundant aromatic polymers on the Earth, lignin has great potential to produce biofuels and aromatic chemicals due to their high carbon content and low oxygen content. Lignin-first biorefinery methods have attracted increasing attention recently for their high-value of aromatic chemicals, and high biofuels productivity from lignocellulosic wastes. Thermostable laccase has proven to be an excellent alternative catalyst in degrading lignin for its versatile catalytic abilities under industrial conditions and pollution-free by-products. Thermostable laccases can be found in native extreme environments or modified by biologically based technologies such as gene recombination expression and enzyme direct evolution. This review demonstrated thermostable laccases and their application in lignin degradation. Future research should focus more on the investigation of the reaction of thermostable laccases with lignin substrates.

Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purifed enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specifcity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharifcation. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignifcation of lignocellulosic biomass. Fil: Navas, Laura Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Martínez, Fernando D.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Taverna, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina. Universidad Tecnológica Nacional. Facultad Regional San Francisco; Argentina Fil: Fetherolf, Morgan M.. University of British Columbia; Canadá Fil: Eltis, Lindsay D.. University of British Columbia; Canadá Fil: Nicolau, Verónica. Universidad Tecnológica Nacional. Facultad Regional San Francisco; Argentina Fil: Estenoz, Diana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Benintende, Graciela Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Berretta, Marcelo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina

Highlights • Production and purification of laccase from Alcaligenes faecalis. • Purified laccase from Alcaligenes faecalis active & stable at high temperature and pH. • Laccase had remarkable specificity to an extensive range of probable substrate and tolerant to various metal ions. • Efficiently decolorization of different synthetic dyes by laccase., A laccase producing new bacterial strain (Alcaligenes faecalis XF1) was isolated from green site of Chandigarh (India) by standard screening method. Nutrient broth medium containing 0.2 mM CuSO4 was used for the production of laccase. Maximum production (110 U/ml) was achieved after four days of incubation. The extracellular laccase from the medium was purified by simple salt precipitation and ion exchange technique to get 3.8 fold purified protein with 1637.8 U/mg specific activity. Purified laccase (named as LAC1*) revealed its optimum activity at pH 8.0 and 80 °C temperature, and displayed remarkable stability in the range of 70–90 °C and in the pH range (pH 7.0–9.0). The single bands on SDS-PAGE represents the purity of LAC1* with molecular weight of ∼71 kDa. The kinetic parameters for 2,6-DMP oxidation was Km, Vmax and kcat were 480 μM, 110 U/mL and 1375 s−1. Enzyme activity of the LAC1* was significantly enhanced by Cu2+, Mg2+, Mn2+, SDS and NaCl, and was slightly inhibited in the presence of conventional inhibitors like cysteine, EDTA and sodium azide. Extracellular nature and significant stability of LAC1* under extreme conditions of temperature, pH, heavy metals, halides and detergents confined its suitability for various biotechnological and industrial applications which required these qualities of laccase. So after recognizing all these properties the purified laccase was studied for its application in decolorization of industrial dyes.

Additional file 1. Additional tables and figures.

7 páginas.-- 3 figuras.-- 2 tablas.-- 34 referencias, Streptomycetes are actually considered one of the main groups of ligninolytic microoganisms producing a lignin-carbohydate complex named APPL (acid precipitable polymeric lignin) when growing on lignocellulosic materials. Although in these conditions the production of hemicellulolytic and oxidative extracellular enzymes were reported the specific role of laccases in lignin degradation is poorly understood. SilA, a thermostable salt-resistant and pH-versatile laccase produced by Streptomyces ipomoeae CECT 3341 was recently discovered and their particular characteristics make attractive to deep in its knowledge for biotechnological and environmental purposes. Pyrolysis/GC-MS was used to analyse the behaviour of the laccase-producing strain (SilA strain) and a laccase-negative mutant (SilA- strain) when growing on wheat straw in solid-state fermentation (SSF). Quantitative yields of APPL and the relative abundance of lignin-derived compounds were much higher for SilA strain than for SilA- showing a higher solubilizing activity of S. ipomoeae wild-type on lignocellulosic residues. Nonetheless the patterns of lignin derived compounds found in the APPL pyrograms were similar for both strains and distinct from the control showing a shortening of lignin propyl side-chains. Our results demonstrate that SilA laccase is a key enzyme in the lignin solubilization by S. ipomoeae and also points to the involvement of other oxidative enzymatic activities distinct to laccase in this process., This work was supported by the Project Grants CTQ2009-10447 and CTQ2014-56038-C3-2-R from the Ministerio de Ciencia e Innovación, Spain.

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (kcat/Km) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co2+ and Mn2+, while activity was strongly inhibited in the presence of Fe2+, Zn2+, and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency.

A native laccase (Lac-Q) with robust cold-adapted and thermostable characteristics from the white-rot fungus Pycnoporus sp. SYBC-L10 was purified, characterized, and used in antibiotic treatments. Degradation experiments revealed that Lac-Q at 10.0 U mL−1 coupled with 1.0 mmol L−1 ABTS could degrade 100% of the tetracycline or oxytetracycline (50 mg L−1) within 5 min with a static incubation at 0 °C (pH 6.0). The presence of the Mn2+ ion inhibited the removal rate of tetracycline and oxytetracycline by the Lac-Q–ABTS system, and the presence of Al3+, Cu2+, and Fe3+ accelerated the removal rate of tetracycline and oxytetracycline by the Lac-Q–ABTS system. Furthermore, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. Furthermore, the growth inhibition of Bacillus altitudinis SYBC hb4 and E. coli by tetracycline antibiotics revealed that the antimicrobial activity was significantly reduced after treatment with the Lac-Q–ABTS system. Finally, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. These results suggest that the Lac-Q–ABTS system shows a great potential for the treatment of antibiotic wastewater containing different metal ions at various temperatures.

Synthetic dyes are widely used in many industries, but they cause serious environmental problems due to their carcinogenic and mutagenic properties. In contrast to traditional physical and chemical treatments, biodegradation is generally considered an environmental-friendly, efficient, and inexpensive way to eliminate dye contaminants. Here, a novel laccase-like enzyme Lac1326 was cloned from a marine metagenomic library. It showed a maximum activity at 60°C, and it retained more than 40% of its maximal activity at 10°C and more than 50% at 20-70°C. Interestingly, the laccase behaved stably below 50°C, even in commonly used water-miscible organic solvents. The enzyme decolorized all tested dyes with high decolorization efficiency. This thermostable enzyme with high decolorization activity and excellent tolerance of organic solvents and salt has remarkable potential for bioremediation of dye wastewater. It is thus proposed as an industrial enzyme.

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest Kcat and Kcat/Km were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 °C, respectively. Mn2+, Cd2+, Fe2+, Cu2+ and Na+ activated laccase while Ca2+ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co2+, Hg2+, DTT, SDS and EDTA showed an inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents; upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes such as indigocarmine and congo red in the absence of any redox mediator.

For the first time, the investigation of Indigo carmine decolorization was done using an atypical Scytalidium thermophilum laccase. Crude and purified laccases required high temperatures and slight acidic pH to achieve maximum Indigo decolorization. Kinetic parameters (Km and kcat) of the homotrimeric laccase toward Indigo carmine were determined and laccase efficacy toward repeated dye decolorization process was studied. For the first time, 5 g l−1 as initial Indigo carmine concentration were efficiently transformed up to 50% within 6 h of incubation using 0.1 U ml−1 of laccase and without presence of any mediators. In this study, we showed that the atypical laccase transformed the indigoid dye structure, confirmed by the color changing from blue to red. This intermediate (red) was a subject to an efficient microbial consortium treatment monitored by measuring the decrease in optical density and the total organic carbon removal efficiencies. Toxicological studies via micro-toxicity test showed that the released enzymatic and adapted consortium degradation products were both non-toxic while the initial product was toxic.

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.

The gene encoding Thermus thermophilus HJ6 laccase (Tt-laccase) was cloned, sequenced, and comprised of 1,389 nucleotides encoding a protein (462 amino acids) with a predicted molecular mass of 51,049 Da. The deduced amino acid sequence of Tt-laccase showed 99.7% and 44.3% identities to the Thermus thermophilus HB27 laccase and Synechococcus sp. RS9917 laccase, respectively. Tt-laccase gene was expressed as a fusion protein with six histidine residues in E. coli Rosetta-gami (DE3) cells, and the recombinant protein was purified to homogeneity. UV-Vis spectrum analysis revealed that the enzyme has copper atoms, a type I Cu(II) and a type III binuclear Cu(II). The optimum pH for the oxidation of guaiacol was 5.0 and the optimum temperature was 90℃ The half-life of heat inactivation was about 120 min at 90℃ The enzyme reaction was inhibited by sodium azide, L-cystein, EDTA, dithiothreitol, tropolone, and kojic acid. The enzyme oxidized various known laccase substrates, its lowest K m value being for 4-hydroxyindole, highest kcat value for syringaldazine, and highest k cat /K m for guaiacol.

Laccase production by the white rot fungus, Ganoderma lucidum, was investigated using submerged fermentation, and the laccase was purified and characterized. The effects of variations in the media nutrient composition or culture conditions and characteristics of purified laccase were provided for large scale production or industrial uses of the laccase. Low concentrations of glucose and yeast extract stimulated G. lucidum laccase production. The addition of lignocellulosic substrates to the media stimulated laccase production: pomelo peel induced the highest laccase activity of 11842.13 U/L and banana peel reduced laccase production, compared to control media containing wheat bran. Native PAGE and SDS PAGE demonstrated the presence of a single isoform with a molecular weight of 68 kDa. The optimum pH and temperature were 3.0 and 60°C, respectively. G. lucidum laccase is thermostable, with a residual activity of 46% after 80 min at 60°C. The Michaelis-Menten constant (Km) and catalytic constant (Kcat) values forG. lucidum laccase using ABTS (2,2'-azino-di-[3-ethyl-benzothiazolin-sulphonate]) as a substrate were 0.114 mM and 74.63 S-1, respectively, with a specificity constant (Kcat /Km) of 654.65 S-1 mM-1. The thermostable properties of the laccase produced by fermentation of G. lucidum in submerged culture had potential for industrial and biotechnology applications. Key words: Laccase, Ganoderma lucidum, submerged fermentation, pomelo peel.

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low Km) when compared with available published data for other laccase isozymes. The specificity constant (kcat/Km) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (kcat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.

The fungus Cladosporium cladosporioides was isolated from a coal sample as a dye decolorizing microorganism. The maximum production of laccase from this fungus was found to be 4160 U/l with 0.05 mM CuSO4, 4% (w/v) glucose and lignin 0.04% (w/v). The laccase was purified from C. cladosporioides to homogeneity in a very active state using ammonium salt precipitation and gel permeation chromatography. Analysis with SDS–PAGE showed that the purified laccase was a monomeric protein of 71.2 kDa, and the apparent molecular mass of this enzyme was 75.17 kDa (m/z = 75,174), which was accurately determined by MALDI/TOF-MS. The UV–vis absorbance and electron paramagnetic resonance spectra of the purified laccase showed the presence of type 1 and type 3 copper ions. The optimum pH and temperature of the purified laccase were 3.5 and 40–70 °C, respectively, and the N-terminal amino acid sequence was determined to be IGPTGDMYIVNEDVS. The purified laccase was effective in the biotransformation of aromatic compounds as well as in the decolorization of various dyes.

Dye decolorizing potential of the white rot fungus Ganoderma lucidum KMK2 was demonstrated for recalcitrant textile dyes. G. lucidum produced laccase as the dominant lignolytic enzyme during solid state fermentation (SSF) of wheat bran (WB), a natural lignocellulosic substrate. Crude enzyme shows excellent decolorization activity to anthraquinone dye Remazol Brilliant Blue R (RBBR) without redox mediator whereas diazo dye Remazol Black-5 (RB-5) requires a redox mediator. Polyacrylamide gel electrophoresis (PAGE) of crude enzyme confirms that the laccase enzyme was the major enzyme involved in decolorization of either dyes. Native and SDS-PAGE indicates that the presence of single laccase with molecular weight of 43 kDa. N-Hydroxybenzotriazole (HBT) at a concentration of 1 mM was found as the best redox mediator. RB-5 (50 mg l−l) was decolorized by 62% and 77.4% within 1 and 2 h, respectively by the crude laccase (25 U ml−1). RBBR (50 mg l−l) was decolorized by 90% within 20 h, however, it was more efficient in presence of HBT showing 92% decolorization within 2 h. Crude laccase showed high thermostability and maximum decolorization activity at 60 °C and pH 4.0. The decolorization was completely inhibited by the laccase inhibitor sodium azide (0.5 mM). Enzyme inactivation method is a good method which averts the undesirable color formation in the reaction mixture after decolorization. High thermostability and efficient decolorization suggest that this crude enzyme could be effectively used to decolorize the synthetic dyes from effluents.

Pycnoporous sanguineus was identified as a laccase producer when grown on diluted molasses. The single laccase was purified with a purification factor of 967 by ammonium sulphate precipitation, ion exchange and dye affinity chromatography, to a specific activity of 32.9 U/mg. The molecular mass of 58,000 Da, the optimum pH range between 3 and 5 with ABTS, DMP and guaiacol as substrates and the isoelectric point of 6.7, was similar to some other laccases of filamentous fungi. The optimum temperature was 55 °C and the enzyme displayed enhanced thermal stability with a half-life of 170 min at 75 °C. In terms of k cat / K m values for ABTS, syringaldazine and DMP, DMP was the best substrate. Kinetic analysis of fifteen more compounds revealed that, the enzyme was a true laccase with a requirement for a free –OH group with an adjacent free or derivatised –OH. The o -diphenols were preferred above their p- counterparts. Compounds with three adjacent –OH groups displayed higher binding affinities but phloroglucinol, an m -substituted phenol was unreactive. The apparent higher stability of this laccase makes it a good candidate for further investigation into it possible application in biotechnology.

Carpophores of mesophilic basidiomycetes were isolated from the Eastern Cape region of South Africa. These isolates were screened for manganese peroxidase and laccase with high temperature optima. This bio-prospecting has led to the discovery of a thermostable laccase, which exhibits an optimum temperature of 70 °C. The enzyme remains fully active after 9 h incubation at 60 °C. The laccase is also capable of catalyzing the oxidative coupling of 3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone, which are characterized as manganese peroxidase specific substrates. The wide temperature range, thermostability, and potentially wider than usual substrate range, make this an excellent candidate enzyme for industrial application.

A blue laccase was purified from a white rot fungus of Trametes trogii, which was a monomeric protein of 64 kDa as determined by SDS-PAGE. The enzyme acted optimally at a pH of 2.2 to 4.5 and a temperature of 70°C and showed high thermal stability, with a half-life of 1.6 h at 60°C. A broad range of substrates, including the non-phenolic azo dye methyl red, was oxidized by the laccase, and the laccase exhibited high affinity towards ABTS and syringaldazine. Moreover, the laccase was fairly metal-tolerant. A high-molecular-weight kraft lignin was effectively polymerized by the laccase, with a maximum of 6.4-fold increase in weight-average molecular weight, as demonstrated by gel permeation chromatography. Notable structural changes in the polymerized lignin were detected by Fourier transform infrared spectroscopy and 1 H NMR spectroscopy. This revealed an increase in condensed structures as well as carbonyl and aliphatic hydroxyl groups. Simultaneously, phenolic hydroxyl and methoxy groups decreased. These results suggested the potential use of the laccase in lignin modification.

Purification and Partial Characterization of a Thermostable Laccase from Pycnoporus sanguineus CS-2 with Ability to Oxidize High Redox Potential Substrates and Recalcitrant Dyes

Laccases are effective biocatalysts for various biotechnological applications. Treatment of effluents containing phenol components such as toluene using fungal laccases is usually limited to the acid to neutral pH range and moderate temperatures. In this research, severalBacilli sp., were isolated from marine environment in toluene enriched media in purpose to obtain high laccase activities in spore forming bacteria. Among all the identified isolates,Bacillus sp. XJT-7 removed more toluene in contrast with other strains. The isolatedBacillus grew on toluene as the sole source of carbon and energy with maximum laccase activity and compared with Bacillus sphaericus (ATCC: CCM2177) as a standard strain which has laccase activity without any growth on toluene. Laccase activities in spore ghosts which mislay their germination power by heat shock have been investigated. The results showed that the vegetative cell free spore ghosts had thermostable laccase activities and remained active at 100°C shock. These heated spores (spore ghosts) are useful in biodegradation due to a high laccase activity. The image of Atomic Force Microscopy (AFM) showed that the spore ghosts have 50 nm pores which are good for drug delivery and drug synthesis with laccase activity without any chances for having spore germination. Key words: Bacillus sp., spore ghost, Laccase, atomic force microscopy (AFM).

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are enzymes that catalyze the oxidation of phenolic compounds and aromatic amines with the simultaneous reduction of molecular oxygen to water [1]. They are widely distributed in many plants and fungi, some insects and bacteria, being particularly abundant in white-rot basidiomycetes [2]. Typical fungal laccases are described as glycosylated multicopper proteins, which are produced as extracellular monomeric forms of around 60-80 kDa, containing four copper atoms and 15-20% carbohydrates. Operatively, they are moderately thermotolerant, showing optima activity at 50-55 °C, and under acidic conditions (pH 3-5); although their maxima stability occurs in the alkaline zone (pH 8-9) [3]. Their copper atoms are distributed in three different sites bringing unique spectroscopic properties: The type 1 copper (CuT1) atom, is responsible of the intense blue color of enzymes by light absorption around 610 nm; The type 2 copper (CuT2) atom exhibits a weak absorption in the visible region; and the two type 3 copper (CuT3) atoms are present as a binuclear center, which has an absorption maximum about 330 nm. Moreover, CuT2 and CuT3 copper atoms are structural and functionally arranged as a trinuclear cluster. The four copper atoms form part of the active site of enzyme contributing directly to reaction. CuT1 is involved in the initial electron subtraction from reducer substrates, while trinuclear

A laccase was purified from Trametes hirsuta. This laccase was classified as a "white" or "yellow" laccase. pH 2.4 was optimal for the oxidation of ABTS and pH 2.5 for DMP. DMP oxidation was optimal at 85 degrees C. The half-life of this laccase was 70 min at 75 degrees C, and 5 h at 65 degrees C. Non-phenolic dyes, such as Methyl Red, were oxidized by purified laccase without mediators. The enzyme was not inhibited by Cu(2+), Mn(2+), or EDTA. These are atypical laccase characteristics that make it a good candidate for theoretical and applied research.

We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0%. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70 degrees C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.

Optimisation of laccase production by Thermoascus aurantiacus microbial type culture collection (MTCC) 375 using paddy straw as substrate and digested biogas slurry as medium was carried out. Laccase production by a thermophilic mould, Thermoascus aurantiacus MTCC 375, was optimised in solid state fermentation (SSF) by a conventional ‘one variable at a time approach ‘andfurther by response surface methodology. Enzyme activity was strongly affected by three independent variables namely incubation temperature, incubation period and inoculum level. The optimised conditions for maximum laccase (715.0 Ug−1 of paddy straw) production were 45°C temperature, 107 spores ml−1 and 4 days incubation period. The enzyme titre (715.0 Ug−1 of paddy straw) attained in the validation experiment of this study is higher than those reported in the literature. An overall (2.65 fold) increase in laccase production was achieved in SSF due to statistical optimisation compared to that of a conventional ‘one variable at a time approach ’. All these cultural conditions were found to have statistically significant (P < 0.05) effect on enzyme production.