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The LitR/CarH family comprises adenosyl B 12 -based photosensory transcriptional regulators that control light-inducible carotenoid production in nonphototrophic bacteria. In this study, we established a blue-green light-inducible hyperexpression system using LitR and its partner ECF-type sigma factor LitS in streptomycin-producing Streptomyces griseus NBRC 13350. The constructed multiple-copy number plasmid, pLit19, carried five genetic elements: pIJ101rep, the thiostrepton resistance gene, litR, litS, and σ LitS -recognized light-inducible crtE promoter. Streptomyces griseus transformants harboring pLit19 exhibited a light-dependent hyper-production of intracellular reporter enzymes including catechol-2,3-dioxygenase and β-glucuronidase, extracellular secreted enzymes including laccase and transglutaminase, and secondary metabolites including melanin, flaviolin, and indigoidine. Cephamycin-producing Streptomyces sp. NBRC 13304, carrying an entire actinorhodin gene cluster, exhibited light-dependent actinorhodin production after the introduction of the pLit19 shuttle-type plasmid with the pathway-specific activator actII-ORF4. Insertion of sti fragment derived from Streptomyces phaeochromogenes pJV1 plasmid into pLit19 increased its light sensitivity, allowing gene expression under weak light irradiation. The two constructed Escherichia coli-Streptomyces shuttle-type pLit19 plasmids were found to have abilities similar to those of pLit19. We successfully established an optogenetically controlled hyperproduction system for S. griseus NBRC 13350 and Streptomyces sp. NBRC 13304., (© 2024. The Author(s).)

The enzymatic biodegradation of mycotoxins in food and feed has attracted the most interest in recent years. In this paper, the laccase gene from Bacillus swezeyi was cloned and expressed in Escherichia coli BL 21(D3). The sequence analysis indicated that the gene consisted of 1533 bp. The purified B. swezeyi laccase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis -12% with an estimated molecular weight of 56.7 kDa. The enzyme is thermo-alkali-tolerant, displaying the optimal degradation of zearalenone (ZEN) and aflatoxin B 1 (AFB 1 ) at pH 8 and 9, with incubation temperatures of 55 and 50 °C, respectively, within 24 h. The degradation potentials of the 50 μg of the enzyme against ZEN (5.0 μg/mL) and AFB 1 (2.5 μg/mL) were 99.60 and 96.73%, respectively, within 24 h. To the best of our knowledge, this is the first study revealing the recombinant production of laccase from B. swezeyi , its biochemical properties, and potential use in ZEN and AFB 1 degradation in vitro and in vivo.

Aflatoxin B 1 (AFB 1 ) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB 1 without redox mediators. However, the use of this CotA-laccase to remove AFB 1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB 1 , twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB 1 , with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies ( k cat /K m ) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB 1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.

Modification of kraft lignin (KL), traditionally uses harsh and energy-demanding physical and chemical processes. In this study, the potential of the bacterial laccase CotA (spore coating protein A) for oxidation of KL under mild conditions was assessed. Thereby, the efficiency of CotA to oxidize both softwood and hardwood KL of varying purity at alkaline conditions was examined. For the respective type of wood, the highest oxidation activity by CotA was determined for the medium ash content softwood KL (MA_S) and the medium ash content hardwood KL (MA_H), respectively. By an up to 95% decrease in fluorescence and up to 65% in phenol content coupling of the structural lignin units was indicated. These results correlated with an increase in viscosity and molecular weight, which increased nearly 2 and 20-fold for MA_H and about 1.3 and 6.0-fold for MA_S, respectively. Thus, this study confirms that the CotA laccase can oxidize a variety of KL at alkaline conditions, while the origin and purity of KL were found to have a major impact on the efficiency of oxidation. Under the herein tested conditions, it was observed that the MA_H KL showed the highest susceptibility to CotA oxidation when compared to the other hardwood KLs and the softwood KLs. Therefore, this could be a viable method to produce sustainable resins and adhesives.

Background: Laccase is one member of the blue multicopper oxidase family. It can catalyze the oxidation of various substrates. The Thermus thermophilus SG0.5JP17-16 laccase (lacTT) is thermostable, pH-stable, and high tolerance to halides, and can decolorize the synthetic dyes. In lacTT, the function of the loop 6 constructing the substrate-binding pocket wasn't clear., Methods: The residues Asp394 and Asp396 located in loop 6, and were used to probe how the loop 6 influenced catalytic properties of the laccase. Site-directed mutagenesis was performed for two amino acids. Kinetic assay was utilized to characterize the catalytic efficiency of mutants. Mutants with different catalytic activities were used to decolorize the synthetic dyes to clarify the relationship between the catalytic efficiency and dye decolorization. Redox potential, structural and spectral analyses were performed to explain the differences in laccase activity between wild type and mutant enzymes., Results: D394M, D394E and D394R mutants with the lower laccase activity displayed a decreased decolorization efficiency, while D396A, D396M and D396E mutant enzymes with higher catalytic efficiency decolorized the synthetic dye more efficiently than the wild type enzyme., Conclusions: The pocket loop 6 might experience a conformational dynamics. The D394 residue controlled this conformation change by amino acid interaction networks containing the D396 residue at the entrance of substrate channel., General Significances: These studies may provide clues to improve the activity of the laccase for the better use in industrial applications, and/or contribute to further understanding the mechanism of laccase oxidation on the substrate., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Thermus thermophilus laccase belongs to the sub-class of multicopper oxidases that is activated by the extra binding of copper to a methionine-rich domain allowing an electron pathway from the substrate to the conventional first electron acceptor, the T1 Cu. In this work, two key amino acid residues in the 1st and 2nd coordination spheres of T1 Cu are mutated in view of tuning their redox potential and investigating their influence on copper-related activity. Evolution of the kinetic parameters after copper addition highlights that both mutations play a key role influencing the enzymatic activity in distinct unexpected ways. These results clearly indicate that the methionine rich domain is not the only actor in the cuprous oxidase activity of CueO-like enzymes., Competing Interests: Declaration of competing interest The authors declined any conflict of interest., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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 (k cat /K m ) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co 2+ and Mn 2+ , while activity was strongly inhibited in the presence of Fe 2+ , Zn 2+ , 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bacterial laccases are very stable at high temperature and high pH values, and have many biotechnological and industrial applications. Here we describe how we cloned, expressed and purified the laccase from Bacillus subtilis (B. subtilis). The enzyme molecular weight has been determined as 34 kDa in SDS-PAGE analysis. The activity of the recombinant enzyme has been proved by guaiacol oxidation. The KM and Vmax values of the enzyme were at 1.1077 mM and at 19.3 μmol/min/mg, respectively. The recombinant laccase was effective in the decolorization of Turquoise blue HF6, Remazol red 106, Remazol brilliant orange 3R, and Brilliant blue, thus, possessing the characteristics necessary for its possible application in textile and environmental industries.

Laccases are multicopper oxidases with important industrial value. In the study, a novel laccase gene (mco) in a Staphylococcus haemolyticus isolate is identified and heterologously expressed in Escherichia coli . Mco shares less than 40% of amino acid sequence identities with the other characterized laccases, exhibiting the maximal activity at pH 4.0 and 60°C with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) as a substrate. Additionally, the Mco is tolerant to a wide range of pH, heavy metal ions and many organic solvents, and it has a high decolorization capability toward textile dyes in the absence of redox mediators. The characteristics of the Mco make this laccase potentially useful for industrial applications such as textile finishing. Based on BLASTN results, mco is found to be widely distributed in both the bacterial genome and bacterial plasmids. Its potential role in oxidative defense ability of staphylococci may contribute to the bacterial colonization and survival.

Laccases are multicopper enzymes that catalyze oxidation of electron-rich substrates coupled to reduction of molecular oxygen to water. Since the Type 1 copper (T1 Cu) is the site where electrons are withdrawn from the substrate, it is assumed that the reduction potential of this copper correlates with enzyme activity. Herein, we studied the correlation of the T1 Cu reduction potential and the enzymatic activity of the small two-domain laccase Ssl1 from Streptomyces sviceus. For a systematic approach, we aimed to minimize any effects other than the reduction potential difference. To this end, we constructed a series of Ssl1 mutants with reduction potentials varying from <290 to 560 mV. Along with the hydrophobicity of the axial ligand of the T1 Cu also structural changes in the substrate binding site and additional hydrogen bonding increased the reduction potential. Enzyme activity experiments demonstrated that the T1 Cu reduction potential has a different effect on oxidation of different substrates. Whereas there was no obvious correlation between the T1 Cu reduction potential and kinetic parameters for the oxidation of syringaldazine (with a reduction potential of 390 mV), a good correlation was observed between the T1 Cu reduction potential and the conversion of substituted phenols with reduction potentials between 660 and 820 mV. This correlation was pronounced for the Ssl1 variants with reduction potentials above 470 mV, which demonstrated increased activities also during the oxidation of two dyes, alizarin red S and indigo carmine., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3 α and Cu3 β ), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Streptomyces griseoflavus Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His165 is a "gateway" at the O 2 -tunnel leading from solvent to the Cu3 β of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His157 that belongs to the first coordination sphere of Cu3 α .

In this study, we aimed to investigate lignin accumulation and its relationship with the composition of bacterial laccase-like genes in three arable lands (i.e., upland limestone soil (UL), upland red soil (UR), and upland-paddy rotation red soil (UPR)), which are subjected to long-term straw incorporation. After 9-13 years of straw incorporation, the lignin content significantly increased from 337.1, 414.5, and 201.6 mg/kg soil to 2096.5, 2092.4, and 1972.2 mg/kg soil in UL, UR, and UPR, respectively. The dominant lignin monomer changed from vanillyl (V)-type to cinnamyl (C)-type in UR. Both V- and C-types were the dominant monomers in UPR, and V-type monomer remained the dominant monomer in UL. Compared with the treatment without straw, straw incorporation significantly promoted the activity of laccase enzyme and the abundance of bacterial laccase-like genes in all soils. The redundancy analysis showed that the main influencing factors on lignin accumulation patterns with straw incorporation were the laccase enzyme activity, nitrogen availability, and some specific bacterial communities possessing the laccase-like genes (e.g., Thermotogae and Acidobacteria). The variation partitioning analysis confirmed that the strongest influencing factor on lignin accumulation was the composition of bacterial laccase-like genes (explained 31.4% of variance). The present study provides novel insights into the importance of bacterial laccase-like genes in shaping lignin monomer accumulation with straw incorporation in arable soils.

Unprecedented 3D nanobiosystems composed of recombinant CotA laccases and citrate-stabilised gold nanoparticles have been successfully achieved by an electrostatic self-assembly strategy. The bioelectrochemical reduction of O2 driven by CotA laccase at the spore coat was mimicked. Consequently key insights into its bioelectrocatalytic function were unravelled.

Sortase-catalyzed ligations have emerged as powerful tools for the site-specific ligation of peptides and proteins in material science and biocatalysis. In this work, a directed sortase evolution strategy (SortEvolve) has been developed as a general high-throughput screening (HTS) platform to improve activity of sortase A (application 1) and to perform directed laccase evolution through a semipurification process in 96-well microtiter plate (MTP) (application 2). A semipurification process in polypropylene MTP (PP-MTP) is achieved through the anchor peptide LCI, which acts as adhesion promoter. To validate the SortEvolve screening platform for both applications, three site-saturation mutagenesis (SSM) libraries of sortase A (Sa-SrtA) from Staphylococcus aureus (application 1) and two SSM libraries of the copper efflux oxidase (CueO laccase) from Escherichia coli (application 2) were generated at literature reported positions. After screening and rescreening, an array of Sa-SrtA variants (including the previously reported P94S, D160N, and D165A) and CueO variants (including the previously reported D439A and P444A) were identified. Further recombinant Sa-SrtA variant P94T/D160L/D165Q and CueO variant D439V/P444V were characterized with 22-fold and 103-fold improvements in catalytic efficiency compared with corresponding wild-types, respectively. An important advantage of the SortEvolve screening platform in comparison to many MTP-based screening systems is that the background noise was minimized (decreased 20-fold; application 2) due to the employed semipurification process. In essence, SortEvolve provides a universal surface-functionalized screening platform for sortases and enzymes in which especially background activity can be minimized to enable successful directed evolution campaigns.

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET&#95;RS10855 was heterologously expressed in Escherichia coli cells. The His 6 -tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO 4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K 4 [Fe(CN) 6 ]. Temperature optimum was 75 °C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.

Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli., Result: Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K m ) and maximum velocity (V max ) at 60 °C and pH 6.0 were determined to be 2.4 × 10 3  µM and 0.04 × 10 3 µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme's ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS., Conclusions: Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.

CotA protein from Bacillus subtilis is of laccase activity. The solubility of recombinant CotA is low, which hinders its application. In this study, histidine tag position optimization and hydrophilic engineering were applied to increase the yield and activity of CotA protein. The results showed that the protein yield of CotA with his tag at C-terminal (CH6-CotA) was four times of that of NH6-CotA (His tag at N-terminal). Then, 23 single mutants were constructed by substitutions of hydrophobic residues with hydrophilic amino acids. Among them, the protein yield of the mutant F207Y was increased by 30%; the catalytic activity (kcat/Km) of V403T and P455S was two and three times higher than that of CH6-CotA, respectively. Finally, triple mutant F2071Y/V403T/P455S with C-terminal his-tag (CH6-TSY) was constructed. When the proteins were expressed in microanaerobic condition, the activities of mutants CH6-P455S and CH6-TSY were enhanced about 48- and 42-folds compared to that of NH6-CotA in non-static culture., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Laccases are multicopper containing enzymes capable of performing one electron oxidation of a broad range of substrates. Using molecular oxygen as the final electron acceptor, they release only water as a by-product, and as such, laccases are eco-friendly, versatile biocatalysts that have generated an enormous biotechnological interest. Indeed, this group of enzymes has been used in different industrial fields for very diverse purposes, from food additive and beverage processing to biomedical diagnosis, and as cross-linking agents for furniture construction or in the production of biofuels. Laccases have also been studied intensely in nanobiotechnology for the development of implantable biosensors and biofuel cells. Moreover, their capacity to transform complex xenobiotics makes them useful biocatalysts in enzymatic bioremediation. This review summarizes the most significant recent advances in the use of laccases and their future perspectives in biotechnology., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Laccases and laccase-like multi-copper oxidases (LMCOs) are versatile and robust biocatalysts applied in a variety of oxidative processes, and various studies have attempted to improve their catalytic activity. Here we report the engineering of a bacterial LMCO for enhanced oxidation of the lignin-related compound guaiacol by a combination of structure-guided mutagenesis and DNA shuffling. Mutant L9 showed a 1.39 mM Km for guaiacol and a 2.5-fold increase in turnover rate (kcat/Km = 2.85·104 M-1s-1)., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (ESU71923) annotated as laccase. However, the gene product did not display any laccase-like activity against the substrates examined. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 was prepared in Escherichia coli. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7-7.5. The purified enzyme was highly tolerant to various halides and organic solvents, thus having a potential for various industrial applications. To the best of our knowledge, this is the first characterization of a laccase from genus Geobacillus which identifies a gene responsible for functional laccase in this genus.

Bacterial laccases are potential enzymes for biotechnological applications because of their remarkable advantages, such as broad substrate spectrum, various reactions, high thermostability, wide pH range, and resistance to strongly alkaline environments. However, the use of bacterial laccases for industrialized applications is limited because of their low expression level and catalytic efficiency. In this study, CotA, a bacterial laccase from Bacillus pumilus, was engineered through presumptive reasoning and rational design approaches to overcome low catalytic efficiency and thermostability. L386W/G417L, a CotA double-mutant, was constructed through site-directed mutagenesis. The catalytic efficiency of L386W/G417L was 4.3 fold higher than that of wild-type CotA-laccase, but the thermostability of the former was decreased than that of the latter and other mutants. The half-life (t 1/2 ) of wild-type and G417L were 1.14 and 1.47 h, but the half-life of L386W/G417L was only 0.37 h when incubating the enzyme at 80 °C. Considering the high catalytic efficiency of L386W/G417L, we constructed L386W/G417L/G57F, another mutant, to improve thermostability. Results showed that the half-life of L386W/G417L/G57F was 0.54 h when incubating the enzyme at 90 °C for 2 h with about 34% residual activity, but the residual activity of L386W/G417L was less than 40% when incubating the enzyme at 90 °C for 5 min. L386W/G417L was more efficient in decolorizing various industrial dyes at pH 10 than other mutants. L386W/G417L/G57F also exhibited an efficient decolorization ability. L386W/G417L/G57F is appropriate for biotechnological applications because of its high activity and thermostability in decolorizing industrial dyes. CotA-laccase may be further subjected to molecular modification and be used as an enhancer to improve decolorization efficiency for the physical and chemical treatment of dye wastewater.

The soil native bacterial strains were screened for laccase activity. Bacterial strain L3.8 with high laccase activity was identified as Sinorhizobium meliloti. The crude intracellular L3.8 enzyme extract was able to oxidize typical diagnostic substrates of plant and fungal laccases. Laccase L3.8 was purified 81-fold with a yield of 19.5%. The molecular mass of the purified bacterial laccase was found to be 70.0kDa and its pI was 4.77. UV-vis spectrum showed that L3.8 protein is a multicopper oxidase. The carbohydrate content of the purified enzyme was estimated at 3.2%. Moreover, the laccase active fraction was characterized in terms of kinetics, temperature, and pH optima as well as the effect of various chemical compounds on the laccase activity, and antioxidant properties, which indicated that the L3.8 laccase had unique properties that might be important in biotechnological applications. The lacc gene encoding S. meliloti laccase was cloned and characterized. The full-length sequence of 1950bp encoded a protein of 649 aa preceded by a signal peptide consisting of 26aa. Laccase L3.8 shared significant structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. Potential biotechnological importance of a newly identified laccase is discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bacterial laccase CueO from Escherichia coli can oxidize polycyclic aromatic hydrocarbons (PAHs); however, its application in the remediation of PAH-contaminated soil mainly suffers from a low oxidation rate and copper dependence. It was reported that a laccase with a higher redox potential tended to have a higher oxidation rate; thus, the present study investigated the oxidation of PAHs using another bacterial laccase CotA from Bacillus subtilis with a higher redox potential (525 mV) than CueO (440 mV). Recombinant CotA was overexpressed in E. coli and partially purified, exhibiting a higher laccase-specific activity than CueO over a broad pH and temperature range. CotA exhibited moderate thermostability at high temperatures. CotA oxidized PAHs in the absence of exogenous copper. Thereby, secondary heavy metal pollution can be avoided, another advantage of CotA over CueO. Moreover, this study also evaluated some unexplained phenomena in our previous study. It was observed that the oxidation of PAHs with bacterial laccases can be promoted by copper. The partially purified bacterial laccase oxidized only two of the 15 tested PAHs, i.e., anthracene and benzo[a]pyrene, indicating the presence of natural redox mediators in crude cell extracts. Overall, the recombinant CotA oxidizes PAHs with high laccase activity and copper independence, indicating that CotA is a better candidate for the remediation of PAHs than CueO. Besides, the findings here provide a better understanding of the oxidation of PAHs using bacterial laccases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In our search for degrading activities of biogenic amines (BAs) in lactic acid bacteria, a protein annotated as laccase enzyme was identified in Lactobacillus plantarum J16 (CECT 8944). In this study, the gene of this new laccase was cloned and heterologously overexpressed in Escherichia coli. The recombinant laccase protein was purified and characterized biochemically. The purified laccase showed characteristic spectroscopic properties of blue multicopper oxidases. The enzyme has a molecular weight of ∼ 62.5 kDa and activity toward typical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP). The pH optima on ABTS and 2,6-DMP were 3.5 and 7.0, respectively. Kinetic constants Km and Vmax were of 0.21 mM and 0.54 U/mg for ABTS and 1.67 mM and 0.095 U/mg for 2,6-DMP, respectively. The highest oxidizing activity toward 2,6-DMP was obtained at 60 °C. However, after a preincubation step at 85 °C for 10 min, no residual activity was detected. It has been demonstrated that recombinant L. plantarum laccase oxidizes biogenic amines, mainly tyramine, and thus presents new biotechnological potential for the enzyme in eliminating toxic compounds present in fermented food and beverages.

Objectives: Isolation and identification of a novel laccase (namely Lac4) with various industrial applications potentials from an endophytical bacterium., Results: Endophyte Sd-1 cultured in rice straw showed intra- and extra-cellular laccase activities. Genomic analysis of Sd-1 identified four putative laccases, Lac1 to Lac4. However, only Lac4 contains the complete signature sequence of laccase and shares at most 64 % sequence identity with other characterized bacterial multi-copper oxidases. Recombinant Lac4 can oxidize non-phenolic and phenolic compounds under acidic conditions and at 30-50 °C; Km values of Lac4 for ABTS at pH 2.5 and for guaiacol at pH 4.5 were 1 ± 0.15 and 6.1 ± 1.7 mM, respectively. The activity of Lac4 was stimulated by 0.8 mM Cu(2+) and 5 mM Fe(2+). In addition, Lac4 could decolorize various synthetic dyes and exhibit the degradation rate of 38 % for lignin., Conclusions: The data suggest that Lac4 possesses promising biotechnological potentials.

An open reading frame of the Thermus thermophilus HJ6 hypothetical laccase, which composed of 729 bases, was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli SoluBL21™ cells. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 6M guanidine HCl. The solubilized protein was refolded by a simple on-column refolding procedure using Ni-chelation affinity chromatography and then the refolded protein was purified by gel filtration chromatography. It showed a single band with a molecular mass of 27kDa in SDS-PAGE. The results from UV-visible absorption and electron paramagnetic resonance (EPR) analysis suggested that the enzyme had the typical copper sites, type-1, 2, and 3 Cu(II) of laccase. The purified enzyme exhibited the laccase activity with the optimal catalytic temperature at 75°C. The optimum pH for the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and syringaldazine was 4.5 and 6.0, respectively. The recombinant protein showed high thermostability, and the half-life of heat inactivation was about 50min at 85°C. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest kcat value for guaiacol, and highest kcat/Km for 2,6-dimethoxy-phenol. The enzyme reaction was strongly inhibited by the metal chelators and the thiol compounds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study investigated the diversity and abundance of bacterial lacasse-like genes in different particle size fractions, namely sand, silt, and clay of sediments in a subtropical mangrove ecosystem. Moreover, the effects of nutrient conditions on bacterial laccase-like communities as well as the correlation between nutrients and, both the abundance and diversity indices of laccase-like bacteria in particle size fractions were also studied. Compared to bulk sediments, Bacteroidetes, Caldithrix, Cyanobacteria and Chloroflexi were dominated in all 3 particle-size fractions of intertidal sediment (IZ), but Actinobacteria and Firmicutes were lost after the fractionation procedures used. The diversity index of IZ fractions decreased in the order of bulk > clay > silt > sand. In fractions of mangrove forest sediment (MG), Verrucomicrobia was found in silt, and both Actinobacteria and Bacteroidetes appeared in clay, but no new species were found in sand. The declining order of diversity index in MG fractions was clay > silt > sand > bulk. Furthermore, the abundance of lacasse-like bacteria varied with different particle-size fractions significantly (p < 0.05), and decreased in the order of sand > clay > silt in both IZ and MG fractions. Additionally, nutrient availability was found to significantly affect the diversity and community structure of laccase-like bacteria (p < 0.05), while the total organic carbon contents were positively related to the abundance of bacterial laccase-like genes in particle size fractions (p < 0.05). Therefore, this study further provides evidence that bacterial laccase plays a vital role in turnover of sediment organic matter and cycling of nutrients.

A green method for phenol spectrophotometric determination was developed based on the color reaction of phenol with 4-aminoantipyrine catalyzed by addition of Bacillus amyloliquefaciens endospores in the presence of O2. The catalytic activity of the endospores may be attributed to the presence of coat protein A on the cell surfaces. This deduction was confirmed by cotA gene knock-out from B. amyloliquefaciens using the homologous double-exchange method. Under optimal conditions, linear responses were obtained over phenol concentrations ranging from 5.0×10(-5)gL(-1) to 1.0×10(-2)gL(-1) (r=0.9984) with a detection limit of 2.1×10(-5)gL(-1) (3σ). Repeatability measurements of 1.0mgL(-1) phenol provided reproducible results with a relative standard deviation of 5.3% (n=11). Standard addition tests indicated recoveries ranging from 92.78% to 107.60%. The proposed whole-cell method was successfully used to detect total phenol in synthetic samples. Results confirmed the potential use of the developed method in practical applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development.

Streptomyces griseorubens JSD-1 is a novel actinomycete that could grow efficiently upon lignin, and the ligninolytic genes active in this biotransformation were expected to be crucial. To investigate the molecular mechanism of utilizing lignin, genome sequencing was carried out to obtain its draft genome, which was deposited at GenBank under the accession No. JJMG00000000. Multiple copper oxidase (MCO) was obtained, which proved to be an extracellular enzyme and have relative high expression with the stimulation of ligninolytic materials. Judging from its putative 3D structure, the N-terminal of MCO was bared, which was fit for the linkage of poly-HIS10 tag. As a result, heterogeneous expression conditions of recombinant laccase was achieved with TransB(DE3) grown in a modified terrific broth (TB) medium with an extra addition of 0.5% glucose at 30 °C until optical density at 600 nm (OD600) reached 0.8 when expression was induced by 25 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and also 100 μM copper sulphate as supplement. Finally, it exhibited special characters of thermal robustness, alkaline activity profiles, high resistance to metallic ions and chemical inhibitors as well as dye decolourization. In summary, our findings illustrated the genetic basic of utilizing lignin in this isolate. Additionally, a novel laccase expected to be potential in agricultural and industrial application was expressed and characterized as well.

Bacterial laccases show low activities but can be of biotechnological interest due to industrially suitable characteristics such as thermostability and tolerance to alkaline pH. In this study, three separate mutations (M298F, V290N and V290A) were introduced at or near the T1 copper site of the small laccase (SLAC) from Streptomyces coelicolor A3(2) and biochemical properties were assessed in comparison with the native enzyme. The mutation, V290N showed approximately double the activity of SLAC when ABTS was used as substrate while the specific activity of SLAC-M298F was 4-5 times higher than that of SLAC when the assays were performed at ≥70°C. There was no significant difference in activity with 2,6-dimethoxyphenol (2,6-DMP); however, there was a significant shift in the optimal pH from pH 9.5 (SLAC) to 7.5 (SLAC-V290N). Optimal temperature for activity was not significantly altered but thermostability was reduced in all three mutants. The substrate range of the mutant variants remained largely unchanged, with the exception of SLAC-M298F which was unable to oxidise veratryl alcohol. Interestingly, the "typical" laccase inhibitor, sodium azide, had no significant inhibitory effect on the activity of SLAC-M298F, which also exhibited increased resistance to inhibition by sulfhydryl compounds. SLAC-V290N showed higher catalytic efficiency for 2,6-DMP (kcat/Km=2.226mM(-1)s(-1)) and ABTS (kcat/Km=1.874mM(-1)s(-1)) compared to SLAC (kcat/Km=1.615mM(-1)s(-1) for 2,6-DMP and kcat/Km=1.611mM(-1)s(-1) for ABTS). This study has shown that three ligands that are closely associated with the T1 copper in SLAC play a key role in maintaining enzymatic activity. Whilst the introduction of mutations at these sites negated favourable characteristics such as thermostability, several favourable effects were observed. This study has also extended the knowledge base on the biochemical characteristics of SLAC, and its suitability as a template for engineering with the aim of widening its potential range of industrial applications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate "green catalysts". This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Copper depletion of bacterial laccases obtained by heterologous expression in Escherichia coli is a common problem in production of these versatile biocatalysts. We demonstrate that coexpression of small soluble copper chaperones can mitigate this problem. The laccase CotA and the copper chaperone CopZ both from Bacillus licheniformis were used as model system. The use of the E. coli BL21(DE3) strain expressing CopZ and CotA simultaneously from two plasmids resulted in an 20% increase in copper occupancy and in 26% higher specific activity. We conclude that not only intracellular copper ion concentration, but also presence of an appropriate copper chaperone influences copper ion insertion into CotA laccase. Moreover, E. coli BL21(DE3) seems to lack such a copper chaperone which can be partially complemented by heterologous expression thereof. The presented system is simple and can routinely be used for improved heterologous production of bacterial laccase in E. coli., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term "laccase-like multi-copper oxidase" (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.

Background: Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations., Results: A bacterial laccase (WlacD) was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ) anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG) 25 and diazo-dye Acid Red (AR) 18. The results showed that decolorization of both dyes is Cu(2+)- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l) with relative decolorization values of 91.2% (3 h) and 97.1% (18 h), as well as high activity to AR18 (1 g/l) by 80.5% (3 h) and 89.0% (18 h), was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l). No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved via a subsequent 4-h cell culturing., Conclusions: This study demonstrates, for the first time, the methodology by which the engineered P. putida with surface-immobilized laccase was successfully used as regenerable biocatalyst for biodegrading synthetic dyes, thereby opening new perspectives in the use of biocatalysis in industrial dye biotreatment.

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu(2+)-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5-9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.

Background: Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product., Results: A novel CotA-type laccase from Bacillus pumilus was cloned, expressed and purified and its biochemical characteristics are presented here. The molecular weight of the purified laccase was estimated to be 58 kDa and the enzyme was found to be associated with four copper atoms. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine (SGZ) was investigated. The kinetic parameters KM and kcat for ABTS were 80 ± 4 μM and 291 ± 2.7 s(-1), for 2,6-DMP 680 ± 27 μM and 11 ± 0.1 s(-1) and for SGZ only kcat could be estimated to be 66 ± 1.5 s(-1). The pH optimum for ABTS was 4, for 2,6-DMP 7 and for SGZ 6.5 and temperature optima for ABTS and 2,6-DMP were found to be around 70°C. The screening of 37 natural and non-natural compounds as substrates for B. pumilus laccase revealed 18 suitable compounds. Three of them served as redox mediators in the laccase-catalyzed decolorization of the dye indigocarmine (IC), thus assessing the new enzyme's biotechnological potential., Conclusions: The fully copper loaded, thermostable CotA laccase from Bacillus pumilus is a versatile laccase with potential applications as an industrial biocatalyst.

The electrochemical properties of a laccase from Thermus thermophilus HB27 (Tth-laccase) were characterized. The gene encoding the laccase was cloned and overexpressed in Escherichia coli. One-step purification of the corresponding apo-enzyme was achieved by nickel-affinity chromatography. Copper was incorporated into the apo-laccase as the cofactor to yield the holo-enzyme. The temperature-dependent catalytic activity of the laccase was investigated by spectrophotometric as well as electrochemical methods. Specifically, the catalytic properties of the enzyme were characterized by employing a photometric assay based on the oxidation of the substrate 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS). The electroactive substrate ABTS can be also monitored by cyclic voltammetry, thus allowing for determination of the enzymatic activity electrochemically. It was found that the recombinant laccase exhibited higher activity as the temperature increased up to 65 °C. Spectroscopic studies of Tth-laccase based on circular dichroism and fluorescence measurements are consistent with a thermally stable secondary structure of the protein.

A unique urate-oxidizing enzyme was identified in a bacterium, strain T-15. Based on its phylogenetic, physiological and biochemical properties, strain T-15 was deemed to be a novel species within the genus Lysobacter. The enzyme expressed in Lysobacter sp. T-15 was composed of 592 amino acids and contained four consensus copper-binding sites, and the recombinant enzyme was, at least in this study, speculated to have three Cu ions per subunit. The primary structure of the enzyme was 33% identical to Marinomonas mediterranea polyphenol oxidase, but it showed no significant similarity to any known urate oxidase. With urate as the substrate, the catalytic efficiency (k(cat)/K(m)) of recombinant enzyme was 4.0 × 10(2) s(-)(1)mM(-)(1), and it was not inhibited by xanthine, a strong urate oxidase inhibitor. The enzyme also showed activity toward 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), 2,6-dimethoxyphenol and bilirubin, with catalytic efficiencies of 4.9 × 10(2), 1.1 × 10(2) and 3.6 × 10(3) s(-)(1)mM(-)(1), respectively. We deemed the enzyme would be a member of laccase from its broad substrate specificity. However, typical laccase and other multi-copper oxidases such as bilirubin oxidase and ascorbate oxidase seldom exhibit urate oxidation activity. These results would expand the laccase substrate range to include urate.

Directed evolution is an effective strategy to engineer and optimize protein properties, and microbial cell-surface display is a successful method to screen protein libraries. Protein surface display on Bacillus subtilis spores is demonstrated as a tool for screening protein libraries for the first time. Spore display offers advantages over more commonly utilized microbe cell-surface display systems, which include gram-negative bacteria, phage and yeast. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; hence, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. The substrate specificity of the enzyme CotA is narrowed. CotA is a laccase and it occurs naturally on the outer coat of B. subtilis spores. A library of CotA genes were expressed in the spore coat, and it was screened for activity toward ABTS [diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] over SGZ (4-hydroxy-3,5-dimethoxy-benzaldehyde azine). A mutant CotA was found to be 120-fold more specific for ABTS. This research demonstrates that B. subtilis spores can be a useful platform for screen protein libraries.

Lac591, a gene encoding a novel multicopper oxidase with laccase activity, was identified through activity-based functional screening of a metagenomic library from mangrove soil. Sequence analysis revealed that lac591 encodes a protein of 500 amino acids with a predicted molecular mass of 57.4 kDa. Lac591 was overexpressed heterologously as soluble active enzyme in Escherichia coli and purified, giving rise to 380 mg of purified enzyme from 1 l induced culture, which is the highest expression report for bacterial laccase genes so far. Furthermore, the recombinant enzyme demonstrated activity toward classical laccase substrates syringaldazine (SGZ), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP). The purified Lac591 exhibited maximal activity at 55 degrees C and pH 7.5 with guaiacol as substrate and was found to be stable in the pH range of 7.0-10.0. The substrate specificity on different substrates was studied with the purified enzyme, and the optimal substrates were in the order of 2, 6-DMP > catechol > alpha-naphthol > guaiacol > SGZ > 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). The alkaline activity and highly soluble expression of Lac591 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and dyestuffs processing.

The multicopper oxidases couple the one-electron oxidation of four substrate molecules to the four electron reductive cleavage of the O-O bond of dioxygen. This reduction takes place at the trinuclear copper centre of the enzyme and the dioxygen approaches this centre through an entrance channel. In this channel, an acidic residue plays a key role in steering the dioxygen to the trinuclear copper site, providing protons for the catalytic reaction and giving overall stability to this site. In this study, the role of the Glu(498) residue, located within the entrance channel to the trinuclear copper centre, has been investigated in the binding and reduction of dioxygen by the CotA-laccase from Bacillus subtilis. The absence of an acidic group at the 498 residue, as in the E498T and E498L mutants, results in a severe catalytic impairment, higher than 99%, for the phenolic and non-phenolic substrates tested. The replacement of this glutamate by aspartate leads to an activity that is around 10% relative to that of the wild-type. Furthermore, while this latter mutant shows a similar K(m) value for dioxygen, the E498T and E498L mutants show a decreased affinity, when compared to the wild-type. X-ray structural and spectroscopic analysis (UV-visible, electron paramagnetic resonance and resonance Raman) reveal perturbations of the structural properties of the catalytic centres in the Glu(498) mutants when compared to the wild-type protein. Overall, the results strongly suggest that Glu(498) plays a key role in the protonation events that occur at the trinuclear centre and in its stabilization, controlling therefore the binding of dioxygen and its further reduction.

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

The axial ligand of the catalytic mononuclear T1 copper site (Met(502)) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant k(cat) in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded.

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E0 =455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the kcat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15-0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.