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Lígia O. Martins - One of the best experts on this subject based on the ideXlab platform.
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Mimicking the bioelectrocatalytic function of recombinant Cota laccase through electrostatically self-assembled bioconjugates
Nanoscale, 2019Co-Authors: David Alba-molina, Lígia O. Martins, Daily Rodríguez-padrón, Alain R. Puente-santiago, Juan J. Giner-casares, María T. Martín-romero, Luis Camacho, Mario J. Muñoz-batista, Manuel Cano, Rafael LuqueAbstract: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.
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Unfolding pathway of Cota-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state.
Biochemical and biophysical research communications, 2012Co-Authors: Auguste Fernandes, Lígia O. Martins, Carlos Lopes, Eduardo P. MeloAbstract:Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase Cota laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-Cota was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-Cota but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-Cota in a non-native coordination promoting Cota aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.
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Insight into stability of Cota laccase from the spore coat of Bacillus subtilis.
Biochemical Society Transactions, 2007Co-Authors: Eduardo P. Melo, Auguste Fernandes, Paulo Durão, Lígia O. MartinsAbstract: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.
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substrate and dioxygen binding to the endospore coat laccase from bacillus subtilis
Journal of Biological Chemistry, 2004Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, D Marcal, Rosa Grenha, P F Lindley, Maria Arménia CarrondoAbstract:The Cota laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.
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crystal structure of a bacterial endospore coat component a laccase with enhanced thermostability properties
Journal of Biological Chemistry, 2003Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, M A CarrondoAbstract:Abstract Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa Cota protein is an abundant component of the outer coat layer. Cota is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of Cota at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of Cota comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in Cota are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that Cota may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, Cota appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of Cota, its assembly and the properties of the bacterial spore coat structure are discussed.
Adriano O Henriques - One of the best experts on this subject based on the ideXlab platform.
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substrate and dioxygen binding to the endospore coat laccase from bacillus subtilis
Journal of Biological Chemistry, 2004Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, D Marcal, Rosa Grenha, P F Lindley, Maria Arménia CarrondoAbstract:The Cota laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.
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crystal structure of a bacterial endospore coat component a laccase with enhanced thermostability properties
Journal of Biological Chemistry, 2003Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, M A CarrondoAbstract:Abstract Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa Cota protein is an abundant component of the outer coat layer. Cota is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of Cota at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of Cota comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in Cota are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that Cota may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, Cota appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of Cota, its assembly and the properties of the bacterial spore coat structure are discussed.
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Spore-coat laccase Cota from Bacillus subtilis: crystallization and preliminary X-ray characterization by the MAD method
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, Pedro M. Matias, Diana Plácido, Maria Arménia CarrondoAbstract:Bacterial endospores are highly resistant structures that allow survival for long periods of time in adverse environments. The spore-forming Gram-positive bacterium Bacillus subtilis synthesizes a coat around the endospore during development composed of several assembled polypeptides. The role of these components of the spore coat remains unclear; however, some of them appear to be enzymes possibly involved in the assembly process or in the final properties of the spore. The outer spore-coat protein Cota is a 65 kDa polypeptide showing a high degree of sequence similarity with copper-dependent oxidases, including fungal and plant laccases, ascorbate oxidase and CueO from Esherichia coli. Cota has been recently characterized as a copper-dependent laccase. Unlike previously reported laccases, Cota shows increased thermostability. Here, the crystallization of a recombinant Cota protein produced in E. coli and the preliminary characterization of the crystals is reported. Structure solution by the MAD method at the copper K edge is also reported.
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molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the bacillus subtilis endospore coat
Journal of Biological Chemistry, 2002Co-Authors: Lígia O. Martins, Claudio M Soares, Manuela M Pereira, Miguel Teixeira, Teresa Costa, George H Jones, Adriano O HenriquesAbstract:Abstract The Bacillus subtilis endospore coat protein Cota shows laccase activity. By using comparative modeling techniques, we were able to derive a model for Cota based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of Cota contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the Cota T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The Cota protein was purified to homogeneity from an overproducingEscherichia coli strain. The purified Cota shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at ≤pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K mvalues for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 μm, respectively, with correspondingk cat values of 16.8 ± 0.8 and 3.7 ± 0.1 s−1. Maximal enzyme activity was observed at 75 °C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 °C of about 4 and 2 h, respectively, indicating that Cota is intrinsically highly thermostable.
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molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the bacillus subtilis endospore coat
Journal of Biological Chemistry, 2002Co-Authors: Lígia O. Martins, Claudio M Soares, Manuela M Pereira, Miguel Teixeira, Teresa Costa, George H Jones, Adriano O HenriquesAbstract:Abstract The Bacillus subtilis endospore coat protein Cota shows laccase activity. By using comparative modeling techniques, we were able to derive a model for Cota based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of Cota contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the Cota T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The Cota protein was purified to homogeneity from an overproducingEscherichia coli strain. The purified Cota shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at ≤pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K mvalues for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 μm, respectively, with correspondingk cat values of 16.8 ± 0.8 and 3.7 ± 0.1 s−1. Maximal enzyme activity was observed at 75 °C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 °C of about 4 and 2 h, respectively, indicating that Cota is intrinsically highly thermostable.
Francisco J Enguita - One of the best experts on this subject based on the ideXlab platform.
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substrate and dioxygen binding to the endospore coat laccase from bacillus subtilis
Journal of Biological Chemistry, 2004Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, D Marcal, Rosa Grenha, P F Lindley, Maria Arménia CarrondoAbstract:The Cota laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.
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crystal structure of a bacterial endospore coat component a laccase with enhanced thermostability properties
Journal of Biological Chemistry, 2003Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, M A CarrondoAbstract:Abstract Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa Cota protein is an abundant component of the outer coat layer. Cota is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of Cota at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of Cota comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in Cota are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that Cota may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, Cota appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of Cota, its assembly and the properties of the bacterial spore coat structure are discussed.
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Spore-coat laccase Cota from Bacillus subtilis: crystallization and preliminary X-ray characterization by the MAD method
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, Pedro M. Matias, Diana Plácido, Maria Arménia CarrondoAbstract:Bacterial endospores are highly resistant structures that allow survival for long periods of time in adverse environments. The spore-forming Gram-positive bacterium Bacillus subtilis synthesizes a coat around the endospore during development composed of several assembled polypeptides. The role of these components of the spore coat remains unclear; however, some of them appear to be enzymes possibly involved in the assembly process or in the final properties of the spore. The outer spore-coat protein Cota is a 65 kDa polypeptide showing a high degree of sequence similarity with copper-dependent oxidases, including fungal and plant laccases, ascorbate oxidase and CueO from Esherichia coli. Cota has been recently characterized as a copper-dependent laccase. Unlike previously reported laccases, Cota shows increased thermostability. Here, the crystallization of a recombinant Cota protein produced in E. coli and the preliminary characterization of the crystals is reported. Structure solution by the MAD method at the copper K edge is also reported.
Maria Arménia Carrondo - One of the best experts on this subject based on the ideXlab platform.
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substrate and dioxygen binding to the endospore coat laccase from bacillus subtilis
Journal of Biological Chemistry, 2004Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, D Marcal, Rosa Grenha, P F Lindley, Maria Arménia CarrondoAbstract:The Cota laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.
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Spore-coat laccase Cota from Bacillus subtilis: crystallization and preliminary X-ray characterization by the MAD method
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Francisco J Enguita, Lígia O. Martins, Adriano O Henriques, Pedro M. Matias, Diana Plácido, Maria Arménia CarrondoAbstract:Bacterial endospores are highly resistant structures that allow survival for long periods of time in adverse environments. The spore-forming Gram-positive bacterium Bacillus subtilis synthesizes a coat around the endospore during development composed of several assembled polypeptides. The role of these components of the spore coat remains unclear; however, some of them appear to be enzymes possibly involved in the assembly process or in the final properties of the spore. The outer spore-coat protein Cota is a 65 kDa polypeptide showing a high degree of sequence similarity with copper-dependent oxidases, including fungal and plant laccases, ascorbate oxidase and CueO from Esherichia coli. Cota has been recently characterized as a copper-dependent laccase. Unlike previously reported laccases, Cota shows increased thermostability. Here, the crystallization of a recombinant Cota protein produced in E. coli and the preliminary characterization of the crystals is reported. Structure solution by the MAD method at the copper K edge is also reported.
Xiangru Liao - One of the best experts on this subject based on the ideXlab platform.
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Extracellular expression of mutant Cota-laccase SF in Escherichia coli and its degradation of malachite green.
Ecotoxicology and environmental safety, 2020Co-Authors: Ya-jing Wang, Xiangru Liao, Yujie Cai, Zhengbing GuanAbstract:Abstract In this study, mutant Cota-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of Cota-laccase SF was 1257.22 U/L. Extracellularly expressed Cota-laccase SF exhibits enzymatic properties similar to intracellular Cota-laccase SF. Cota-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of Cota-laccase SF to MG were 39.6 mM and 18.36 s−1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by Cota-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, Cota-laccase SF had a good application prospect for degrading malachite green.
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Enhancement in catalytic activity of Cota-laccase from Bacillus pumilus W3 via site-directed mutagenesis
Journal of bioscience and bioengineering, 2019Co-Authors: Hao-ran Wang, Xiangru Liao, Ya-jing Wang, Yujie Cai, Xia Jing, Zhengbing GuanAbstract:Cota-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel Cota-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type Cota-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7–11. Mutant S208G/F227A showed optimal temperature at 80°C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type Cota-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of Cota-laccase through site-directed mutagenesis.
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hydrogen peroxide resistant Cota and yjqc of bacillus altitudinis spores are a promising biocatalyst for catalyzing reduction of sinapic acid and sinapine in rapeseed meal
PLOS ONE, 2016Co-Authors: Yanzhou Zhang, Xunhang Li, Ruchun Xi, Xiangru LiaoAbstract:For the more efficient detoxification of phenolic compounds, a promising avenue would be to develop a multi-enzyme biocatalyst comprising peroxidase, laccase and other oxidases. However, the development of this multi-enzyme biocatalyst is limited by the vulnerability of fungal laccases and peroxidases to hydrogen peroxide (H2O2)-induced inactivation. Therefore, H2O2-resistant peroxidase and laccase should be exploited. In this study, H2O2-stable Cota and YjqC were isolated from the outer coat of Bacillus altitudinis SYBC hb4 spores. In addition to the thermal and alkali stability of catalytic activity, Cota also exhibited a much higher H2O2 tolerance than fungal laccases from Trametes versicolor and Trametes trogii. YjqC is a sporulation-related manganese (Mn) catalase with striking peroxidase activity for sinapic acid (SA) and sinapine (SNP). In contrast to the typical heme-containing peroxidases, the peroxidase activity of YjqC was also highly resistant to inhibition by H2O2 and heat. Cota could also catalyze the oxidation of SA and SNP. Cota had a much higher affinity for SA than B. subtilis Cota. Cota and YjqC rendered from B. altitudinis spores had promising laccase and peroxidase activities for SA and SNP. Specifically, the B. altitudinis spores could be regarded as a multi-enzyme biocatalyst composed of Cota and YjqC. The B. altitudinis spores were efficient for catalyzing the degradation of SA and SNP in rapeseed meal. Moreover, efficiency of the spore-catalyzed degradation of SA and SNP was greatly improved by the presence of 15 mM H2O2. This effect was largely attributed to synergistic biocatalysis of the H2O2-resistant Cota and YjqC toward SA and SNP.
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Efficient secretory production of Cota-laccase and its application in the decolorization and detoxification of industrial textile wastewater.
Environmental science and pollution research international, 2015Co-Authors: Zhengbing Guan, Ning Zhang, Yujie Cai, Shui Yan, Chen-meng Song, Xiangru LiaoAbstract:Fungal laccases are typically unstable at high pH and temperature conditions, which limit their application in the decolorization of textile wastewater. By contrast, the highly stable bacterial laccases can function within a wider pH range and at high temperatures, thus have significant potential in treatment for textile wastewater. In our previous work, a thermo-alkali-stable Cota-laccase gene was cloned from Bacillus pumilus W3 and overexpressed in Escherichia coli. In this study, the robust Cota-laccase achieved efficient secretory expression in Bacillus subtilis WB600 by screening a suitable signal peptide. A maximum Cota-laccase yield of 373.1 U/mL was obtained at optimum culture conditions in a 3-L fermentor. Furthermore, the decolorization and detoxification of textile industry effluent by the purified recombinant Cota-laccase in the presence and absence of redox mediators were investigated. Among the potential mediators that enhanced effluent decolorization, acetosyringone (ACS) was the most effective. The toxicity of the Cota-laccase-ACS-treated effluent was greatly reduced compared with that of the crude effluent. To the best of our knowledge, this study is the first to report on the heterologous expression of Cota-laccase in B. subtilis. The recombinant strain B. subtilis WB600-5 has a great potential in the industrial production of this bacterial enzyme, and the Cota-laccase-ACS system is a promising candidate for the biological treatment of industrial textile effluents.
