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Giovanni Sannia - One of the best experts on this subject based on the ideXlab platform.
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Beyond natural Laccases: extension of their potential applications by protein engineering
Applied Microbiology and Biotechnology, 2020Co-Authors: Ilaria Stanzione, Cinzia Pezzella, Paola Giardina, Giovanni Sannia, Alessandra PiscitelliAbstract:Laccases bring exciting promises into the green industries, and the development of enzymes with improved properties is further raising their exploitation potential. Molecular engineering methods to build highly efficient catalysts both through rational and random mutagenesis were extensively applied. Moreover, computational approaches are becoming always more reliable in aiding proper design of efficient and tailored catalyst for specific applications. In this review, the results of the last 10 years about industrial application of engineered Laccases in different fields are analyzed. Tailoring laccase towards a target substrate and defining a proper screening strategy for the selection of the “jackpot mutant” represent the keys of a winning mutagenesis pathway. Likewise, laccase chimerae, built by the fusion of Laccases with relevant proteins, emerged as an added value in the designing of flexible and well-rounded biocatalysts. Despite being promising in most of the reported examples, evolved Laccases are currently tested at a laboratory scale and a feedback from the industry world is continuously required to strengthen the biotechnological exploitation of these improved enzymes.
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induction and transcriptional regulation of Laccases in fungi
Current Genomics, 2011Co-Authors: Alessandra Piscitelli, Cinzia Pezzella, Paola Giardina, Giovanni Sannia, Vincenzo Lettera, Vincenza FaracoAbstract:Fungal Laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native Laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.
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identification of a new member of pleurotus ostreatus laccase family from mature fruiting body
Fungal Biology, 2010Co-Authors: Vincenzo Lettera, Cinzia Pezzella, Alessandra Piscitelli, Leila Birolo, Giovanni SanniaAbstract:Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. Most of the known Laccases have fungal or plant origins, although few Laccases have been also identified in bacteria and insects. Most of the fungal Laccases reported thus far are extra-cellular enzymes, whereas only few enzymes from fruiting bodies have been described so far. Multiple isoforms of Laccases are usually secreted by each fungus depending on species and environmental conditions. As a fact, a laccase gene family has been demonstrated in the white-rot fungus Pleurotus ostreatus. This work allowed identification and characterization of the first laccase isoenzyme from the fruiting body of P. ostreatus. Discovery through mass spectrometry of LACC12 proves the expression of a functional protein by the related deduced encoding transcript. The topology of phylogenetic tree of fungal Laccases proves that LACC12 falls in cluster with the members of P. ostreatus LACC10 (=POXC) subfamily, although lacc12 deduced intron–exon structure differs from that of the subfamily members and the related locus is located in a different chromosome. Results show that the evolutionary pattern of lacc12 and that of the other laccase isozyme genes may have evolved independently, possibly through duplication–divergence events. The reported data add a new piece to the knowledge about P. ostreatus laccase multigene family and shed light on the role(s) played by individual laccase isoforms in P. ostreatus.
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The Pleurotus ostreatus laccase multi-gene family: isolation and heterologous expression of new family members
Current Genetics, 2009Co-Authors: Cinzia Pezzella, Flavia Autore, Paola Giardina, Alessandra Piscitelli, Giovanni Sannia, Vincenza FaracoAbstract:This work was aimed at identifying and at characterizing new Pleurotus ostreatus Laccases, in order to individuate the most suitable biocatalysts for specific applications. The existence of a laccase gene clustering was demonstrated in this basidiomycete fungus, and three new laccase genes were cloned, taking advantage of their closely related spatial organization on the fungus genome. cDNAs coding for two of the new Laccases were isolated and expressed in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis , in order to optimize their production and to characterize the recombinant proteins . Analysis of the P. ostreatus laccase gene family allowed the identification of a “laccase subfamily” consisting of three genes. A peculiar intron–exon structure was revealed for the gene of one of the new Laccases, along with a high instability of the recombinant enzyme due to lability of its copper ligand. This study allowed enlarging the assortment of P. ostreatus Laccases and increasing knowledge to improve laccase production.
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overlap of Laccases cellobiose dehydrogenase activities during the decolourisation of anthraquinonic dyes with close chemical structures by pycnoporus strains
Enzyme and Microbial Technology, 2007Co-Authors: Sophie Vanhulle, Giovanni Sannia, Estelle Enaud, Marie Trovaslet, Nancy Nouaimeh, Christianmarie Bols, Tajalli Keshavarz, Thierry Tron, Annemarie CorbisierAbstract:Pycnoporus strains were used as model to understand the role of Laccases in the in vivo decolourisation of three anthraquinonic dyes. The decolourisation capability of Pycnoporus sanguineus MUCL 41582 (PS7), which produces Laccases as the main oxidative enzyme, was assayed and compared with the decolourisation capability of a control strain, Pycnoporus cinnabarinus MUCL 39533 (PC330) described as laccase-deficient strain. In absence of dye, laccase activity was observed during the trophophase and the idiophase with PS7, while no laccase activity was observed with PC330. Acid Blue 62 (ABu62), Acid Blue 281 (ABu281) and Reactive Blue 19 (RBu 19) caused an increase in laccase activity and surprisingly laccase activity was detected with PC330. In vitro, oxidation of all three anthraquinones by a laccase preparation was obtained to a lesser extent than the whole cell process; suggesting that other factor(s) could be required for a complete decolourisation. As the time space of laccase production in the tested fungi was not perfectly coincidental with the decolourisation process, the activity of cellobiose dehydrogenase (CDH) was monitored. Present early in the broth during the growth of the fungi, CDH displayed in vitro a synergism with Laccases in the decolourisation of ABu62, and an antagonism with Laccases in the decolourisation of ABu281 and RBu19. (c) 2006 Elsevier Inc. All rights reserved.
Miguel Alcalde - One of the best experts on this subject based on the ideXlab platform.
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New colorimetric screening assays for the directed evolution of fungal Laccases to improve the conversion of plant biomass
BMC Biotechnology, 2013Co-Authors: Isabel Pardo, Xiomara Chanagá, Ana Isabel Vicente, Miguel Alcalde, Susana CamareroAbstract:Background Fungal Laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λ_max of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential Laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λ_max of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential Laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal Laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering Laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring Laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc Laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
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new colorimetric screening assays for the directed evolution of fungal Laccases to improve the conversion of plant biomass
BMC Biotechnology, 2013Co-Authors: Isabel Pardo, Xiomara Chanagá, Ana Isabel Vicente, Miguel Alcalde, Susana CamareroAbstract:Fungal Laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential Laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential Laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal Laccases secreted by yeast. The high-throughput screening methods reported in this work could be useful for engineering Laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring Laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc Laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
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development of chimeric Laccases by directed evolution
Biotechnology and Bioengineering, 2012Co-Authors: Isabel Pardo, Miguel Alcalde, Ana I Vicente, Diana M Mate, Susana CamareroAbstract:DNA recombination methods are useful tools to generate diversity in directed evolution protein engineer- ing studies. We have designed an array of chimeric Laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal Laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previ- ously tailored by laboratory evolution for functional expres- sion in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved a-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid Laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012;109: 2978-2986.
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Engineering and Applications of fungal Laccases for organic synthesis
Microbial Cell Factories, 2008Co-Authors: Adinarayana Kunamneni, Susana Camarero, Francisco J Plou, Antonio Ballesteros, Carlos García-burgos, Miguel AlcaldeAbstract:Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal Laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis. Thus, Laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, Laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal Laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.
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in vitro evolution of a fungal laccase in high concentrations of organic cosolvents
Chemistry & Biology, 2007Co-Authors: Miren Zumarraga, Thomas Bulter, Sergey Shleev, Julio Polaina, A Martinezarias, Francisco J Plou, Antonio Ballesteros, Miguel AlcaldeAbstract:Fungal Laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which Laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase-R2 variant-was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.
Susana Camarero - One of the best experts on this subject based on the ideXlab platform.
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heterologous expression engineering and characterization of a novel laccase of agrocybe pediades with promising properties as biocatalyst
Journal of Fungi, 2021Co-Authors: Pablo Aza, Gonzalo Molpeceres, Francisco J Ruizduenas, Susana CamareroAbstract:Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, Laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than Laccases from Polyporales, despite the fact that the former fungi are excellent sources of Laccases. Here, the gene of a novel laccase of Agrocybe pediades, that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in Saccharomyces cerevisiae using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three N-glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme.
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identification and characterization of laccase type multicopper oxidases involved in dye decolorization by the fungus leptosphaerulina sp
BMC Biotechnology, 2015Co-Authors: Xiomara Chanagá, Maria Jesus Martinez, Ledys S Copete, Jorge Barriuso, Maria F Lopezlucendo, Susana CamareroAbstract:Fungal Laccases are multicopper oxidases (MCOs) with high biotechnological potential due to their capability to oxidize a wide range of aromatic contaminants using oxygen from the air. Albeit the numerous laccase-like genes described in ascomycete fungi, ascomycete Laccases have been less thoroughly studied than white-rot basidiomycetous Laccases. A variety of MCO genes has recently been discovered in plant pathogenic ascomycete fungi, however little is known about the presence and function of Laccases in these fungi or their potential use as biocatalysts. We aim here to identify the laccase-type oxidoreductases that might be involved in the decolorization of dyes by Leptosphaerulina sp. and to characterize them as potential biotechnological tools. A Leptosphaerulina fungal strain, isolated from lignocellulosic material in Colombia, produces laccase as the main ligninolytic oxidoreductase activity during decolorization of synthetic organic dyes. Four laccase-type MCO genes were partially amplified from the genomic DNA using degenerate primers based on laccase-specific signature sequences. The phylogenetic analysis showed the clustering of Lac1, Lac4 and Lac3 with ascomycete Laccases, whereas Lac2 grouped with fungal ferroxidases (together with other hypothetical Laccases). Lac3, the main laccase produced by Leptosphaerulina sp. in dye decolorizing and laccase-induced cultures (according to the shotgun analysis of both secretomes) was purified and characterized in this study. It is a sensu-stricto laccase able to decolorize synthetic organic dyes with high efficiency particularly in the presence of natural mediator compounds. The searching for laccase-type MCOs in ascomycetous families where their presence is poorly known, might provide a source of biocatalysts with potential biotechnological interest and shed light on their role in the fungus. The information provided by the use of genomic and proteomic tools must be combined with the biochemical evaluation of the enzyme to prove its catalytic activity and applicability potential.
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New colorimetric screening assays for the directed evolution of fungal Laccases to improve the conversion of plant biomass
BMC Biotechnology, 2013Co-Authors: Isabel Pardo, Xiomara Chanagá, Ana Isabel Vicente, Miguel Alcalde, Susana CamareroAbstract:Background Fungal Laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λ_max of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential Laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λ_max of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential Laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal Laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering Laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring Laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc Laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
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new colorimetric screening assays for the directed evolution of fungal Laccases to improve the conversion of plant biomass
BMC Biotechnology, 2013Co-Authors: Isabel Pardo, Xiomara Chanagá, Ana Isabel Vicente, Miguel Alcalde, Susana CamareroAbstract:Fungal Laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential Laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential Laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal Laccases secreted by yeast. The high-throughput screening methods reported in this work could be useful for engineering Laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring Laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc Laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
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development of chimeric Laccases by directed evolution
Biotechnology and Bioengineering, 2012Co-Authors: Isabel Pardo, Miguel Alcalde, Ana I Vicente, Diana M Mate, Susana CamareroAbstract:DNA recombination methods are useful tools to generate diversity in directed evolution protein engineer- ing studies. We have designed an array of chimeric Laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal Laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previ- ously tailored by laboratory evolution for functional expres- sion in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved a-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid Laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012;109: 2978-2986.
Alessandra Piscitelli - One of the best experts on this subject based on the ideXlab platform.
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Beyond natural Laccases: extension of their potential applications by protein engineering
Applied Microbiology and Biotechnology, 2020Co-Authors: Ilaria Stanzione, Cinzia Pezzella, Paola Giardina, Giovanni Sannia, Alessandra PiscitelliAbstract:Laccases bring exciting promises into the green industries, and the development of enzymes with improved properties is further raising their exploitation potential. Molecular engineering methods to build highly efficient catalysts both through rational and random mutagenesis were extensively applied. Moreover, computational approaches are becoming always more reliable in aiding proper design of efficient and tailored catalyst for specific applications. In this review, the results of the last 10 years about industrial application of engineered Laccases in different fields are analyzed. Tailoring laccase towards a target substrate and defining a proper screening strategy for the selection of the “jackpot mutant” represent the keys of a winning mutagenesis pathway. Likewise, laccase chimerae, built by the fusion of Laccases with relevant proteins, emerged as an added value in the designing of flexible and well-rounded biocatalysts. Despite being promising in most of the reported examples, evolved Laccases are currently tested at a laboratory scale and a feedback from the industry world is continuously required to strengthen the biotechnological exploitation of these improved enzymes.
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induction and transcriptional regulation of Laccases in fungi
Current Genomics, 2011Co-Authors: Alessandra Piscitelli, Cinzia Pezzella, Paola Giardina, Giovanni Sannia, Vincenzo Lettera, Vincenza FaracoAbstract:Fungal Laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native Laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet.
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identification of a new member of pleurotus ostreatus laccase family from mature fruiting body
Fungal Biology, 2010Co-Authors: Vincenzo Lettera, Cinzia Pezzella, Alessandra Piscitelli, Leila Birolo, Giovanni SanniaAbstract:Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. Most of the known Laccases have fungal or plant origins, although few Laccases have been also identified in bacteria and insects. Most of the fungal Laccases reported thus far are extra-cellular enzymes, whereas only few enzymes from fruiting bodies have been described so far. Multiple isoforms of Laccases are usually secreted by each fungus depending on species and environmental conditions. As a fact, a laccase gene family has been demonstrated in the white-rot fungus Pleurotus ostreatus. This work allowed identification and characterization of the first laccase isoenzyme from the fruiting body of P. ostreatus. Discovery through mass spectrometry of LACC12 proves the expression of a functional protein by the related deduced encoding transcript. The topology of phylogenetic tree of fungal Laccases proves that LACC12 falls in cluster with the members of P. ostreatus LACC10 (=POXC) subfamily, although lacc12 deduced intron–exon structure differs from that of the subfamily members and the related locus is located in a different chromosome. Results show that the evolutionary pattern of lacc12 and that of the other laccase isozyme genes may have evolved independently, possibly through duplication–divergence events. The reported data add a new piece to the knowledge about P. ostreatus laccase multigene family and shed light on the role(s) played by individual laccase isoforms in P. ostreatus.
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The Pleurotus ostreatus laccase multi-gene family: isolation and heterologous expression of new family members
Current Genetics, 2009Co-Authors: Cinzia Pezzella, Flavia Autore, Paola Giardina, Alessandra Piscitelli, Giovanni Sannia, Vincenza FaracoAbstract:This work was aimed at identifying and at characterizing new Pleurotus ostreatus Laccases, in order to individuate the most suitable biocatalysts for specific applications. The existence of a laccase gene clustering was demonstrated in this basidiomycete fungus, and three new laccase genes were cloned, taking advantage of their closely related spatial organization on the fungus genome. cDNAs coding for two of the new Laccases were isolated and expressed in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis , in order to optimize their production and to characterize the recombinant proteins . Analysis of the P. ostreatus laccase gene family allowed the identification of a “laccase subfamily” consisting of three genes. A peculiar intron–exon structure was revealed for the gene of one of the new Laccases, along with a high instability of the recombinant enzyme due to lability of its copper ligand. This study allowed enlarging the assortment of P. ostreatus Laccases and increasing knowledge to improve laccase production.
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recombinant expression of pleurotus ostreatus Laccases in kluyveromyces lactis and saccharomyces cerevisiae
Applied Microbiology and Biotechnology, 2005Co-Authors: Alessandra Piscitelli, Paola Giardina, Cristina Mazzoni, Giovanni SanniaAbstract:Heterologous expression of Pleurotus ostreatus POXC and POXA1b Laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active Laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant Laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure–function relationships and allows the development of new oxidative catalysts through molecular evolution techniques.
Luka Ausec - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp
Applied Microbiology and Biotechnology, 2015Co-Authors: Luka Ausec, Miha Črnigoj, Marko Šnajder, Nataša Poklar Ulrih, Ines Mandic-mulecAbstract:Laccases are oxidoreductases mostly studied in fungi, while bacterial Laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial Laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli . The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.
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two and three domain bacterial laccase like genes are present in drained peat soils
Soil Biology & Biochemistry, 2011Co-Authors: Luka Ausec, Jan Dirk Van Elsas, Ines MandicmulecAbstract:Laccases of fungal origin have been intensively studied due to their importance in various biotechnological applications. There is a constant demand for new Laccases with improved properties such as stability at higher temperatures or at an alkaline pH. Growing molecular evidence suggests that Laccases may also be widespread in bacteria. While only a handful of bacterial Laccases have been purified and characterized, several novel traits have already been discovered (e.g. pH-stability and 2-domain organization of the enzyme as opposed to the usual 3-domain structure of fungal Laccases). The aim of this study was to examine the diversity of bacterial laccase-like genes in two types of high-organic peat soil using a cloning and sequencing approach. Gene libraries prepared of small fragments (150 base pairs) revealed an amazing diversity of bacterial Laccases. The fragments clustered in 11 major lineages, and one third of the 241 sequences resembled laccase-like genes of Acidobacteria. Additionally, a new primer was used to retrieve several larger fragments of the putative bacterial laccase genes that spanned all four copper-binding sites. Both “conventional” 3-domain Laccases and the recently described 2-domain small Laccases have been obtained using this approach, demonstrating the potential of the primer. The present study thus contributes to the understanding of the diversity of bacterial Laccases and provides a new tool for finding laccase-like sequences in bacterial strains and soil samples.
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Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes
2011Co-Authors: Luka Ausec, Martha Zakrzewski, Er Goesmann, Andreas Schlüter, Ines MAbstract:Fungal Laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial Laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of Laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and threedomain Laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76 % of the genes, signal peptides were predicted, indicating that these bacterial Laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications
