José Toca-herrera | University of Natural Resources and Life Sciences, Vienna (BOKU) (original) (raw)

Papers by José Toca-herrera

International Journal of Molecular Sciences

Stress-associated changes in the mechanical properties at the single-cell level of Escherichia co... more Stress-associated changes in the mechanical properties at the single-cell level of Escherichia coli (E. coli) cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. The cell growth, peptide content, and cell lysis were analysed, and changes in the mechanical properties were investigated using atomic force microscopy. Recombinant-tagged somatostatin-28 was expressed as soluble up to 197 ± 11 mg g−1. The length of both cultivated strains increased throughout the cultivation by up to 17.6%, with nearly constant diameters. The peptide-producing cells were significantly softer than the non-producers throughout the cultivation, and respective Young’s moduli decreased by up to 57% over time. A minimum Young’s modulus of 1.6 MPa was observed after 23 h of the fed-batch. Furthermore, an analysis of the viscoelastic properties revealed that pepti...

Scientific Reports

Cell mechanical properties have been proposed as label free markers for diagnostic purposes in di... more Cell mechanical properties have been proposed as label free markers for diagnostic purposes in diseases such as cancer. Cancer cells show altered mechanical phenotypes compared to their healthy counterparts. Atomic Force Microscopy (AFM) is a widely utilized tool to study cell mechanics. These measurements often need skilful users, physical modelling of mechanical properties and expertise in data interpretation. Together with the need to perform many measurements for statistical significance and to probe wide enough areas in tissue structures, the application of machine learning and artificial neural network techniques to automatically classify AFM datasets has received interest recently. We propose the use of self-organizing maps (SOMs) as unsupervised artificial neural network applied to mechanical measurements performed via AFM on epithelial breast cancer cells treated with different substances that affect estrogen receptor signalling. We show changes in mechanical properties due...

International Journal of Molecular Sciences, 2013

Crystalline S(urface)-layers are the most commonly observed cell surface structures in prokaryoti... more Crystalline S(urface)-layers are the most commonly observed cell surface structures in prokaryotic organisms (bacteria and archaea). S-layers are highly porous protein meshworks with unit cell sizes in the range of 3 to 30 nm, and thicknesses of ~10 nm. One of the key features of S-layer proteins is their intrinsic capability to form self-assembled mono-or double layers in solution, and at interfaces. Basic research on S-layer proteins laid foundation to make use of the unique self-assembly properties of native and, in particular, genetically functionalized S-layer protein lattices, in a broad range of applications in the life and non-life sciences. This contribution briefly summarizes the knowledge about structure, genetics, chemistry, morphogenesis, and function of S-layer proteins and pays particular attention to the self-assembly in solution, and at differently functionalized solid supports.

Macromolecules, 2002

ABSTRACT

Health education research, 2015

The relationship among physical activity, physical fitness and academic achievement in adolescent... more The relationship among physical activity, physical fitness and academic achievement in adolescents has been widely studied; however, controversy concerning this topic persists. The methods used thus far to analyse the relationship between these variables have included mostly traditional lineal analysis according to the available literature. The aim of this study was to perform a visual analysis of this relationship with self-organizing maps and to monitor the subject's evolution during the 4 years of secondary school. Four hundred and forty-four students participated in the study. The physical activity and physical fitness of the participants were measured, and the participants' grade point averages were obtained from the five participant institutions. Four main clusters representing two primary student profiles with few differences between boys and girls were observed. The clustering demonstrated that students with higher energy expenditure and better physical fitness exhib...

Microscopy Research and Technique, 2014

This review reports on the use of the atomic force microscopy in the investigation of the mechani... more This review reports on the use of the atomic force microscopy in the investigation of the mechanical properties of cells. It is shown that the technique is able to deliver information about the cell surface properties (e.g., topography), the Young modulus, the viscosity, and the cell the relaxation times. Another aspect that this short review points out is the utilization of the atomic force microscope to investigate basic questions related to materials physics, biology, and medicine. The review is written in a chronological way to offer an overview of phenomenological facts and quantitative results to the reader. The final section discusses in detail the advantages and disadvantages of the Hertz and JKR models. A new implementation of the JKR model derived by Dufresne is presented.

Biotechnology Advances, 2006

Laccases have received much attention from researchers in last decades due to their ability to ox... more Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and nonphenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

The search for efficient and green oxidation technologies has increased the interest in the use o... more The search for efficient and green oxidation technologies has increased the interest in the use of enzymes to replace the conventional non-biological methods. Among the different existing oxidant enzymes, laccases (benzenediol: oxygen oxidoreductases; EC 1.10.3.2) have been subject of intensive research in the last decades due to their low substrate specificity. The use of laccases in the textile industry is growing very fast, since besides to decolourise textile effluents, laccases are used to bleach textiles, modify the surface of fabrics and synthetise dyes. Therefore, laccase-based processes might replace the traditionally high chemical, energy and water-consuming textile operations. The present

Opiorphin (Oph) is a naturally produced endogenous peptide with a strong analgesic effect, superi... more Opiorphin (Oph) is a naturally produced endogenous peptide with a strong analgesic effect, superior to that of morphine, and without the severe side effects that morphine and morphine-like drugs exert. However, despite its strong therapeutic potential, the short duration of action, probably due to its low chemical stability and rapid degradation by the peptidases in the bloodstream, represents a serious obstacle to the Oph use into clinical practice. In this work a novel approach to construct Oph-loaded particles as a platform for its delivery has been developed. Gel beads loaded with Oph were synthesized from alginate, a naturally occurring biodegradable anionic polysaccharide, and coated with polyelectrolyte multilayers (from natural polyelectrolytes (chitosan and hyaluronic acid) and synthetic polyelectrolytes (poly(allylamine hydrochloride) and poly(styrene sulfonate)) or hybrid polyelectrolyte-graphene oxide multilayers. All coated Oph-loaded alginate beads show prolonged drug ...

Coatings, Jan 27, 2019

The well-known bacterial S-layer protein SbpA from Lysinibacillus sphaericus CCM2177 induces spon... more The well-known bacterial S-layer protein SbpA from Lysinibacillus sphaericus CCM2177 induces spontaneous crystal formation via cooperative self-assembly of the protein subunits into an ordered supramolecular structure. Recrystallization occurs in the presence of divalent cations (i.e., Ca 2+) and finally leads to producing smooth 2-D crystalline coatings composed of squared (p4) lattice structures. Among the factors interfering in such a process, the rate of protein supply certainly plays an important role since a limited number of accessible proteins might turn detrimental for film completion. Studies so far have mostly focused on high SbpA concentrations provided under stopped-flow or dynamic-flow conditions, thus omitting the possibility of investigating intermediate states, in which dynamic flow is applied for more critical concentrations of SbpA (i.e., 25, 10, and 5 µg/mL). In this work, we have characterized both physico-chemical and topographical aspects of the assembly and recrystallization of SbpA protein in such low concentration conditions by means of in situ Quartz Crystal Microbalance with Dissipation (QCMD) and atomic force microscopy (AFM) measurements, respectively. On the basis of these experiments, we can confirm how the application of a dynamic flow influences the formation of a closed and crystalline protein film from low protein concentrations (i.e., 10 µg/mL), which otherwise would not be formed.

Polymers, 2018

New strategies in regenerative medicine include the implantation of stem cells cultured in bio-re... more New strategies in regenerative medicine include the implantation of stem cells cultured in bio-resorbable polymeric scaffolds to restore the tissue function and be absorbed by the body after wound healing. This requires the development of appropriate micro-technologies for manufacturing of functional scaffolds with controlled surface properties to induce a specific cell behavior. The present report focuses on the effect of substrate topography on the behavior of human mesenchymal stem cells (MSCs) before and after co-differentiation into adipocytes and osteoblasts. Picosecond laser micromachining technology (PLM) was applied on poly (L-lactide) (PLLA), to generate different microstructures (microgrooves and microcavities) for investigating cell shape, orientation, and MSCs co-differentiation. Under certain surface topographical conditions, MSCs modify their shape to anchor at specific groove locations. Upon MSCs differentiation, adipocytes respond to changes in substrate height and ...

Methods and Applications in Fluorescence, 2014

ABSTRACT Fluorescence proteins are widely used as markers for biomedical and technological purpos... more ABSTRACT Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

Microscopy Research and Technique, 2014

Materials, 2012

Models for the organization of sterols into regular arrays within phospholipid bilayers have been... more Models for the organization of sterols into regular arrays within phospholipid bilayers have been proposed previously. The existence of such arrays in real systems has been supported by the fact that concentration-dependent sterol properties show discontinuities at the cholesterol mole fractions corresponding to regular lattice arrangements. Experimental results presented here are based on a surface plasmon resonance assay that was used to analyze rates of cyclodextrin-mediated removal of cholesterol from adsorbed liposomes at cholesterol mole fractions up to χ C = 0.55. Two kinetic pools of cholesterol were detected; there was a fast pool present at χ C > 0.25, and a slow pool, with a removal rate that was dependent on the initial χ C but that did not vary as χ C decreased during the course of one experiment. The cholesterol activity therefore seems to be affected by sample history as well as local concentration, which could be explained in terms of the formation of superlattices that are stable for relatively long times. We also describe a variation on the traditional lattice models, with phosphatidylcholine (PC) being treated as an arrangement of hexagonal tiles; the cholesterol is then introduced at any vertex point, without increasing the total area occupied by all the lipid molecules. This model is consistent with Langmuir trough measurements of total lipid area and provides a simple explanation for the maximum solubility of cholesterol in the PC bilayer.

Journal of Molecular Biology, 2002

Journal of Biotechnology, 2007

Journal of Biotechnology, 2007

Engineering in Life Sciences, 2009

ABSTRACT In this paper an eco-friendly strategy for the removal of synthetic dyes from aqueous so... more ABSTRACT In this paper an eco-friendly strategy for the removal of synthetic dyes from aqueous solutions was proposed. Thus, in a first step the dyes were adsorbed onto sunflower seed shells (SS) by using a batch technique. Subsequently, in a second step, these dyed SS were used as support-substrates to produce laccase by the white-rot fungus Trametes pubescens under semi-solid-state conditions. The effect of inducer addition on laccase production was studied. The optimum conditions (addition of both 0.5 mM Cu+2 and 50 μM tannic acid on the 3rd day of cultivation) led to a maximum laccase activity of 30272 U/L. Further, the system was efficiently scaled-up to laboratory bioreactors producing a maximum activity of 40172 U/L together with a total decolouration of the adsorbed dye. In addition, SDS-PAGE showed that laccases were the only enzymes present in the extracellular fluid. Therefore, apart from dye removal this approach allowed the production of high titres of laccase, which was obtained almost pure (only needed to be ultra-filtrated).

International Journal of Molecular Sciences

Stress-associated changes in the mechanical properties at the single-cell level of Escherichia co... more Stress-associated changes in the mechanical properties at the single-cell level of Escherichia coli (E. coli) cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. The cell growth, peptide content, and cell lysis were analysed, and changes in the mechanical properties were investigated using atomic force microscopy. Recombinant-tagged somatostatin-28 was expressed as soluble up to 197 ± 11 mg g−1. The length of both cultivated strains increased throughout the cultivation by up to 17.6%, with nearly constant diameters. The peptide-producing cells were significantly softer than the non-producers throughout the cultivation, and respective Young’s moduli decreased by up to 57% over time. A minimum Young’s modulus of 1.6 MPa was observed after 23 h of the fed-batch. Furthermore, an analysis of the viscoelastic properties revealed that pepti...

Scientific Reports

Cell mechanical properties have been proposed as label free markers for diagnostic purposes in di... more Cell mechanical properties have been proposed as label free markers for diagnostic purposes in diseases such as cancer. Cancer cells show altered mechanical phenotypes compared to their healthy counterparts. Atomic Force Microscopy (AFM) is a widely utilized tool to study cell mechanics. These measurements often need skilful users, physical modelling of mechanical properties and expertise in data interpretation. Together with the need to perform many measurements for statistical significance and to probe wide enough areas in tissue structures, the application of machine learning and artificial neural network techniques to automatically classify AFM datasets has received interest recently. We propose the use of self-organizing maps (SOMs) as unsupervised artificial neural network applied to mechanical measurements performed via AFM on epithelial breast cancer cells treated with different substances that affect estrogen receptor signalling. We show changes in mechanical properties due...

International Journal of Molecular Sciences, 2013

Crystalline S(urface)-layers are the most commonly observed cell surface structures in prokaryoti... more Crystalline S(urface)-layers are the most commonly observed cell surface structures in prokaryotic organisms (bacteria and archaea). S-layers are highly porous protein meshworks with unit cell sizes in the range of 3 to 30 nm, and thicknesses of ~10 nm. One of the key features of S-layer proteins is their intrinsic capability to form self-assembled mono-or double layers in solution, and at interfaces. Basic research on S-layer proteins laid foundation to make use of the unique self-assembly properties of native and, in particular, genetically functionalized S-layer protein lattices, in a broad range of applications in the life and non-life sciences. This contribution briefly summarizes the knowledge about structure, genetics, chemistry, morphogenesis, and function of S-layer proteins and pays particular attention to the self-assembly in solution, and at differently functionalized solid supports.

Macromolecules, 2002

ABSTRACT

Health education research, 2015

The relationship among physical activity, physical fitness and academic achievement in adolescent... more The relationship among physical activity, physical fitness and academic achievement in adolescents has been widely studied; however, controversy concerning this topic persists. The methods used thus far to analyse the relationship between these variables have included mostly traditional lineal analysis according to the available literature. The aim of this study was to perform a visual analysis of this relationship with self-organizing maps and to monitor the subject's evolution during the 4 years of secondary school. Four hundred and forty-four students participated in the study. The physical activity and physical fitness of the participants were measured, and the participants' grade point averages were obtained from the five participant institutions. Four main clusters representing two primary student profiles with few differences between boys and girls were observed. The clustering demonstrated that students with higher energy expenditure and better physical fitness exhib...

Microscopy Research and Technique, 2014

This review reports on the use of the atomic force microscopy in the investigation of the mechani... more This review reports on the use of the atomic force microscopy in the investigation of the mechanical properties of cells. It is shown that the technique is able to deliver information about the cell surface properties (e.g., topography), the Young modulus, the viscosity, and the cell the relaxation times. Another aspect that this short review points out is the utilization of the atomic force microscope to investigate basic questions related to materials physics, biology, and medicine. The review is written in a chronological way to offer an overview of phenomenological facts and quantitative results to the reader. The final section discusses in detail the advantages and disadvantages of the Hertz and JKR models. A new implementation of the JKR model derived by Dufresne is presented.

Biotechnology Advances, 2006

Laccases have received much attention from researchers in last decades due to their ability to ox... more Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and nonphenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

The search for efficient and green oxidation technologies has increased the interest in the use o... more The search for efficient and green oxidation technologies has increased the interest in the use of enzymes to replace the conventional non-biological methods. Among the different existing oxidant enzymes, laccases (benzenediol: oxygen oxidoreductases; EC 1.10.3.2) have been subject of intensive research in the last decades due to their low substrate specificity. The use of laccases in the textile industry is growing very fast, since besides to decolourise textile effluents, laccases are used to bleach textiles, modify the surface of fabrics and synthetise dyes. Therefore, laccase-based processes might replace the traditionally high chemical, energy and water-consuming textile operations. The present

Opiorphin (Oph) is a naturally produced endogenous peptide with a strong analgesic effect, superi... more Opiorphin (Oph) is a naturally produced endogenous peptide with a strong analgesic effect, superior to that of morphine, and without the severe side effects that morphine and morphine-like drugs exert. However, despite its strong therapeutic potential, the short duration of action, probably due to its low chemical stability and rapid degradation by the peptidases in the bloodstream, represents a serious obstacle to the Oph use into clinical practice. In this work a novel approach to construct Oph-loaded particles as a platform for its delivery has been developed. Gel beads loaded with Oph were synthesized from alginate, a naturally occurring biodegradable anionic polysaccharide, and coated with polyelectrolyte multilayers (from natural polyelectrolytes (chitosan and hyaluronic acid) and synthetic polyelectrolytes (poly(allylamine hydrochloride) and poly(styrene sulfonate)) or hybrid polyelectrolyte-graphene oxide multilayers. All coated Oph-loaded alginate beads show prolonged drug ...

Coatings, Jan 27, 2019

The well-known bacterial S-layer protein SbpA from Lysinibacillus sphaericus CCM2177 induces spon... more The well-known bacterial S-layer protein SbpA from Lysinibacillus sphaericus CCM2177 induces spontaneous crystal formation via cooperative self-assembly of the protein subunits into an ordered supramolecular structure. Recrystallization occurs in the presence of divalent cations (i.e., Ca 2+) and finally leads to producing smooth 2-D crystalline coatings composed of squared (p4) lattice structures. Among the factors interfering in such a process, the rate of protein supply certainly plays an important role since a limited number of accessible proteins might turn detrimental for film completion. Studies so far have mostly focused on high SbpA concentrations provided under stopped-flow or dynamic-flow conditions, thus omitting the possibility of investigating intermediate states, in which dynamic flow is applied for more critical concentrations of SbpA (i.e., 25, 10, and 5 µg/mL). In this work, we have characterized both physico-chemical and topographical aspects of the assembly and recrystallization of SbpA protein in such low concentration conditions by means of in situ Quartz Crystal Microbalance with Dissipation (QCMD) and atomic force microscopy (AFM) measurements, respectively. On the basis of these experiments, we can confirm how the application of a dynamic flow influences the formation of a closed and crystalline protein film from low protein concentrations (i.e., 10 µg/mL), which otherwise would not be formed.

Polymers, 2018

New strategies in regenerative medicine include the implantation of stem cells cultured in bio-re... more New strategies in regenerative medicine include the implantation of stem cells cultured in bio-resorbable polymeric scaffolds to restore the tissue function and be absorbed by the body after wound healing. This requires the development of appropriate micro-technologies for manufacturing of functional scaffolds with controlled surface properties to induce a specific cell behavior. The present report focuses on the effect of substrate topography on the behavior of human mesenchymal stem cells (MSCs) before and after co-differentiation into adipocytes and osteoblasts. Picosecond laser micromachining technology (PLM) was applied on poly (L-lactide) (PLLA), to generate different microstructures (microgrooves and microcavities) for investigating cell shape, orientation, and MSCs co-differentiation. Under certain surface topographical conditions, MSCs modify their shape to anchor at specific groove locations. Upon MSCs differentiation, adipocytes respond to changes in substrate height and ...

Methods and Applications in Fluorescence, 2014

ABSTRACT Fluorescence proteins are widely used as markers for biomedical and technological purpos... more ABSTRACT Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

Microscopy Research and Technique, 2014

Materials, 2012

Models for the organization of sterols into regular arrays within phospholipid bilayers have been... more Models for the organization of sterols into regular arrays within phospholipid bilayers have been proposed previously. The existence of such arrays in real systems has been supported by the fact that concentration-dependent sterol properties show discontinuities at the cholesterol mole fractions corresponding to regular lattice arrangements. Experimental results presented here are based on a surface plasmon resonance assay that was used to analyze rates of cyclodextrin-mediated removal of cholesterol from adsorbed liposomes at cholesterol mole fractions up to χ C = 0.55. Two kinetic pools of cholesterol were detected; there was a fast pool present at χ C > 0.25, and a slow pool, with a removal rate that was dependent on the initial χ C but that did not vary as χ C decreased during the course of one experiment. The cholesterol activity therefore seems to be affected by sample history as well as local concentration, which could be explained in terms of the formation of superlattices that are stable for relatively long times. We also describe a variation on the traditional lattice models, with phosphatidylcholine (PC) being treated as an arrangement of hexagonal tiles; the cholesterol is then introduced at any vertex point, without increasing the total area occupied by all the lipid molecules. This model is consistent with Langmuir trough measurements of total lipid area and provides a simple explanation for the maximum solubility of cholesterol in the PC bilayer.

Journal of Molecular Biology, 2002

Journal of Biotechnology, 2007

Journal of Biotechnology, 2007

Engineering in Life Sciences, 2009

ABSTRACT In this paper an eco-friendly strategy for the removal of synthetic dyes from aqueous so... more ABSTRACT In this paper an eco-friendly strategy for the removal of synthetic dyes from aqueous solutions was proposed. Thus, in a first step the dyes were adsorbed onto sunflower seed shells (SS) by using a batch technique. Subsequently, in a second step, these dyed SS were used as support-substrates to produce laccase by the white-rot fungus Trametes pubescens under semi-solid-state conditions. The effect of inducer addition on laccase production was studied. The optimum conditions (addition of both 0.5 mM Cu+2 and 50 μM tannic acid on the 3rd day of cultivation) led to a maximum laccase activity of 30272 U/L. Further, the system was efficiently scaled-up to laboratory bioreactors producing a maximum activity of 40172 U/L together with a total decolouration of the adsorbed dye. In addition, SDS-PAGE showed that laccases were the only enzymes present in the extracellular fluid. Therefore, apart from dye removal this approach allowed the production of high titres of laccase, which was obtained almost pure (only needed to be ultra-filtrated).