Ana P.M. Tavares

Ionic liquids as alternative co-solvents for laccase: Study of enzyme activity and stability

The activity and stability of commercial laccase (DeniLite base) in three different water soluble ionic liquids (ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [emim][[MDEGSO4], 1-ethyl-3-methylimidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3]) have been studied and compared to that in two organic solvents (acetonitrile and dimethyl sulfoxide). Initial enzyme activities were similar among the ILs if the same conditions were used. A high reduction on initial enzyme activity was found with acidic pH (5.0). The effect of pH and solvent concentration on enzyme stability were investigated in more detail for 1 week. The enzyme maintained a high stability at pH 9.0 for all ILs tested. [emim][MDEGSO4] was the most promising IL for laccase with an activity loss of about 10% after 7 days of incubation. The kinetic studies in the presence of ABTS as substrate allowed to calculate the Michaelis- Menten parameters. Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.

Application of statistical experimental methodology to optimize reactive dye decolourization by commercial laccase.

Three-level Box-Behnken factorial design with three factors (pH, temperature and enzyme concentration) combined with response surface methodology (RSM) was applied to optimize the dye degradation of reactive red 239 (RR239), reactive yellow 15 (RY15) and reactive blue 114 (RB114) dyes by commercial laccase. Mathematical models were developed for each dye showing the effect of each factor and their interactions on colour removal. The model predicted for RY15 that a decolourization above 90% (after 24h) could be obtained when the enzyme concentration, temperature and pH were set at 109.8U/L, 39.2 degrees C and 6.6, respectively; whilst for RB114 and RR239 the temperature and enzyme concentration did not affect the decolourization (>90%) in the considered range and optimum pH value was found at 5.5-7.0 and 7.0-7.5, respectively. These predicted values were also experimentally validated. Average final values of responses were in good agreement with calculated values, thus confirming the reliability of the models of RY15, RB114 and RR239 decolourization.

Optimization and Modeling of Laccase Production by Trametes versicolor in a Bioreactor Using Statistical Experimental Design

Experimental design and response surface methodologies were applied to optimize laccase production by Trametes versicolor in a bioreactor. The effects of three factors, initial glucose concentration (0 and 9 g/L), agitation (100 and 180 rpm), and pH (3.0 and 5.0), were evaluated to identify the significant effects and its interactions in the laccase production. The pH of the medium was found to be the most important factor, followed by initial glucose concentration and the interaction of both factors. Agitation did not seem to play an important role in laccase production, nor did the interaction agitation x medium pH and agitation x initial glucose concentration. Response surface analysis showed that an initial glucose concentration of 11 g/L and pH controlled at 5.2 were the optimal conditions for laccase production by T. versicolor. Under these conditions, the predicted value for laccase activity was >10,000 U/L, which is in good agreement with the laccase activity obtained experimentally (11,403 U/L). In addition, a mathematical model for the bioprocess was developed. It is shown that it provides a good description of the experimental profile observed, and that it is capable of predicting biomass growth based on secondary process variables.

Laccase immobilization over multi-walled carbon nanotubes: Kinetic, thermodynamic and stability studies.

The biocatalytic performance of immobilized enzyme systems depends mostly on the intrinsic properties of both biomolecule and support, immobilization technique and immobilization conditions. Multi-walled carbon nanotubes (MWCNTs) possess unique features for enzyme immobilization by adsorption. Enhanced catalytic activity and stability can be achieved by optimization of the immobilization conditions and by investigating the effect of operational parameters. Laccase was immobilized over MWCNTs by adsorption. The hybrid material was characterized by Fourier transformed infrared (FTIR) spectroscopy, scanning and transmission electron microscopy (SEM and TEM, respectively). The effect of different operational conditions (contact time, enzyme concentration and pH) on laccase immobilization was investigated. Optimized conditions were used for thermal stability, kinetic, and storage and operational stability studies. The optimal immobilization conditions for a laccase concentration of 3.75μL/mL were a pH of 9.0 and a contact time of 30min (522 Ulac/gcarrier). A decrease in the thermal stability of laccase was observed after immobilization. Changes in ΔS and ΔH of deactivation were found for the immobilized enzyme. The Michaelis-Menten kinetic constant was higher for laccase/MWCNT system than for free laccase. Immobilized laccase maintained (or even increased) its catalytic performance up to nine cycles of utilization and revealed long-term storage stability.

Modeling the discoloration of a mixture of reactive textile dyes by commercial laccase.

Degradation of a mixture of three reactive textile dyes (Reactive Black 5, Reactive Yellow 15 and Reactive Red 239), simulating a real textile effluent, by commercial laccase, was investigated in a batch reactor. The discoloration was appraised as a percentage of the absorbance reduction at the wavelength of maximum absorbance for each dye and as total color removal based in all visible spectrum. A significantly high discoloration was achieved in both cases, indicating the applicability of this method for textile wastewater treatment. Mathematical models were developed to simulate the kinetics of laccase catalyzed degradation of reactive dyes in mixtures. Like in single dye degradation, some of the reactions present an unusual kinetic behavior, corresponding to the activation of the laccase-mediator system. The kinetic constants of the models were estimated by minimizing the difference between the predicted and the experimental time courses. Although not perfect, the ability of the models in representing the experimental results suggests that they could be used in design and simulation applications.

Decolorization of Dyes from textile wastewater by Trametes versicolor

The white rot fungus Trametes versicolor was applied to the decolourisation of three synthetic textile dyes in the presence and absence of glucose. Different initial dye concentrations were tested and approximately 97% decolourisation was achieved. It was found that fungal metabolism induced by the glucose as well as the pH play an important role in the decolourisation process. This treatment was also applied to a real wastewater from a textile industry-dyeing sector leading to 92% decolourisation.

Studies of laccase from Trametes versicolor in aqueous solutions of several methylimidazolium ionic liquids

Stability and kinetic behavior of laccase from Trametes versicolor in the presence of several ionic liquids from the methylimidazolium family have been investigated. In general laccase stability diminished as the size of the alkylic substitute in the methylimidazolium ring increased. Higher concentrations of ionic liquids caused more destabilization than lower ones. Thus, low concentrations of [C(2)mim(+)][EtSO(4)(-)] allowed maintaining enzymatic stability. [C(4)mim(+)][Cl(-)] appeared to have a stabilizing effect on laccase, as little activity decay was observed within three weeks. Kinetic studies indicated that both [C(2)mim(+)][EtSO(4)(-)] and [C(4)mim(+)][Cl(-)] inhibited laccase activity, although 10-fold more [C(2)mim(+)][EtSO(4)(-)] than [C(4)mim(+)][Cl(-)] was required to cause the same degree of inhibition. A kinetic model was developed to represent the experimental data.

Treatment and kinetic modelling of a simulated dye house effluent by enzymatic catalysis

Biocatalytic treatment of a synthetic dye house effluent, simulating a textile wastewater containing various reactive dyestuffs (Reactive Yellow 15, Reactive Red 239 and Reactive Black 5) and auxiliary chemicals, was investigated in a batch reactor using a commercial laccase. A high decolourisation (above 86%) was achieved at the maximum wavelength of Reactive Black 5. The decolourisation at the other dyes wavelengths (above 63% for RY15 and around 41% for RR239) and the total decolourisation based on all the visible spectrum (around 55%) were not so good, being somewhat lower than in the case of a mixture of the dyes (above 89% for RB5, 77% for RY15, 68% for RR239 and above 84% for total decolourisation). Even so, these results suggest the applicability of this method to treat textile dyeing wastewaters. Kinetic models were developed to simulate the synthetic effluent decolourisation by commercial laccase. The kinetic constants of the models were estimated by minimizing the difference between the predicted and the experimental time courses. The close correlation between the experimental data and the simulated values seems to demonstrate that the models are able to describe with remarkable accuracy the simulated effluent degradation. Water quality parameters such as TOC, COD, BOD(5) and toxicity were found to be under the maximum permissible discharge limits for textile industries wastewaters.

The release of light metals from a brown seaweed (Sargassum sp.) during zinc biosorption in a continuous system

The biosorption of zinc and calcium was investigated with a biomass of Sargassum sp., a brown seaweed, in a continuous system consisting of three serial tubular fixed-bed laboratory reactors. Results indicated that zinc was efficiently recovered by the biomass. After treatment of 9.0 liters of a mixed solution containing 130.0 mg/l zinc and 260.0 mg/l calcium, the first column of the system saturated with zinc; the remaining columns did not saturate with zinc as a result of the pre treatment performed by the first reactor. Calcium was also efficiently biosorbed by the biomass, saturating the system much faster than zinc. X-ray fluorescence spectrum indicated the presence of various elements in the structure of the Sargassum sp. biomass, especially alkaline and alkaline-earth elements. Alkaline and alkaline earth elements played a key role in the biosorption of zinc, being responsible for ion-exchange reactions performed during zinc biosorption.

New Generations of Ionic Liquids Applied to Enzymatic Biocatalysis

Ionic liquids are salts in a liquid state, combinations of cations and anions that are liquid at temperatures below 100 oC. Thus, they have been called Room-Temperature Ionic Liquids (RTILs, or just ILs) in order to differentiate them from traditional salts, which melt at much higher temperatures and receive the name of “molten salts”. In contrast to conventional or‐ ganic solvents, ILs usually have extremely low volatility. Indeed, vapor pressures for ILs are scarce in the literature exactly because they are extremely low (< 1 Pa) and have to be ob‐ tained at high temperatures (400-500 K) [1]. For this ”negligible” vapor pressure, ILs are of‐ ten said to be “green“ solvents when compared to traditional, environmentally harmful volatile organic compounds (VOCs). A big goal in the use of ILs in enzymatic reactions is the replacement of VOCs by ILs. In addition, ILs have other potential advantageous proper‐ ties such as reasonable thermal stability; ability to dissolve a wide range of organic, inorgan‐ ic and organometallic compounds; controlled miscibility with organic solvents (which is relevant for applications in biphasic systems) among others. All these properties make them very attractive non-aqueous solvents for biocatalysis. As they have been extensively descri‐ bed, ILs offer new possibilities for the application of solvent engineering to enzymatic reac‐ tions. Biocatalysis with ILs as reaction medium was first showed in the beginning of 2000 [2-4]. During the last decade, ILs have fast increased their attention as reaction media for en‐ zymes with some remarkable results [2-4]. The advantage of using ILs in enzymatic biocatal‐ ysis, as compared to VOCs, is the enhancement in the solubility of substrates or products without inactivation of the enzymes, high conversion rates and high activity and stability [5]. ILs are also being used as co-solvents in aqueous biocatalytic reactions, since ILs help to