Diego Moldes

Recent developments and applications of immobilized laccase

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.

Improving laccase production by employing different lignocellulosic wastes in submerged cultures of Trametes versicolor.

Laccase production by the white-rot fungus Trametes versicolor (CBS100.29) grown in submerged cultures was studied. Addition of different insoluble lignocellulosic materials into the culture medium in order to enhance laccase production was investigated. The lignocellulosic materials were grape seeds, grape stalks and barley bran, selected because of their availability and low cost, since they are agro-industrial wastes abundant in most countries. Barley bran gave the highest activities, a maximum value of 639U/l, which was 10 times the value attained in the cultures without lignocellulosics addition. The decolourisation of a model dye, Phenol Red, by the ligninolytic fluids obtained in the above-mentioned cultures was investigated. Grape stalk and barley bran cultures showed the highest ability to decolourise the dye, attaining a percentage of decolourisation of around 60% in 72 h.

Different proportions of laccase isoenzymes produced by submerged cultures of Trametes versicolor grown on lignocellulosic wastes.

The white-rot fungus Trametes versicolor grown in submerged culture produced two laccase isoenzymes, LacI and LacII. Addition of insoluble lignocellulosic materials into the culture medium increased the total laccase activity. The proportion of laccase isoenzymes also changed depending on the lignocellulosic material employed, with ratios of activity LacII/LacI from 0.9 (barley straw) to 4.4 (grape stalks). Besides, this proportion played an important role in the dye decolourisation.

Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp

The natural phenolic compounds syringaldehyde and vanillin were compared to the synthetic mediators 1-hydroxybenzotriazole, violuric acid and promazine in terms of boosting efficiency in a laccase-assisted biobleaching of eucalyptus kraft pulp. Violuric acid and 1-hydroxybenzotriazole revealed to be the most effective mediators of the bioprocess. Nevertheless, laccase-syringaldehyde system also improved the final pulp properties (28% delignification and 63.5% ISO brightness) compared to the process without mediator (23% and 61.5% respectively), in addition to insignificant denaturation effect over laccase. The efficiency of the biobleaching process was further related to changes in non-conventionally used optical and chromatic parameters of pulp, such as (L*), chroma (C*) and dye removal index (DRI) showing good correlation. Adverse coupling reactions of the natural phenolic mediators on pulp lignin were predicted by electrochemical studies, demonstrating the complexity of the laccase-mediator reaction on pulp.

Study of the degradation of dyes by MnP of Phanerochaete chrysosporium produced in a fixed-bed bioreactor.

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium in a fixed-bed tubular bioreactor, filled with cubes of nylon sponge, operating in semi-solid-state conditions, was studied. Maximum individual manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1293 and 225 U/l were detected. The in vitro decolourisation of two structurally different dyes (Poly R-478, crystal violet) by the extracellular liquid obtained in the above-mentioned bioreactor was monitored in order to determine its degrading capability. The concentration of some compounds (sodium malonate, manganese sulphate) from the reaction mixture was optimised in order to maximise the decolourisation levels. A percentage of Poly R-478 decolourisation of 24% after 15 min of dye incubation was achieved. On the other hand, a methodology for a long treatment of these dyes based on the continuous addition of MnP enzyme and H(2)O(2) was developed. Moreover, this enzymatic treatment was compared with a photochemical decolourisation process. The former allowed to maintain the degradation rate almost constant for a long time, resulting in a decolourisation percentage of 70% and 30% for crystal violet and Poly R-478, respectively, after 2 h of treatment. As for the latter, it was not able to degrade Poly R-478, whereas crystal violet reached a degradation of 40% in 2 h.

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.

Investigation of several bioreactor configurations for laccase production by Trametes versicolor operating in solid-state conditions

Abstract In the present paper, the production of laccase by Trametes versicolor (CBS 100.29) in laboratory-scale bioreactors, operating in semi-solid-state conditions, was studied. Three bioreactor configurations were investigated in order to determine the most suitable one for laccase production: immersion, expanded-bed and tray. In addition, the nature of support employed (inert or non-inert) on laccase production was also evaluated. According to the results attained in the previous work by our research group, nylon sponge and barley bran was employed as an inert and as a lignocellulosic support, respectively. Higher laccase activities were produced operating with barley bran than with nylon sponge as a support in all the configurations tested. As regards bioreactor design, the tray configuration led to the highest laccase activities especially operating with barley bran as a support, where activities of about 10-fold higher than that found in the corresponding cultivation with nylon sponge were attained. Therefore, it could be asserted that the tray bioreactor is a very appropriate bioreactor configuration to produce laccase by T. versicolor in solid-state conditions operating with lignocellulosic supports.

Laccase-HBT bleaching of eucalyptus kraft pulp: influence of the operating conditions.

Different operating conditions (viz. pulp consistency, oxygen pressure and treatment time) in the biobleaching of eucalyptus kraft pulp with the laccase-HBT system was tested in order to describe their effect and normalize a biobleaching protocol. A high O(2) pressure (0.6MPa) was found to result in improved laccase-assisted delignification of the pulp. Also, a high pulp consistency (10%) and a short treatment time (2h) proved the best choices with a view to obtaining good pulp properties (kappa number and ISO brightness) under essentially mild conditions. The laccase-HBT treatment was found to result in slight delignification (in the form of a 20-27% decrease in kappa number); however, an alkaline extraction stage raised delignification to 41-45%, a much higher level than those obtained in the control tests (16-23%). Also, the use of hydrogen peroxide in the extraction stage resulted in improved brightness (14-19%), but in scarcely improved delignification (4-7%). Treating the pulp with the laccase-HBT system reduced the amount of hydrogen peroxide required for subsequent alkaline bleaching by a factor of 3-4 relative to control tests.

Biobleaching of eucalypt kraft pulp with a two laccase-mediator stages sequence

A new biobleaching sequence, with two enzymatic stages based on the application of laccase-mediator systems, was tested (L(1)EL(2)QPo) in order to increase the effectiveness of enzyme delignification on eucalypt kraft pulp. Different synthetic -1-hydroxybenzotriazole (HBT) and violuric acid (VA) - and natural - syringaldehyde (SyAl) - mediators were used in the laccase stages and the biobleached pulp were compared in terms of chemical, optical and physico-mechanical properties. The pulp bleached with HBT or VA showed similar delignification (64.1% and 65.9% respectively) and optical properties (86.4% and 86.1% ISO brightness respectively) than an industrial TCF pulp (68.3% delignification and 84.8% ISO brightness). SyAl improved these properties in a lower extent (56.71% delignification and 80.52% ISO brightness). Regarding physico-mechanical properties of pulp, the biobleaching sequence had no a negative effect, even some slight improvements were observed in very specific cases.

Enzymatic polymerisation and effect of fractionation of dissolved lignin from Eucalyptus globulus Kraft liquor

The potential ability of the laccase from Myceliophthora thermophila, either alone or with low molecular weight (LMW) additives, to polymerise a dissolved lignin from Kraft liquor of eucalypt cooking was investigated. A previous study of enzymatic performance (activity and stability) was carried out using a design experiment methodology. In addition, Kraft dissolved lignin (KDL) was fractionated according to two different protocols (solvent extraction and acidic fractionation) in order to identify possible lignin fractions with noticeable polymerisation ability. KDL and its corresponding lignin fractions were treated with laccase and analysed by size exclusion chromatography and Fourier transform infrared spectroscopy. The results provide conclusive evidence of notable lignin modifications after incubation with laccase. Moreover, lignin fractionation allows to obtain lignin fractions with different chemical characteristics and polymerisation capability. Depending on the type of raw lignin, molecular weight can increase from 4- to 21-fold by means of laccase polymerisation.