Soluble Laccase Research Articles

Immobilization of laccase on Sepharose-linked antibody support for decolourization of phenol red

Laccases which are considered as “green tools” in biotechnology have potential to degrade toxic contaminants/synthetic dyes present in industrial effluents. The loss in activity and stability of laccases are key challenges faced in their potential industrial applications. Here, laccase from Trametes versicolor (polypore mushroom) was immobilized on Sepharose-linked antibody support to carry out the decolourization of phenol red. This support was prepared by covalent linking of anti-laccase antibodies to CNBr activated Sepharose at pH 8.5, and then laccase was immobilized on this affinity support at pH 5.0. The amount of laccase immobilized was approximately 33 mg per gram of the affinity support, giving an immobilization yield of 83.4%. The immobilized enzyme displayed an activity of 3.88 U with an effectiveness factor (η) of 0.90. Immobilization of laccase led to significant enhancement in thermal and storage stability. The immobilized enzyme retained 44% of its activity after 10 cycles of continuous use. The decolourization of phenol red dye obtained by immobilized and soluble laccase after 6 h of incubation at 50 °C was 80 and 56%, respectively. Thus, immobilization of laccase on Sepharose-linked antibody support leads to remarkable improvement in its various properties, making it more versatile for industrial applications.

Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW).

The enzymatic factory of ligninolytic fungi has proven to be a powerful tool in applications regarding the degradation of various types of pollutants. The degradative potential of fungi is mainly due to the production of different types of oxidases, of which laccases is one of the most prominent enzymatic activities. In the present work, crude laccases from the supernatant of Pleurotus citrinopileatus cultures grown in olive oil mill wastewater (OOMW) were immobilized in crosslinked enzyme aggregates (CLEAs), aiming at the development of biocatalysts suitable for the enzymatic treatment of OOMW. The preparation of laccase CLEAs was optimized, resulting in a maximum of 72% residual activity. The resulting CLEAs were shown to be more stable in the presence of solvents and at elevated temperatures compared to the soluble laccase preparation. The removal of the phenolic component of OOMW catalyzed by laccase-CLEAs exceeded 35%, while they were found to retain their activity for at least three cycles of repetitive use. The described CLEAs can be applied for the pretreatment of OOMW, prior to its use for valorization processes, and thus, facilitate its complete biodegradation towards a consolidated process in the context of circular economy.

Delignification of Lignocellulose Biomasses by Alginate–Chitosan Immobilized Laccase Produced from Trametes versicolor IBL-04

An extracellular laccase was produced from a lignin-degrading white-rot fungus Trametes versicolor IBL-04 using the solid-state bioprocessing of corncobs. The enzyme thus produced was effectively immobilized on alginate–chitosan beads. Immobilized laccase furnished the highest immobilization yield (81.43 ± 2.98) under optimized working conditions of 3.0% (w/v) sodium alginate concentration, 2.4% CaCl2, 0.25% chitosan concentration following an immobilization time of 5 h. The immobilization process altered the optimum pH from 5.0 to 4.0 (an acidic shift), whereas the temperature optima were displaced from 40 to 50 °C (towards higher temperature). The Michaelis–Menten constant (K m) and the maximum reaction rate (V max) of the free and immobilized laccase were 0.14 mM, and 780 µmol/mL and 0.10 mM and 814 µmol/mL, respectively. The alginate–chitosan immobilized laccase was more stable and tolerant against the activity inhibition caused by the denaturants tested, concerning its soluble laccase. Delignification results revealed that the enzyme led to a marked reduction in lignin content of all the lignocellulosic wastes with the highest delignification of 78.29% in sugarcane bagasse, followed by wheat bran and corn stover after 15 h. It is demonstrated that the immobilized laccase could be a promising option as a green catalyst for the efficient delignification of lignocellulosic biomass residues.

Continuous decomposition of Acid Blue 74 in a membrane reactor with soluble laccase

Continuous decomposition of Acid Blue 74 in a membrane reactor with soluble laccase

Bioinspired Multifunctional Membrane for Aquatic Micropollutants Removal.

Micropollutants present in water have many detrimental effects on the ecosystem. Membrane technology plays an important role in the removal of micropollutants, but there remain significant challenges such as concentration polarization, membrane fouling, and variable permeate quality. The work reported here uses a multifunctional membrane with rejection, adsorption, and catalysis functions to solve these problems. On the basis of mussel-inspired chemistry and biological membrane properties, a multifunctional membrane was prepared by applying "reverse filtration" of a laccase solution and subsequent "dopamine coating" on a nanofiltration (NF) membrane support, which was tested on bisphenol A (BPA) removal. Three NF membranes were chosen for the preparation of the multifunctional membranes on the basis of the membrane properties and enzyme immobilization efficiency. Compared with the pristine membrane, the multifunctional membrane exhibited significant improvement of BPA removal (78.21 ± 1.95%, 84.27 ± 7.30%, and 97.04 ± 0.33% for NT103, NF270, and NF90, respectively), all of which are clearly superior to the conventional Fenton treatment (55.0%) under similar conditions and comparable to soluble laccase coupled with NF270 membrane filtration (89.0%). The improvement would appear to be due to a combination of separation (reducing the enzymatic burden), adsorption (enriching the substrate concentration as well as prolonging the residence time), and lastly, catalysis (oxidizing the pollutants and breaking the "adsorption saturation limits"). Furthermore, the synergistic effect of the polydopamine (PDA) layer on the enzymatic oxidation of BPA was confirmed, which was due to its enhanced adsorption and electron transfer performance. The multifunctional membrane could be reused for at least seven cycles with an acceptable activity loss, demonstrating good potential for removal of micropollutants.

A strategy for soluble overexpression and biochemical characterization of halo-thermotolerant Bacillus laccase in modified E. coli

An efficient method was introduced for soluble expression of recombinant laccase (rpCotA(SL-1)) from a newly isolated halo-thermotolerant Bacillus sp. SL-1 in modified Escherichia coli, trxB2/gor2 mutant (Origami™ B (DE3)). The yield of purified soluble laccase in Origami strain under micro-aerobic condition was ∼20mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46μM and 23s−1 for ABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6μM and 24s−1 for SGZ (syringaldazine) substrates, respectively. The rpCotA(SL-1) displayed thermostability at 70°C and tolerance to specified concentrations of NaCl, NaN3, EDTA and SDS as inhibitors. The enzyme was relatively stable in the presence of different concentration of organic solvents, however the residual activity was adversely affected as the dipole moment of the solvents increase. Here we successfully report the production of soluble and functional laccase in Origami at the expression level suitable for industrial application.

Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi's L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO4 of 0.5 mM to the medium, and shaking off while cell mass reached to OD(600nm) of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2'-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.

Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris

Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58kDa. The K (m) values of GlLCCI for 2-2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122mM, respectively. The V (max) of GlLCCI for both substrates was 3,024 and 82.13μMmg(-1)min(-1). When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected over pH values from 2 to 8. The enzyme was strongly activated by K(+), Na(+), Cu(2+) and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe(2+), Fe(3+), sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62mgl(-1) of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment of industrial effluent containing azo dye MO.

Non-isothermal bioremediation of waters polluted by phenol and some of its derivatives by laccase covalently immobilized on polypropylene membranes

In view of the heath problems induced by the presence into the environment of endocrine disruptors, laccase from Trametes versicolor was covalently immobilized on a chemically modified polypropylene membrane in order to remove phenol and its derivatives from polluted waters. Using phenol as substrate model the optimal immobilization conditions were determined. The immobilized laccase exhibited maximal enzyme activity at pH 5.5 and optimal temperature at 55 °C. These operative parameters have been compared with those obtained with the soluble laccase in order to ascertain the immobilization effect. When employed in a bioreactor operating under isothermal conditions the immobilized laccase was able to oxidize a wide range of phenolic substrates. In particular it was found that some phenol derivatives (2-CP, 3-CP, 4-CP, NP and chlorophene) were oxidized at a similar rate than phenol, other derivatives (paracetamol, 3-MP and chloroxyphenol) at a smaller rate, while others (2,4-DCP and BPA) at higher rate. When the catalytic membrane was employed in a non-isothermal reactor the reaction rate increased with the increase of the applied temperature difference. Practically the increase of the laccase oxidative power under the non-isothermal conditions followed the same sequence observed under isothermal conditions. Interesting enough, the percentage increase of enzyme reaction rate under non-isothermal conditions resulted higher in the cases in which the isothermal reaction rate was smaller. When the reduction of the production times by the presence of a temperature gradient is considered, the measured values strongly candidate the technology of non-isothermal bioreactors as a useful tool in processes of detoxification of waste waters polluted by endocrine disruptors of phenolic origin.

Biofuel Cell Controlled by Enzyme Logic Systems

An enzyme-based biofuel cell with a pH-switchable oxygen electrode, controlled by enzyme logic operations processing in situ biochemical input signals, has been developed. Two Boolean logic gates (AND/OR) were assembled from enzyme systems to process biochemical signals and to convert them logically into pH-changes of the solution. The cathode used in the biofuel cell was modified with a polymer-brush functionalized with Os-complex redox species operating as relay units to mediate electron transport between the conductive support and soluble laccase biocatalyzing oxygen reduction. The electrochemical activity of the modified electrode was switchable by alteration of the solution pH value. The electrode was electrochemically mute at pH > 5.5, and it was activated for the bioelectrocatalytic oxygen reduction at pH < 4.5. The sharp transition between the inactive and active states was used to control the electrode activity by external enzymatic systems operating as logic switches in the system. The enzyme logic systems were decreasing the pH value upon appropriate combinations of the biochemical signals corresponding to the AND/OR Boolean logic. Then the pH-switchable electrode was activated for the oxygen reduction, and the entire biofuel cell was switched ON. The biofuel cell was also switched OFF by another biochemical signal which resets the pH value to the original neutral value. The present biofuel cell is the first prototype of a future implantable biofuel cell controlled by complex biochemical reactions to deliver power on-demand responding in a logical way to the physiological needs.

Oxygen cathode based on a layer-by-layer self-assembled laccase and osmium redox mediator

Trametes trogii laccase has been studied as biocatalyst for the oxygen electro-reduction in three different systems: (i) soluble laccase was studied in solution; (ii) an enzyme monolayer was tethered to a gold surface by dithiobis N-succinimidyl propionate (DTSP), with a soluble osmium pyridine-bipyridine redox mediator in both cases. The third case (iii) consisted in the sequential immobilization of laccase and the osmium complex derivatized poly(allylamine) self-assembled layer-by-layer (LbL) on mercaptopropane sulfonate modified gold to produce an all integrated and wired enzymatic oxygen cathode. The polycation was the same osmium complex covalently bound to poly-(ally-lamine) backbone (PAH-Os), the polyanion was the enzyme adsorbed from a solution of a suitable pH so that the protein carries a net negative charge. The adsorption of laccase was studied by monitoring the mass uptake with a quartz crystal microbalance and the oxygen reduction electrocatalysis was studied by linear scan voltammetry. While for the three cases, oxygen electrocatalysis mediated by the osmium complex was observed, for tethered laccase direct electron transfer in the absence of redox mediator was also apparent but no electrocatalysis for the oxygen reduction was recorded in the absence of mediator in solution. For the fully integrated LbL self-assembled laccase and redox mediator (case iii) a catalytic reduction of oxygen could be recorded at different oxygen partial pressures and different electrolyte pH. The tolerance of the reaction to methanol and chloride was also investigated.

Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases

Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1-1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].

Covalent immobilization of laccase on activated carbon for phenolic effluent treatment

Laccase was covalently immobilised to activated carbon using four derivatisation methods. The highest bound activity was obtained using diimide coupling of laccase to carboxyl groups on the carbon. The maximum bound activity was reached at 11.5 mg laccase/g carbon. The carbon-immobilised laccase (CIL) was stable at pH values from 4.0 to 9.0. CIL stored at 4°C lost 38± 5% activity in the first 4 days, then a further 22±5% in 126 days. CIL showed increased stability to low pH although the pH optimum was unchanged. The activation energy of CIL was lower than soluble laccase. Oxidation of 2,6-dimethoxyphenol (DMP) by CIL in a packed-bed system was only 30±10% of that in a fluidised bed system. Of the initial activity 10–30% was retained after oxidation of seven batches of DMP. CIL removed colour from two industrial effluents. Colour was removed from pulp mill bleach plant effluent at 115 colour units per enzyme unit per hour and the removal rate increased with increasing effluent concentration.

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml−1) precipitated 1.2 g l−1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.

Immobilization of enzymes on clays and soils

Various enzymes such as glucose oxidase, β- d-glucosidase, acid phosphatase. tyrosinase and laccases were immobilized on clays and soils activated with 3-aminopropyltriethoxysilane and glutaraldehyde. After immobilization the enzymes retained a large amount of their original activities. The retention of laccase activity increased with the increase of clay content in soils, whereas the activity of β- d-glucosidase, tyrosinase and acid phosphatase decreased. The immobilized enzymes showed varying degrees of resistance to proteolysis and storage at high temperatures. After addition to soil suspensions, soluble laccase and glucose oxidase were rapidly inactivated (100 and 85% loss of activity in 15 days for laccase and glucose oxidase, respectively) whereas after immobilization, these enzymes were extraordinarily stable (12 and 25% loss of activity in 15 days for laccase and glucose oxidase, respectively). The possibility of incorporating the stabilized enzymes into soil for the improvement of numerous desired biochemical processes is discussed.