Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal.

BACKGROUND: Azo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes. METHODS: Trichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett-Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange. RESULTS: Eight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 µmol guaiacol and 3.82 µmol guaiacol/min. Its activity was stable in the pH range of 5-7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%. CONCLUSION: Trichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.

Isolation and Characterization of a Novel Laccase for Lignin Degradation, LacZ1.

Lignin is a complex natural organic polymer and is one of the primary components of lignocellulose. The efficient utilization of lignocellulose is limited because it is difficult to degrade lignin. In this study, we screened a lacz1 gene fragment encoding laccase from the macrotranscriptome data of a microbial consortium WSC-6, which can efficiently degrade lignocellulose. The reverse transcription-quantitative PCR (RT-qPCR) results demonstrated that the expression level of the lacz1 gene during the peak period of lignocellulose degradation by WSC-6 increased by 30.63 times compared to the initial degradation period. Phylogenetic tree analysis demonstrated that the complete lacz1 gene is derived from a Bacillus sp. and encoded laccase. The corresponding protein, LacZ1, was expressed and purified by Ni-chelating affinity chromatography. The optimum temperature was 75°C, the optimum pH was 4.5, and the highest enzyme activity reached 16.39 U/mg. We found that Cu2+ was an important cofactor needed for LacZ1 to have enzyme activity. The molecular weight distribution of lignin was determined by gel permeation chromatography (GPC), and changes in the lignin structure were determined by 1H nuclear magnetic resonance (1H NMR) spectra. The degradation products of lignin by LacZ1 were determined by gas chromatography and mass spectrometry (GC-MS), and three lignin degradation pathways (the gentian acid pathway, benzoic acid pathway, and protocatechuic acid pathway) were proposed. This study provides insight into the degradation of lignin and new insights into high-temperature bacterial laccase. IMPORTANCE Lignin is a natural aromatic polymer that is not easily degraded, hindering the efficient use of lignocellulose-rich biomass resources, such as straw. Biodegradation is a method of decomposing lignin that has recently received increasing attention. In this study, we screened a gene encoding laccase from the lignocellulose-degrading microbial consortium WSC-6, purified the corresponding protein LacZ1, characterized the enzymatic properties of laccase LacZ1, and speculated that the degradation pathway of LacZ1 degrades lignin. This study identified a new, high-temperature bacterial laccase that can degrade lignin, providing insight into lignin degradation by this laccase.

Extracellular laccase from Monilinia fructicola: isolation, primary characterization and application.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.

Purification and properties of a laccase from the mushroom Agaricus sinodeliciosus.

A homogeneous monomeric laccase (ASL) from Agaricus sinodeliciosus, with a molecular mass of 65 kDa, was isolated using ion-exchange chromatography (CM-cellulose and Q-Sepharose) and gel-filtration chromatography (Superdex 75). This laccase exhibited maximum activity at 50 °C and pH 5.0. Hg2+ and Cd2+ significantly inhibited its activity. The laccase displayed a Km value of 0.9 mM toward 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS). In addition to ABTS, ASL exhibited higher affinity toward o-toluidine and benzidine than other substrates. ASL is able to decolorize malachite green and Eriochrome black T.

Laccase and Tyrosinase Biosensors Used in the Determination of Hydroxycinnamic Acids.

In recent years, researchers have focused on developing simple and efficient methods based on electrochemical biosensors to determine hydroxycinnamic acids from various real samples (wine, beer, propolis, tea, and coffee). Enzymatic biosensors represent a promising, low-cost technology for the direct monitoring of these biologically important compounds, which implies a fast response and simple sample processing procedures. The present review aims at highlighting the structural features of this class of compounds and the importance of hydroxycinnamic acids for the human body, as well as presenting a series of enzymatic biosensors commonly used to quantify these phenolic compounds. Enzyme immobilization techniques on support electrodes are very important for their stability and for obtaining adequate results. The following sections of this review will briefly describe some of the laccase (Lac) and tyrosinase (Tyr) biosensors used for determining the main hydroxycinnamic acids of interest in the food or cosmetics industry. Considering relevant studies in the field, the fact has been noticed that there is a greater number of studies on laccase-based biosensors as compared to those based on tyrosinase for the detection of hydroxycinnamic acids. Significant progress has been made in relation to using the synergy of nanomaterials and nanocomposites for more stable and efficient enzyme immobilization. These nanomaterials are mainly carbon- and/or polymer-based nanostructures and metallic nanoparticles which provide a suitable environment for maintaining the biocatalytic activity of the enzyme and for increasing the rate of electron transport.

Partial purification and characterization of a thermophilic and alkali-stable laccase of Phoma herbarum isolate KU4 with dye-decolorization efficiency.

Production of an extracellular thermophilic and alkali stable laccase from Phoma herbarum isolate KU4 was reported for the first time, both in submerged fermentation (SmF, highest 1590 U/mL) and solid state fermentation (SSF, highest 2014.21 U/mL) using agro-industrial residues. The laccase was partially purified to 7.93 fold with the apparent molecular weight of 298 kDa. The enzyme had pH optimum at 5.0 and temperature optimum at 50 °C, with maximum stability at pH 8.0. It showed activity towards various phenolic and non-phenolic compounds. The kinetic parameters, Km, Vmax and Kcat of the laccase for DMP were 0.216 mM, 270.27 U/mg and 506.69 s-1, respectively. Laccase activity was inhibited by various metal ions and conventional inhibitors, however, it was slightly increased by Zn2+. The laccase showed good decolorization efficiency towards four industrial dyes, namely, methyl violet (75.66%), methyl green (65%), indigo carmine (58%) and neutral red (42%) within 24 h. FTIR analysis of the decolorized products confirmed the degradation of the dyes. The decolorization efficiency of the enzyme suggests that the partially purified laccase could be used to decolorize synthetic dyes present in industrial effluents and for waste water treatments. The thermophilic and alkali stable laccase may also have wider potential industrial applications.

Isolation, identification and optimization of enhanced production of laccase from Galactomyces geotrichum under solid-state fermentation.

Laccases are a group of oxidases that catalyze the oxidation of a wide range of electron rich substrates like phenolic compounds, lignin and aromatic amines. They are of interest because of their potential to be used in environmental and industrial applications. In this research, potent laccase producer fungi were screened and isolated from olive mill wastewater (OMW). One of the 23 isolated fungi was identified as Galactomyces geotrichum based on 18S rDNA sequence analysis that detected good laccase activity. Produced laccase had a molecular weight of 55 kDa that was confirmed by zymogram analysis. This is the first report about the optimization of laccase Production by G. geotrichum under solid-state fermentation. The optimization was made by the Taguchi design of experiments (DOE) methodology. An orthogonal array (L25) was designed using Minitab 19 software to study four effective process factors in five levels for laccase production. The optimum condition derived was; moisture content (80%), fermentation time (14 day), CuSO4⋅5H2O as the inducer (300 µM), glucose as a co-substrate (5 g/L). Maximum laccase activity of 52.86 (U/g of dry substrate) was obtained using optimum fermentation condition. This study aimed to better understand the laccase producing microorganisms in OMW and take them to OMW treatment that is rich in phenolic compounds.

Refolding, characterization, and dye decolorization ability of a highly thermostable laccase from Geobacillus sp. JS12.

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (kcat/Km) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co2+ and Mn2+, while activity was strongly inhibited in the presence of Fe2+, Zn2+, and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency.

Purification, characterization, and gene cloning of two laccase isoenzymes (Lac1 and Lac2) from Trametes hirsuta MX2 and their potential in dye decolorization.

In this study, two laccase isoenzymes (Lac1 and Lac2) from the culture supernatant of Trametes hirsuta MX2 were purified, and the genes (Lac1 and Lac2) coding the isoenzymes were cloned. Both Lac1 and Lac2 contained an open reading frame of 1563 bp with an identity of 79%. The two isoenzymes showed significant biochemical differences. The maximal activities of Lac1 and Lac2 were at pH 2.5 with 2-2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and the optimal temperatures for the activities of Lac1 and Lac2 were 60 and 50 °C, respectively. Lac1 exhibited excellent resistance to acidic conditions and retained 62.17% of its initial activity at pH 2.5 after a 72-h incubation. Lac2 was more thermostable than Lac1 with half-lives (t1/2) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t1/2 of Lac1 were only 4.19 and 0.88 h, respectively. Both Lac1 and Lac2 isoenzymes have a strong tolerance to Mg2+, Mn2+, Cu2+, and EDTA (50 mM). At a low concentration of 0.05 U mL-1, the enzymes could decolorize towards Remazol Brilliant Blue R, Acid Red 1, Crystal Violet, and Neutral Red in the presence of ABTS. These unusual properties demonstrated that the two laccases have strong potential for specific industrial applications.

Blue Copper Peroxidase and Phthalocyanine Conjugate: Synthesis, Characterization, and Applications.

Trametes versicolor can degrade bark as a source for carbon necessity. Therefore, it secretes lignin peroxidase, mangan peroxidase, and laccase. The laccase enzyme was produced in high yield at pH of 5 and glucose concentration of 10 g L-1. In optimized medium, the enzyme activity was between 200 and 250 U L-1 when an inducer was absent. It was seen that the activity reached 400 U L-1 when phenol was used as an inducer. The molecular weight of purified laccase was found to be 80 kDa with SDS-PAGE, and kinetic constant Km and Vmax values for 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonate were determined to be 3.66 × 10-4 µM and 1652 U L-1, respectively. Because of these properties, these enzymes are widely used, free or immobilized, in industrial areas. Laccase enzyme decolorization of six different dyes was carried out. A decolorization capacity of 50-99% was achieved by cultivation for 20 days using a beginning dye concentration of 20 ppm. The removal of color with an active enzyme was obtained around 90%. Also, the laccase enzyme was conjugated, amine-functionalized, low-symmetry phthalocyanine. This conjugate was examined by both photodynamic therapy and chemosensor application. This conjugate fluorescence had a quantum yield of 0.32 (lifetime 3.59 ns) and efficiently generated singlet oxygen (quantum yield 0.4). The conjugate successfully displayed photodamage in HeLa and HuH-7 cells in the photodynamic therapy application. These results indicate that the conjugate represents an interesting agent with potential applications in photodynamic therapy. In addition, the chemosensor behavior of this compound to different metal ions has been studied, and this conjugate is displayed as a fluorescence chemosensor for the determination of Fe3+ions.

Comparison of downstream processing methods in purification of highly active laccase.

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae.

Laccase is used in various industrial fields, and it has been the subject of numerous studies. Trametes versicolor laccase has one of the highest redox potentials among the various forms of this enzyme. In this study, we optimized the expression of laccase in Saccharomyces cerevisiae. Optimizing the culture conditions resulted in an improvement in the expression level, and approximately 45 U/L of laccase was functionally secreted in the culture. The recombinant laccase was found to be a heavily hypermannosylated glycoprotein, and the molecular weight of the carbohydrate chain was approximately 60 kDa. These hypermannosylated glycans lowered the substrate affinity, but the optimum pH and thermo-stability were not changed by these hypermannosylated glycans. This functional expression system described here will aid in molecular evolutionary studies conducted to generate new variants of laccase.

Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris.

AIMS: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. METHODS AND RESULTS: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat /Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. CONCLUSIONS: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens.

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75 °C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.

Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli.

Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET-30a expression plasmid. The recombinant laccase was purified and visualized on SDS-PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg-1 , the optimum temperature and pH value were 60 °C and 4.0 respectively, measured by oxidation of 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Purified laccase activity under different metal ions and an inhibitor were tested, revealing that laccase activity increased by approximately 434.8% with Fe3+ , and 217.4% with Cu2+ at 10 mmol · L-1 concentrations, Mn2+ increased the laccase activity only at 5 mmol · L-1 , while Na+ increased activity at 1 mmol · L-1 but inhibited activity at 5 and 10 mmol · L-1 . SDS increased laccase activity at 1 mmol · L-1 , and inhibited activity at 5 and 10 mmol · L-1 .

Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments.

BACKGROUND: Fungal laccase has profound applications in different fields of biotechnology due to its broad specificity and high redox potential. Any successful application of the enzyme requires large scale production. As laccase production is highly dependent on medium components and cultural conditions, optimization of the same is essential for efficient product production. RESULTS: Production of laccase by fungal strain Marasmiellus palmivorus LA1 under solid state fermentation was optimized by the Taguchi design of experiments (DOE) methodology. An orthogonal array (L8) was designed using Qualitek-4 software to study the interactions and relative influence of the seven selected factors by one factor at a time approach. The optimum condition formulated was temperature (28 °C), pH (5), galactose (0.8%w/v), cupric sulphate (3 mM), inoculum concentration (number of mycelial agar pieces) (6Nos.) and substrate length (0.05 m). Overall yield increase of 17.6 fold was obtained after optimization. Statistical optimization leads to the elimination of an insignificant medium component ammonium dihydrogen phosphate from the process and contributes to a 1.06 fold increase in enzyme production. A final production of 667.4 ± 13 IU/mL laccase activity paves way for the application of this strain for industrial applications. CONCLUSION: Study optimized lignin degrading laccases from Marasmiellus palmivorus LA1. This laccases can thus be used for further applications in different scales of production after analyzing the properties of the enzyme. Study also confirmed the use of taguchi method for optimizations of product production.

Variants of PpuLcc, a multi-dye decolorizing laccase from Pleurotus pulmonarius expressed in Pichia pastoris.

A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (-2, -5, -13 AA) of the target protein were combined with different secretion signals. Heterologous expression of codon optimized constructs resulted in extracellular activities of the PpuLcc variants of up to 7000 U L-1 (substrate ABTS) which was six times higher than non-codon optimized constructs. In contrast to previous works, altering the C-terminal end of the protein did not influence kinetic parameters or the rate of expression. The His-Tag purified enzymes showed high temperature optima (50-70 °C) and thermo stability. All of the recombinant variants degraded triarylmethane and azo dyes. Rapid bleaching of ß-carotene (E 160a) and the polyene acid norbixin (E 160b) using a laccase was found for the first time. Thus, the enzyme may be useful in decolorizing unwanted polyene pigments, for example from the processing of cheese, bakery, desserts, ice cream or coloured casings.

Biosynthesis of nanoparticles of metals and metalloids by basidiomycetes. Preparation of gold nanoparticles by using purified fungal phenol oxidases.

The work shows the ability of cultured Basidiomycetes of different taxonomic groups-Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum, and Grifola frondosa-to recover gold, silver, selenium, and silicon, to elemental state with nanoparticles formation. It examines the effect of these metal and metalloid compounds on the parameters of growth and accumulation of biomass; the optimal cultivation conditions and concentrations of the studied ion-containing compounds for recovery of nanoparticles have been identified. Using the techniques of transmission electron microscopy, dynamic light scattering, X-ray fluorescence and X-ray phase analysis, the degrees of oxidation of the bioreduced elements, the ζ-potential of colloidal solutions uniformity, size, shape, and location of the nanoparticles in the culture fluid, as well as on the surface and the inside of filamentous hyphae have been determined. The study has found the part played by homogeneous chromatographically pure fungal phenol-oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) in the recovery mechanism with formation of electrostatically stabilized colloidal solutions. A hypothetical mechanism of gold(III) reduction from HAuCl4 to gold(0) by phenol oxidases with gold nanoparticles formation of different shapes and sizes has been introduced.

A simple strategy for extracellular production of CotA laccase in Escherichia coli and decolorization of simulated textile effluent by recombinant laccase.

Laccases are green oxidases with a number of potential industrial applications. In this study, recombinant Bacillus subtilis CotA laccase was secreted by Escherichia coli via both the α-hemolysin secretion system and the YebF secretion system after microaerobic induction. Meanwhile, we discovered a much simpler approach for extracellular production of recombinant CotA laccase from E. coli, involving alternation of induction conditions to release recombinant CotA following intracellular expression. By optimizing the induction parameters, the extracellular yield of recombinant CotA laccase was improved from 157.4 to 2401.3 U/L after 24 h of induction. This strategy could be suitable for large-scale production of CotA laccase for industrial use. Recombinant CotA laccase was purified by Ni2+ affinity chromatography in a single step and showed similar biochemical properties to wild-type laccase. Purified as well as crude recombinant CotA laccase efficiently decolorized seven structurally different dyes. The decolorization capability of recombinant CotA laccase under harsh conditions was investigated by incubation of the enzyme with a simulated textile effluent (STE) with pH 11.6, 3.5 % salinity and peak absorbance of 10.42. Recombinant CotA laccase efficiently decolorized 77.0 % of STE after 48 h reaction, demonstrating the potential of this enzyme for industrial dye effluent treatment.

Induction, purification, and characterization of a thermo and pH stable laccase from Abortiporus biennis J2 and its application on the clarification of litchi juice.

A fungus J2 producing laccase with high yield was screened in soils and identified as Abortiporus biennis. The production of laccase was induced by 0.1 mM Cu2+, 0.1 mM tannic acid, and 0.5 M ethanol. The laccase from Abortiporus biennis J2 was purified to electrophoretic homogeneity by a couple of steps. The N-terminal amino acid sequence of the enzyme was AIGPTADLNISNADI. The properties of the purified laccase were investigated. The result showed the laccase from Abortiporus biennis J2 is a thermo and pH stable enzyme. The laccase activity was inhibited by Hg2+, Cd2+, Fe2+, Ag+, Cu2+, and Zn2+, while promoted by Mg2+, Mn2+ at 10 mM level. Purified laccase was used to the clarification of litchi juice. After treatment with this laccase, the phenolic content of litchi juice had been found to be greatly reduced along with an increase in the clarity of the juice. The result indicated the potential of this laccase for application in juice procession.