Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis

A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t_1/2 at pH 10 = 30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein.     

Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis

A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t_1/2 at pH 10 = 30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein.     

Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation

Aims: To select better performing laccase variants among the 2300 randomly mutated variants of Pleurotus ostreatus POXA1b laccase to develop improved laccase‐based biocatalysts. Methods and Results: Screening of collections of 2300 randomly mutated variants of POXA1b was performed by assaying activity towards the phenolic substrate 2,6‐dimethoxyphenol. Two new variants endowed with higher enzyme activity than the wild‐type laccase were characterized, and their ability to decolourize industrial dyes with complex trisazo‐, polyazo‐ and stilbene‐type structures, in the absence of mediators, was demonstrated. One of the mutants (2L4A) was also proved to be highly stable at both acidic and alkaline pH values (displaying a half‐life of around 1 month at the pH levels of both 5 and 10). Conclusions: In comparison with the wild‐type laccase, the new selected 2L4A mutant shows a significant increase in stability at acidic pH, whilst storing its high stability at alkaline pH. This variant also represents a more versatile enzyme with respect to both the variety of xenobiotics degraded and the operative conditions. Significance and Impact of the Study: This work represents the first example of improvement of a basidiomycete laccase for industrial effluents bioremediation by directed evolution.     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.     

Expression,characterization and 2,4,6-trichlorophenol degradation of laccase from <Emphasis Type="Italic">Monilinia fructigena</Emphasis>

A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V _max values are 243.9 and 10.55 Um min⁻¹ mg⁻¹, respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Characterization of the Alkaline Laccase Ssl1 from Streptomyces sviceus with Unusual Properties Discovered by Genome Mining

Fungal laccases are well investigated enzymes with high potential in diverse applications like bleaching of waste waters and textiles, cellulose delignification, and organic synthesis. However, they are limited to acidic reaction conditions and require eukaryotic expression systems. This raises a demand for novel laccases without these constraints. We have taken advantage of the laccase engineering database LccED derived from genome mining to identify and clone the laccase Ssl1 from Streptomyces sviceus which can circumvent the limitations of fungal laccases. Ssl1 belongs to the family of small laccases that contains only few characterized enzymes. After removal of the twin-arginine signal peptide Ssl1 was readily expressed in E. coli. Ssl1 is a small laccase with 32.5 kDa, consists of only two cupredoxin-like domains, and forms trimers in solution. Ssl1 oxidizes 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and phenolic substrates like 2,6-dimethoxy phenol, guaiacol, and syringaldazine. The k_cat value for ABTS oxidation was at least 20 times higher than for other substrates. The optimal pH for oxidation reactions is substrate dependent: for phenolic substrates the highest activities were detected at alkaline conditions (pH 9.0 for 2,6-dimethoxy phenol and guaiacol and pH 8.0 for syringaldazine), while the highest reaction rates with ABTS were observed at pH 4.0. Though originating from a mesophilic organism, Ssl demonstrates remarkable stability at elevated temperatures (T_1/2,60°C = 88 min) and in a wide pH range (pH 5.0 to 11.0). Notably, the enzyme retained 80% residual activity after 5 days of incubation at pH 11. Detergents and organic co-solvents do not affect Ssl1 stability. The described robustness makes Ssl1 a potential candidate for industrial applications, preferably in processes that require alkaline reaction conditions.     

Laboratory evolution of laccase for substrate specificity

A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity, and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild-type CotA oxidizes ABTS (ABTS = diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ = 4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS. Based on the ABTS-bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half-life for the heat inactivation (t_1/2) at 80 °C was increased by 62 min for the mutant. Laccases have several applications in biotechnology, which include pulp bleaching, biosensors, bioremediation, and biofuel cells. The substrate specificity of CotA is moldable and the enzyme can be tailored to oxidize a variety of target molecules for specific purposes.     

Kinetic studies of laccase enzyme of Coriolus versicolor MTCC 138 in an inexpensive culture medium

An attempt was made to use cyanobacterial biomass of water bloom, groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme production by Coriolus versicolor. Laccase production was found to be 10.15 ± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial pH 5.0 and 28 ± 2 °C temperature. Half life of enzyme was found to be 74 min at 60 °C. Kinetic analysis of laccase when made with substrate ABTS, K_m and V_max were found to be 0.29 mM and 9.49 μmol/min respectively. Azide and hydroxylamine were found to exert significant inhibition on thermostable laccase. Inhibitor constant (k_i) for azide and hydroxylamine were 1.33 and 0.18 mM respectively. This study forms the first report on the potential application of waste water cyanobacterial bloom and dyeing effluent as a medium for laccase production by C. versicolor MTCC138.     

Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds

The extracellular laccase produced by the ascomycete Trichoderma atroviride was purified and characterized and its ability to transform phenolic compounds was determined. The purified laccase had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 °C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 °C. Under standard assay conditions, K_m values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator.     

Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 °C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (k_cat/K_m = 18.5 s^?1 μM^?1) and 2,6-dimethoxyphenol (k_cat/K_m = 13.9 s^?1 μM^?1) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield.     

Biochemical characterization of laccase from hairy root culture of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. and role of redox mediators to enhance its potential for the decolorization of textile dyes

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg⁻¹ of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg⁻¹ specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.     

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 (t_1/2) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t_1/2 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.

     

Purification and characterization of a thermostable laccase with unique oxidative characteristics from <Emphasis Type="Italic">Trametes hirsuta</Emphasis>

A laccase was purified from Trametes hirsuta. This laccase was classified as a “white” or “yellow” laccase. pH 2.4 was optimal for the oxidation of ABTS and pH 2.5 for DMP. DMP oxidation was optimal at 85°C. The half-life of this laccase was 70 min at 75°C, and 5 h at 65°C. Non-phenolic dyes, such as Methyl Red, were oxidized by purified laccase without mediators. The enzyme was not inhibited by Cu²⁺, Mn²⁺, or EDTA. These are atypical laccase characteristics that make it a good candidate for theoretical and applied research.     

Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization

Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K _M and k _cat were calculated 535 μM and 127 s⁻¹ for ABTS, 53 μM and 3 s⁻¹ for 2, 6-DMP and 5 μM and 20 s⁻¹ for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80°C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70°C and 2.4-fold after 10 min preincubation at 80°C. Preincubation of the enzyme in 70°C for 30 min raised the activity four-fold with ABTS as the substrate. Also, l-dopa was used as a substrate. The enzyme was able to oxidize l-dopa with the K _M and k _cat of 1,493 μM and 194 s⁻¹, respectively.     

Secretory expression and characterization of a soluble laccase from the <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> strain 7071-9 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

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 58 kDa. The K _m values of GlLCCI for 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM, respectively. The V _max of GlLCCI for both substrates was 3,024 and 82.13 μM mg⁻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⁺, Cu²⁺ 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 Fe²⁺, Fe³⁺, sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l⁻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.     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Isolation and Study of Some Properties of Laccase from the Basidiomycetes Cerrena maxima

A new strain producing extracellular laccase (Cerrena maxima 0275) was found by screening of isolates of Basidiomycetes, and the dynamics of laccase biosynthesis by this strain was studied. The enzyme was purified to homogeneity. The molecular weight of the enzyme is 57 kD, and its pI is 3.5. The activity is constant at pH values in the range 3.0-5.0. The temperature optimum for activity is 50°C. The thermal stability of the laccase was studied. The catalytic and Michaelis constants for catechol, hydroquinone, sinapinic acid, and K₄ Fe(CN)₆ were determined. The standard redox potential of type 1 copper in the enzyme is 750 ± 5 mV. Thus, the investigated laccase is a high redox potential laccase.     

Catalytic behavior and detoxifying ability of a laccase from the fungal strain Cerrena unicolor

The kinetics and stability of a laccase isolated and purified from the fungal strain Cerrena unicolor were studied. The enzyme was produced in a great yield without inducers. Kinetic parameters were determined by using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate. At high ABTS concentrations (> 10 mM) a substrate inhibition phenomenon appeared and an inhibition constant K_i of 24 mM was determined. The pH- and temperature-profiles as well as the sensitivity of the enzyme to several deactivation agents were almost similar to those observed with laccase from different origins. Freezing-thawing treatment, high temperature, acidic pH (< 3.0) and acetonitrile strongly affected laccase activity. The laccase showed a good ability to oxidize different phenolic substances; a significant enhancing effect was showed by ABTS acting as co-substrate. These results seem to suggest that this new laccase preparation may be suitable for environmental purposes.