Laccase Isoforms Research Articles

Thermostable laccases produced by a white-rot fungus from Peniophora species

A strain of Peniophora species was shown to produce laccase, which is a new feature for this species. Peniophora secreted several laccase isoforms with p I -values 3.7–4.2 when grown on glucose and soya meal medium. The Peniophora laccases were typical for Basidiomycetes with respect to pH-dependence, specific activity and inhibitors. The interesting exceptional feature of Peniophora laccases was their good thermostability. At 60 °C, the half-life of the isoforms in the culture filtrate was approximately 5 h and at 70 °C for 15 min. Also, the isolated main isoform with p I -value 4.1 and molecular mass 63 kDa was similarly thermostable. The N-terminal amino acid sequence, altogether 22 amino acid residues, of the separated isoform was 50–63% identical with those of the Basidiomycete laccases analyzed recently.

Production of laccase isoforms by Pleurotus pulmonarius in response to presence of phenolic and aromatic compounds

The effect of several phenolic and aromatic monomers structurally-related to lignin on production of laccase by the white rot fungus P. pulmonarius (Fr.) Quélet has been studied. In the absence of an inducer, laccase was maximally produced after depletion of carbon and nitrogen sources. Among 15 phenolic and aromatic compounds tested, ferulic acid and vanillin were the most efficient inducers, increasing the production of laccase activity up to 10 times. A mixture of ferulic acid and vanillin was more efficient to induce the production of laccase than the isolated phenolics. At least three laccase isoforms designated as lcc1, lcc2 and lcc3 were identified by eletrophoretic analysis of P. pulmonarius culture filtrates. The lcc1 and lcc2 isoforms were produced by non-induced cultures, while lcc3 was found only in induced-culture filtrates.

Purification of a laccase from Marasmius quercophilus induced with ferulic acid: reactivity towards natural and xenobiotic aromatic compounds

In this study, we describe the biochemical features and the ability of a new laccase isoform from a Marasmius quercophilus strain collected on evergreen oak litter to transform various aromatic compounds. This laccase, induced with ferulic acid, exhibits interesting enzymatic properties, such as great thermal stability at 30 and 40 °C (24 h), no inhibition with EDTA, Cystein or SDS. These biochemical features are quite different from those observed with the laccase of another M. quercophilus strain collected in a different area of the South of France. Thus, this study shows the degree of variation in the properties of this enzymatic system within the same species. Furthermore we demonstrate that several natural aromatic compounds were transform leading to polymerization. The transformation of chlorophenols such as 2-chlorophenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol is also observed without adding mediators (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) or 1-hydroxybenzotriazole) to the reaction mixture. Thus, the mediators used do not seem to extend the substrate range for this laccase under these experimental conditions.

Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium.

The wood-degrading fungus Pleurotus pulmonarius produces at least two laccase isoforms, Lcc1 and Lcc2, when grown on wheat bran solid state medium. The main laccase, Lcc2, was purified to apparent electrophoretic homogeneity by using acetone precipitation, anion-exchange chromatography and gel filtration. Lcc2 had been purified 5.9-fold with a yield of 49%. A specific activity of 19750 U/mg protein was found using syringaldazine as a substrate under standard assay conditions. The enzyme is a homodimeric glycoprotein containing 44% glycosilation and an apparent molecular mass of 46 kDa. Type I and type III Cu(2+) centers were identified by spectrophotometry. The laccase showed optimal activity at pH 6.2-6.5, 4.0-5.5, and 6.0-8.0 with syringaldazine, ABTS and guaiacol as substrates, respectively. For all substrates, the highest oxidation rates were obtained at 50 degrees C. The enzyme was stable over a large range of pH (4.5-8.0) and at temperatures up to 50 degrees C. Under standard assay conditions, the apparent K(M) values were 12, 210 and 550 microM for syringaldazine, ABTS and guaiacol, respectively. Purified Lcc2 was strongly inhibited by sodium azide, 2-mercaptoethanol and Hg(2+), and slightly inhibited by Mn(+2) and the chelant agents, EDTA and EGTA. The enzyme was activated by Cu(2+) and it retained a high percentage of its activity in the presence of organic solvents, such as acetonitrile and acetone.

Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus suber L)

A new isoform of laccase from Marasmius quercophilus is described in this study. The strain of this white-rot fungus was isolated for the first time on a cork oak litter. This isoform exhibited certain common properties of laccases (a molecular weight of 65 Kda, an optimum pH of 6.2 with syringaldazine). But this laccase has also particularly novel features: the best activity measured was observed at high temperatures (80 C) and this isoform was not inhibited with EDTA. Furthermore, this induced laccase was able to transform most of the aromatic compounds tested without the addition of mediators to the reaction mixture, and the transformation of certain chlorophenols (2-chlorophenol and 2,4-dichlorophenol) by a laccase isoform from M. quercophilus is reported here for the first time. We also demonstrate the importance of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator since it allowed veratryl alcohol and p-hydroxybenzoic acid transformation. Moreover, new products of transformation were observed using the combination of ABTS with this isoform of laccase.

Purification of a New Isoform of Laccase from a Marasmius quercophilus Strain Isolated from a Cork Oak Litter (Quercus suber L.)

Purification of a New Isoform of Laccase from a Marasmius quercophilus Strain Isolated from a Cork Oak Litter (Quercus suber L.)

Purification and characterization of a laccase from the white-rot fungus Trametes multicolor.

The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.

Effect of phenolic monomers on the production of laccases by the edible mushroom Pleurotus sajor-caju, and partial characterization of a major laccase component

The effects of several phenolic monomers on the growth and laccase production by the edible mushroom Pleurotus sajor-caju have been compared. Of the phenols tested, 4-hydroxybenzaldehyde and vanillin were most inhibitory to fungal growth, and cultures supplemented with these two compounds also exhibited the largest increases in laccase specific activity compared with unsupplemented controls. Up to five laccase isoforms were detected in culture fluids when P. sajor-caju was grown in submerged culture in the presence of several phenolic monomers. A major laccase component (laccase IV) present in P. sajor-caju culture supernatants was purified 152-fold with an overall recovery of 4.9%. Laccase IV was shown to be homogeneous by SDS-PAGE and isoelectric focusing, and had a molecular weight of 55 kD and an isoelectric point of 3.6. It displayed a maximum reaction velocity at pH 2.1 and 45 C, and the activation energy of the enzyme reaction between 25 and 45 C as determined by an Arrhenius plot was 12.4 kJ mol-1. The Km of laccase IV, as determined using ABTS as the substrate, was 0.092 mM. Laccase IV was glycosylated with mannose as the major sugar component together with galactose, glucosamine and fucose. Comparison of up to thirteen N-terminal amino acids of laccase IV with those of other fungal laccases revealed the highest similarity (85%) with a laccase from Pleurotus ostreatus. Considerable similarity (46–50%) was also observed with laccases from Coriolus versicolor, Pycnoporus cinnabarinus, Phlebia radiata, Coriolus hirsutus and Ceriporiopsis subvermispora but not Agaricus bisporus.

Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3.

Two extracellular laccase isoforms (Lac I and Lac II) produced by the white-rot fungus Pycnoporus cinnabarinus from the monokaryotic strain ss3 were purified from ferulic-acid-induced liquid culture medium using ammonium sulphate precipitation, followed by anion-exchange chromatography on a Mono Q column. Strain ss3 is the first generation of the parental strain P. cinnabarinus I-937. The new isolated isoform, Lac II, consists of an 86,000 molecular weight protein as determined by SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of both isoforms were determined, and compared to known laccase protein sequences of other organisms.

Studies of some biochemical and physicochemical properties of an inducible form of extracellular laccase from the basidiomyceteCoriolus hirsutus

An inducible form of extracellular laccase (EC 1.14.18.1) was isolated from the basidiomyceteCoriolus hirsutus. The induction was performed with 0.11 μM syringaldazine, a substrate of laccase. The inducible form of the enzyme consisted of two isoforms, laccase II and laccase 12, whose molecular weights were 69 ±2 and 67 ±2 kDa, respectively. The isoelectric points of these isoenzymes were found to be 3.5 and 4.2, respectively. The optimum pH range for both laccases was 4.4–4.6, and the optimum temperature was 50°C. The thermal stability of these isoenzymes was examined, andK m values for the substrates syringaldazine and pyrocatechol were determined. Our biochemical and physicochemical studies demonstrated that inducible laccase isoforms differed from constitutive forms in molecular weight, IEP,K m, and thermal stability. However, their optimum pH ranges and temperatures were identical.

Electrochemical studies of a truncated laccase produced in Pichia pastoris.

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI-->LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (k(het) for LCCIa = 1.3 x 10(-4) cm s(-1)). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.

Laccase-less mutants of the white-rot fungus Pycnoporus cinnabarinus cannot delignify kraft pulp

The white-rot fungus Pycnoporus cinnabarinus produces only one isoform of laccase and traces of a peroxidase which is neither manganese peroxidase (MnP) nor lignin peroxidase (LiP). To test the impact of laccase on pulp bleaching we performed studies with P. cinnabarinus wild type (WT) and its laccase-less mutants on softwood kraft pulp (SWKP) in both static and shake flask cultures. In the static pulp cultures (at 20% pulp consistency in water), after 25 days incubation, P. cinnabarinus wild type increased the pulp brightness from 35 to 59% ISO, and decreased the kappa number from 15 to 5.7. However P. cinnabarinus laccase-less mutants 51 and 85 increased the brightness to only 40.6 and 40.7, and decreased the kappa number to 12 and 13.4, respectively. In the shake flask cultures with pulp suspended in water (at 2% consistency), the wild type increased pulp brightness from 38 to 52.4% ISO, and decreased the kappa number from 12 to 5.9. The laccase-less mutants 51, and 85 increased the brightness only to 43.8 and 40, and decrease the kappa number to 9.2 and 10.6, respectively. These results showed that both in the static and shaking cultures, the laccase-less mutants were very inefficient or unable to bleach pulp which is a clear indication of the importance of laccase for lignin degradation and pulp bleaching by P. cinnabarinus.

A comparative study of two isoforms of laccase secreted by the “white-rot” fungus Rigidoporus lignosus, exhibiting significant structural and functional differences

Two isoforms of laccase were obtained as the predominant phenol-oxidases in defined medium liquid cultures of the “white-rot” fungus Rigidoporus lignosus ( R. lignosus). A characterization of the two laccases was made in terms of molecular mass, isoelectric point, metal content and N-terminal sequence. Furthermore, in order to gain information on the structural features related to the metal centers, a study of their geometric arrangement and their redox ability was made. It turned out that the two isoenzymes greatly differed with regard to pH stability, catalytic and copper centers features. It is proposed that all such differences are dependent on the amino acid sequences, which cause a distortion of the copper sites, thus accounting for the redox potential values and kinetic properties.

Production of multiple laccase isoforms by Phanerochaete chrysosporium grown under nutrient sufficiency

Phanerochaete chrysosporium strain ME446 produced laccase when grown in a medium with high nitrogen (24 mM), glucose, acetate buffer and high copper (0.4 mM CuSO 4). Copper appeared to serve as an inducer. The highest amount of laccase activity was found in 2–3 day old cultures. Both intracellular and extracellular laccases eluted as four peaks (L1–L4) on a MonoQ (anion exchange) column. All four peaks from intracellular fluid appear to comprise multiple polypeptides that gave rise to a ∼400 kDa peak on a gel filtration column. Extracellular laccases had a non-denaturing M r of 100 kDa.

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju

Pleurotus sajor-caju, strain Pl-27, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP activity was detected in fungal cultures supplemented with both high (26 mM-N) and low (2.6 mM-N) nutrient nitrogen although higher specific activity values were recorded under the latter conditions. Conversely, laccase production was not influenced by nutrient nitrogen levels under the growth conditions adopted. Both the titre and time of appearence of MnP were also affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 15 ppm Mn. Two MnP and five laccase isoforms were identified by FPLC and gel electrophoresis.

Phenoloxidases in Portabella Mushrooms

ABSTRACTTyrosinase, laccase, and peroxidase activities were detected in crude extracts from commercial Portabella mushrooms. Tyrosinase was present in larger amounts than either laccase or peroxidase. Catechol showed the greatest activity as a substrate for tyrosinase while diaminobenzidine was found to be a good substrate for estimating laccase and peroxidase activity. Two tyrosinase and three laccase isoforms were detected after native electrophoresis. At least 10 tyrosinase isoforms were observed after isoelectric focusing. The distribution of these forms varied in different tissues. Browning reactions in Portabella mushrooms are a result of tyrosinase rather than laccase or peroxidase.

Lignin-degrading activity and ligninolytic enzymes of different white-rot fungi: effects of manganese and malonate

Ten species of white-rot fungi, mainly belonging to the family Polyporaceae (Basidiomycotina), were studied in terms of their ability to degrade14C-ring labelled synthetic lignin and secrete ligninolytic enzymes in liquid cultures under varying growth conditions. Lignin mineralization by the fungi in an air atmosphere did not exceed 14% within 29 days. Different responses to the elevated Mn2+concentration and the addition of a manganese chelator (sodium malonate) were observed among various fungal species. This could be related with the utilization of either lignin peroxidase (LiP) or manganese peroxidase (MnP) for lignin depolymerization, i.e., some fungi apparently had an LiP-dominating ligninolytic system and others an MnP-dominating ligninolytic system. The LiP isoforms were purified from Trametes gibbosa and Trametes trogii. Isoelectric focusing of purified ligninolytic enzymes revealed the expression of numerous MnP isoforms in Trametes gibbosa, Trametes hirsuta, Trametes trogii, and Abortiporus biennis grown under a high (50-fold) Mn2+level (120 μM) with the addition of the chelator. In addition, two to three laccase isoforms were detected. Key words: white-rot fungi, lignin degradation, lignin peroxidase, manganese peroxidase, manganese, malonate.

Identification of Specific Laccase Isoforms Capable of Polymerizing Monolignols by an “In-Gel” Procedure

Identification of Specific Laccase Isoforms Capable of Polymerizing Monolignols by an “In-Gel” Procedure

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi.

Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pI values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pI 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II), mainly the MnPs with higher pI values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pI values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pI values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pI values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF.(ABSTRACT TRUNCATED AT 250 WORDS)