Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.10.3.2 (laccase)
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Two chromatographic forms of laccase c1 and c2 were purified approximately 225-fold from the extracellular culture fluid of ligninolytic cultures of Dichomitus squalens, using DEAE-Sepharose and Mono-Q fast protein liquid chromatography. Each homogeneous laccase (c1 and c2) has a molecular mass of approximately 66 kDa as determined by SDS-PAGE. Both forms are glycoproteins, and each contains four copper atoms per molecule of protein. The first 20 amino acids of the N-terminal sequences of these two laccases are identical and are similar to those of laccases from other lignin-degrading fungi. The electronic absorption spectra of these laccases exhibit bands at 610 and 330 nm, indicative of type I and type III copper. The EPR spectrum of laccase c1 exhibits bands indicative of type I and type II copper. Each laccase oxidizes a variety of phenolic substrates, has a pH optimum of 3.0 for the oxidation of 2,6-dimethoxyphenol, and is inhibited strongly by fluoride and azide.
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PMID:Purification and characterization of laccases from the white-rot basidiomycete Dichomitus squalens. 960 69

Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, DEAE Sepharose CL-6B, Sephacryl S-200 HR, Hitrap SP, and Mono S chromatography. The purification was 14.5-fold with an overall yield of 32.3%. The enzyme is a monomeric glycoprotein with 11% carbohydrate content, an isoelectric point of 7.4, and a molecular mass of 73 kDa. The N-terminal amino acid sequence showed low homology to those of the laccases of other white-rot basidiomycetes. Spectroscopic analyses revealed a typical laccase active site in the C. hirsutus enzyme, as all three Cu centers were identified. The absorption spectrum showed a type 1 signal at around 600 nm and a type 3 signal near 330 nm. Type 3 Cu showed fluorescence emission near 418 nm and an excitation maximum at 332 nm. The EPR spectrum yielded parameters for the type 1 and type 2 Cu of gII = 2.191 and AII = 0.0097 cm(-1), and gII = 2.222 and AII = 0.0198 cm(-1), respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for the enzyme was reached at 45 degrees C, and the pH optima of the enzyme varied and was substrate dependent in the range of 2.5 to 4.0. The enzyme oxidized a variety of the usual laccase substrates, including lignin-related phenols and had highest affinity toward guaiacol. Under standard assay conditions, the apparent Km value of the enzyme toward guaiacol was 10.9 microM. The enzyme catalyzed single electron transfer via the phenoxy radical as an intermediate and was completely inhibited by L-cysteine and sodium azide but not by EDTA.
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PMID:Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus. 1114 21

Pleurotus florida (ITCC 3308) produces two laccase enzymes (L1 and L2) in potato-dextrose media containing 0.5% yeast extract. Concentrated culture filtrate was separated on DEAE-Sephadex (A-50) column into two enzyme peaks, subsequently named L1 and L2. The L1 enzyme has been purified to homogeneity by ion-exchange and gel-permeation chromatography. L1 is a monomeric glycoprotein with a molar mass of 77 and 82 kDa as determined by SDS-PAGE and gel-filtration chromatography, respectively. The pI value of L1 has been determined to be 4.1. The optimum reaction temperature of the enzyme is 50 degrees C. The Km and some other kinetic parameters of L1 have been determined. Cyanide and azide completely inhibit the enzyme activity. The enzyme was fully active in 1:1 (V/V) buffer-chloroform for at least 2 h. Spectroscopic analysis revealed that the enzyme has four copper atoms, a type 1 copper, a type 2 copper and a type 3 binuclear copper.
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PMID:Purification and characterization of laccase-1 from Pleurotus florida. 1134 72

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.
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PMID:Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. 1266 49

The aim of the present study was to isolate a laccase from fruiting bodies of the yellow mushroom Cantharellus cibarius. The fruiting body extract was subjected to a purification protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on Con A-Sepharose. The laccase was composed of two identical subunits each with a molecular mass of 46 kDa. The laccase exhibited a temperature-dependent rise in activity over the temperature range 20-50 degrees C. When the temperature was raised above 60 degrees C there was a fall in enzyme activity. The enzyme manifested maximal activity at pH 4. At and above pH 6 there was a dramatic reduction in activity. The unique features of this fruiting body laccase compared with previously reported mycelial laccases include homodimeric nature, a distinctive N-terminal sequence, a higher optimal pH, and adsorption on only ConA-Sepharose among the various chromatographic media tested.
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PMID:A homodimeric laccase with unique characteristics from the yellow mushroom Cantharellus cibarius. 1467 94

A laccase with a novel N-terminal sequence, a low molecular mass of 43 kDa smaller than those of previously reported laccases, a pH optimum of 4, and a temperature optimum at 70 degrees C was isolated from fresh fruiting bodies of the mushroom Tricholoma giganteum. The activity of the enzyme rose steadily from 20 to 50 degrees C, increased very slowly from 50 to 70 degrees C, and fell slightly when the temperature was further increased to 80 degrees C. The activity of the laccase underwent little changes over the pH range 3.0-5.0. However, the enzyme activity dwindled to nothing after exposure to 100 degrees C for 10 min and when the ambient pH was 7 or above. The procedure used for purifying the enzyme included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and FPLC-gel filtration on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. It inhibited HIV-1 reverse transcriptase with an IC(50) of 2.2 microM.
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PMID:Purification of a novel low-molecular-mass laccase with HIV-1 reverse transcriptase inhibitory activity from the mushroom Tricholoma giganteum. 1476 29

A laccase with a novel N-terminal sequence was purified from fresh fruiting bodies of the edible wild mushroom Albatrella dispansus using a procedure that entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. In contrast to most of the previously reported laccases from mushroom mycelia, the laccase was unadsorbed on DEAE-cellulose. Although it was also unadsorbed on Affi-gel blue gel, it was adsorbed on Con A-Sepharose, indicating that it is a glycoprotein. It exhibited a molecular mass of 62kDa in gel filtration and SDS-PAGE. The activity of the laccase increased with temperature from 20 to 70 degrees C, and notably remained high at 80 degrees C. The pH optimum for the enzyme was around 4. Enzyme activity was indiscernible at pH 8 and pH 9. The laccase did not exert any inhibitory activity toward HIV-1 reverse transcriptase at a concentration of 1mg/ml, unlike some previously reported mushroom proteins.
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PMID:A novel laccase with fair thermostability from the edible wild mushroom (Albatrella dispansus). 1517 17

A laccase with a novel N-terminal sequence, a molecular mass of 63kDa, and inhibitory activity toward HIV-1 reverse transcriptase (IC(50)=9.5microM) was isolated from dried fruiting bodies of the monkey head mushroom Hericium erinaceum. A chromatographic procedure involving ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75 was employed. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose but unadsorbed on CM-cellulose. High activity of the enzyme was observed at pH 3-5 and at 50-80 degrees C. Its activity was completely abolished at pH 8 and 9 and after boiling for 10min. A temperature of 50 degrees C and a pH of 5.0 were optimal for its activity.
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PMID:A new laccase from dried fruiting bodies of the monkey head mushroom Hericium erinaceum. 1531 67

Two polyphenol oxidases (EC 1.14.18.1), P-1 and P-2, were purified as electrophoretically homogeneous proteins from the culture filtrate of Trametes sp. MS39401 by acetone precipitation and column chromatographies on DEAE-Sephadex A-50, Sephadex G-150 and hydroxylapatite. P-1 was purified 34-fold with a yield of 4.2%, while P-2 was purified 37-fold with a yield of 20.7%. The molecular masses of P-1 and P-2 were estimated to be 61 kDa and 90 kDa, respectively, by gel filtration. The isoelectric points of P-1 and P-2 were 3.4 and 2.7, respectively. The optimum pH range of both enzymes was 4.5-5.0 at 45 degrees C. The optimum temperature of both enzymes was 55 degrees C at pH 5.0. P-1 was stable at pH 5.0-7.5 and temperatures up to 60 degrees C. P-2 was stable at pH 3.0-7.5 and temperatures up to 50 degrees C. The thermostability of P-1 was comparable to that of the PM1 laccase of basidiomycetes, which was reported to be the most stable among basidiomycete laccases. Both enzymes were active toward various phenolic compounds and aminophenols. However, they lacked activity toward l-tyrosine. The K(m) values for (+)-catechin were 0.19 mM for P-1 and 0.67 mM for P-2. Both enzymes were appreciably inactivated by Hg(2+) and Sn(2+). Significant activation of neither enzyme was observed in the presence of metal ions and reagents. Both enzymes were significantly inhibited by copper-chelating agents, reducing agents and N-bromosuccinimide. Carbon monoxide caused appreciable inactivation of neither enzyme, so it is suggested that P-1 and P-2 belong to the group of laccases.
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PMID:Purification and characterization of polyphenol oxidase from Trametes sp. MS39401. 1623 40

Laccase produced by Basidiomycete was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation, DEAE-cellulose and hydrophobic interaction column chromatography. Purification of about 318.4 fold was achieved with an overall yield of 18.6%. Its molecular weight was estimated to be about 60.3 kD by SDS-PAGE, and that of it was 55.94 kD by mass spectrum. The optimum temperature and pH of the enzyme activity were 65 degrees C and 2.2 - 2.8 respectively. The isoelectric point was 4.02 (room temperature). Its N-terminal sequence was AIGPVTDL. The carbohydrate content was 49.2% by the phenol-sulfuric acid method. Michaelis constant of the enzyme for ABTS was 17.5 micromol/L. The enzyme activity was stable under 45 degrees C and in the pH range of 3.0 - 9.5. The activity was enhanced by Cu2+, and was strongly inhibited by Fe2+. While Mn2+ and Ag+ had no effect on laccase activity. Dithiothreitol and sodium azide inhibited completely the activity. Trp was possible essential residue for enzyme activity.
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PMID:[Purification and properties of laccase from Basidiomycete]. 1627 74


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