Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.10.3.2 (laccase)
 4,656 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.
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PMID:Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila). 136 69

The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a pI of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not aryl-alcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by aryl-alcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol (Km = 0.84 mM, Vmax = 52 U/mg) to 4-methoxybenzyl alcohol (Km = 0.04 mM, Vmax = 208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron-withdrawing groups.
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PMID:Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. 142 67

Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42.9-fold with an overall yield of 10.8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12.5% carbohydrate by weight and 3.9 mol copper (mol protein)-1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g parallel = 2.197 and A parallel = 0.009 cm-1, and g parallel = 2.263 and A parallel = 0.0176 cm-1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pKa values were 5.60-5.70 and 6.70-6.85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6.0-6.5 and 30-35 degrees C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the Km values for ferulic acid and syringic acid were 48 and 89 microM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.
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PMID:Single electron transfer by an extracellular laccase from the white-rot fungus Pleurotus ostreatus. 770 70

Laccase is one of the ligninolytic enzymes found in liquid cultures of the fungus Ceriporiopsis subvermispora in defined medium. As an approach to a clarification of the role of laccases during the attack on lignin by the fungus, the enzyme has been characterized further. The levels of this phenol oxidase increase 2-fold in the presence of p-anisidine and are severely affected when addition of either Mn(II) or Cu(II) ions to the medium is omitted. Isoelectrofocusing allowed the resolution of two laccase isoenzymes, with pIs of 3.65 and 3.59. In rich medium, laccase activity is 10-fold higher than in salt medium, and it is not affected by the external addition of p-anisidine or Mn(II). Four isoenzymes were detected in these cultures, with pIs between 3.76 and 3.60. In a wheat bran medium, four isoenzymes with pIs in the range 3.63-3.46, plus a fifth isoenzyme of high pI (4.82), were also identified. The absorption spectrum of a pool containing the four isoenzymes from rich medium shows a maximum at 600 nm, typical of laccase possessing a type I copper atom. The molecular mass of the isoenzyme with pI 3.60 is 79 kDa, as determined by SDS/PAGE. Upon treatment with endoglycosidase F, the molecular mass of this isoform decreases to 63 kDa, indicating a high degree of glycosylation. Substrate specificity studies conducted with the four isoenzymes from rich medium and a combination of isoenzymes from salt medium showed marked differences among them. The amino-terminal sequences (24 residues) of three isoenzymes isolated from rich medium were determined. Two of them are identical, whereas the third one differs from these in three amino acid residues. The consensus sequence reveals clear homology with laccases from other microorganisms.
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PMID:Properties of laccase isoenzymes produced by the basidiomycete Ceriporiopsis subvermispora. 779 34

Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.
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PMID:The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus. 809 17

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

The degradation of the disazo dye Chicago Sky Blue 6B by a purified laccase from Pycnoporus cinnabarinus was investigated. Laccase was purified to homogeneity and characterized. The enzyme had a molecular size of 63 kDa as determined by SDS-PAGE and an isoelectric point at pH 3. Amino acid composition and N-terminal amino acid sequence was shown to be similar to other fungal laccases. The purified laccase was stable for 1 h at 60 degrees C and was irreversibly inactivated by sodium azide at 0.1 mM. Laccase was shown to initiate destruction of the chromophore of the disazo dye Chicago Sky Blue, resulting in the formation of two intermediate products with absorption intensities about one order of magnitude lower than the parent molecule. The rate at which the dye was transformed by purified laccase was shown to increase with increasing concentrations of the enzyme.
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PMID:Transformation and degradation of the disazo dye Chicago Sky Blue by a purified laccase from Pycnoporus cinnabarinus. 1086 8

Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (> 300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with different relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens.
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PMID:Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish. 1093 21

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.
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PMID:Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3. 1094 25

Soluble and cell wall-associated proteins were extracted from the developing xylem of the compression and non-compression sides of branches of Sitka spruce (Picea sitchensis (Bong) Carr.) by an identical procedure. Equal amounts of proteins were separated by SDS-PAGE, and polypeptides were identified that were more abundant in soluble and cell wall-associated extracts from the developing xylem of either compression or non-compression wood. Two polypeptides (at apparent M(r)s of 48 kDa and 120 kDa) that were more abundant in cell wall-associated extracts of the developing xylem of the compression tissues were selected for amino-terminal protein sequencing. The 48 kDa polypeptide yielded an amino-terminal sequence that had no homology with known protein, gene or EST database sequences. The amino-terminal sequence of the 120 kDa polypeptide was homologous to a number of laccase-type polyphenol oxidases (EC 1.10.3.2) thought to be involved in lignin biosynthesis in trees. Using non-denaturing SDS-PAGE, the 120 kDa laccase was confirmed as a major oxidase activity in extracts of lignifying compression xylem but it was barely detectable in the non-compression extracts where an 85 kDa oxidase was the predominant activity. The differential expression of oxidases in compression and non-compression xylem is discussed.
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PMID:A comparison of proteins from the developing xylem of compression and non-compression wood of branches of sitka spruce (Picea sitchensis) reveals a differentially expressed laccase. 1094 53


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