Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

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.
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PMID:Purification and characterization of a laccase from the white-rot fungus Trametes multicolor. 1201 76

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.
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PMID:A key structural role for active site type 3 copper ions in human ceruloplasmin. 1217 70

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 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.
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PMID:Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium. 1287 9

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 double-stranded RNA virus of the chestnut blight pathogen, Cryphonectria parasitica, has been shown previously to reduce accumulation of mRNAs of extracellular laccase (laccase A) produced by this fungus. Both extra- and intracellular laccases have been detected after growth of the fungus in liquid culture. In addition to cellular localization, the two laccases are distinguishable by time of appearance during growth and electrophoretic mobility. Laccase A was purified from the culture filtrate by standard protein purification procedures. The enzyme was characterized as a glycoprotein with a molecular mass of approximately 77 kDa. Both laccase A and laccase B activities were significantly reduced in the hypovirulent (double-stranded RNA-infected) strain UEP1 compared with the isogenic virulent (double-stranded RNA-free) strain EP155/2.
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PMID:Extra- and Intracellular Laccases of the Chestnut Blight Fungus, Cryphonectria parasitica. 1634 81

An isolate of Botrytis cinerea (strain 61-34) constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The enzyme has been purified to homogeneity by a facile operational sequence, the last stage of which involves hydrophobic interaction chromatography. By these means, over 80 mg of laccase liter(sup-1) can be obtained from aerated fermentor reaction broths. The enzyme, with an estimated M(infr) of 74,000 and pI of 4.0, is a monomeric glycoprotein containing 49% carbohydrate predominantly as hexose. With 2,6-dimethoxyphenol, it exhibits a pH optimum of 3.5 and a temperature optimum of 60(deg)C, and its K(infm) is 100 (mu)M. The purified enzyme with this substrate has a specific activity of 9.1 mkat mg of protein(sup-1). Taken together with a broad substrate range and its stability in 4% sodium dodecyl sulfate or 2 M urea solutions, several biotechnology transfers are suggested.
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PMID:Production and Characterization of Laccase from Botrytis cinerea 61-34. 1653 74

Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.
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PMID:Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detection. 1737 47

We describe the construction of enzymatic nanoreactors through noncovalent envelopment of a glycoprotein by amphiphilic linear-dendritic AB or ABA copolymers. The synthetic procedure is based on the regioselective adsorption of dendritic poly(benzyl ether)-block-linear poly(ethylene glycol)-block-dendritic poly(benzyl ether) or linear poly(ethylene oxide)-block-dendritic poly(benzyl ether) copolymers onto the oxidative enzyme laccase from Trametes versicolor in aqueous medium. The complexes formed have improved catalytic activity compared with the native enzyme (77-85 nkat/mL vs 60 nkat/mL, respectively) and are more stable at elevated temperatures up to 70 degrees C. Experiments with deglycosylated laccase confirm that the glycoside fragments in the native enzyme serve as the anchor sites for the linear-dendritic copolymers. The enzymatic nanoreactors are able to effectively oxidize series of substrates: phenolic compounds (syringaldazine) and hydrophobic polyaromatic hydrocarbons (anthracene and benzo[a]pyrene) under "green" chemistry conditions.
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PMID:Enzymatic nanoreactors for environmentally benign biotransformations. 1. Formation and catalytic activity of supramolecular complexes of laccase and linear-dendritic block copolymers. 1825 55

Proteins and certain carbohydrates contain phenolic moieties, which are potential sites for modification of the function of the biopolymers. In this study, the capability of two different fungal oxidative enzymes, laccase from Trametes hirsuta (ThL) and tyrosinase from Trichoderma reesei (TrT), to catalyze formation of hetero-cross-linking between tyrosine side chains of alpha-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. Formation of reaction products was followed by size exclusion chromatography (SEC), fluorescence spectroscopy, and SDS-PAGE, using specific staining methods for proteins and protein-carbohydrate conjugates. ThL and TrT were observed to differ significantly in their ability to catalyze the formation of protein-carbohydrate conjugates or the linking of the small molecular weight phenolic compounds to alpha-casein. The efficiency of these enzymes to directly cross-link protein also differed notably. TrT was able to cross-link alpha-casein more efficiently than ThL. ThL-catalyzed casein cross-linking was significantly enhanced by ferulic acid, p-coumaric acid, and also hOSX. The main reaction products by ThL appeared to be phenolic acid-bridged alpha-caseins. Indications of hetero-cross-link formation between alpha-casein and hOSX by both oxidative enzymes could be visualized by glycoprotein-specific staining in the SDS-PAGE analysis, although ThL was observed to be more effective in the heteroconjugate formation than TrT.
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PMID:Formation of protein-oligosaccharide conjugates by laccase and tyrosinase. 1842 26


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