• Korean Journal of Microbiology
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• v.44 no.2
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• pp.110-115
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• 2008

Acridine was not a substrate for fungal laccase but it was oxidized to acridone in the culture medium of P. cinnabarinus SCH-3. During the cultivation of P. cinnabarinus SCH-3, Laccase was the predominant extracellular phenoloxidase, and 3-hydroxyanthranilic acid (3-HAA) was produced in the early culture. Cinnabarinic acid (CA) was observed to accumulate in the culture medium. When P. cinnabarinus was grown in the culture medium containing acridine, acridine was oxidized to acridone. But when the laccase purified from the culture medium of P. cinnabarinus directly reacted with acridine in sodium tartrate buffer (pH 3.0), The oxidation of acridine did not happen. In contrast, when 3-HAA was added to the buffer that was mixed with laccase and acridine, the acridine was oxidized to acridone. While in vitro studies, the CA was formed from 3-HAA in the presence of purified laccase. The results suggest that the acridine should be oxidized to the acridone through the mediation of 3-HAA by the laccase in the culture medium of P. cinnabarinus SCH-3.

• The Korean Journal of Mycology
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• v.32 no.2
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• pp.112-118
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• 2004

Laccase produced by Trametes hirsuta S1 isolated from Korea was partially purified and characterized using ultrafiltration, anion exchange chromatography and affinity chromatography. The laccase was produced as the predominant extracellular enzyme during primary metabolism. Neither lignin peroxidase nor veratryl alcohol oxidase (VAO) were detected in the culture fluid. Addition of 2,5-xylidine enhanced 4-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 12%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to $51.2\;{\mu}M\;and\;56.8\;{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $50^{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 20 min at $70^{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the T. hirsuta S1 laccase showed 100% of homology to those of laccase from C. hirsutus.

• The Korean Journal of Mycology
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• v.31 no.2
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• pp.59-66
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• 2003

Laccase produced by Pycnoporus cinnabarinus SCH-3 isolated from Korea was partially purified using ultrafiltration, anion exchange chromatography and affinity chromatography, The laccase was produced as the predominant extracellular phenoloxidase during primary metabolism. Neither lignin peroxidase nor manganese-dependent peroxidase were detected in the culture fluid. In order to examine the effect of inducers in laccase production, 2,5-xylidine was added in the culture of Pycnoporus cinnabarinus SCH-3. Addition of 2,5-xylidine enhanced 25-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 9%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to be $44.4{\mu}M\;and\;56.0{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $60{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 10 min at $80{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus SCH-3 laccase showed 94% of homology to the N-terminal sequences of laccases from P. cinnabarinus PB and P. coccineus.

• Korean Journal of Agricultural Science
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• v.18 no.2
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• pp.157-163
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• 1991

The cultural conditions in shake flasks were investigated under which maximum amounts of laccase produced by a strain of white-rot fungus Coriolus versicolor CV3. The enzyme yields on potato-malt extract medium by the fungus were higher than on other media consisted of onion infusion or malt extract, with maximum activity of $1.50unit/m{\ell}$ culture or 119.5 unit/g mycelium at 11 days of incubation. Maximum yields of laccase and growth were obtained by supplementation of yeast extract or potassium nitrate to the potato-malt extract medium. Addition of 2.5-xylidine at $4{\times}10^{-4}M$ concentration to the medium induced the laccase production 3.1-fold higher than the basal level, while the mycelial growth was somewhat repressed. The pH optimum for the growth and laccase formation by the fungus was between pH 4 to 4.5.

• The Korean Journal of Mycology
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• v.31 no.3
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• pp.181-186
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• 2003

The production of laccase by Fomitella fraxinea was studied. The addition of minerals were necessary far laccase production by Fomitella fraxinea. Jar fermentor and Air-sparging fermentor performed high productivity In laccase activity by F. fraxinea. Laccase activity reached 3,540 in 8 days (Jar fermentor) and 3,100 in 6 days (Air-sparging fermentor) respectively.

• Journal of the Korean Electrochemical Society
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• v.2 no.1
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• pp.31-37
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• 1999

Copper-containig enzyme, laccase (Rhus vernicifera) was immobilized onto gold electrode using self-assembly technique and its surface properties and catalytic activities were examined. Laccase is an oxidoreductase capable to oxidize diphenols or diamines by 4-electron reduction of molecular oxygen without superoxide or peroxide intermediates. The electrode surface were modified by $\beta-mercaptopropionate$ to have a net negative charge in neutral solution and positively charged laccase (pI=9) was immobilized by electrostatic interaction. The successful immobilization was confirmed by cyclic voltammograms which showed typical surface-confined shapes and behaviors. The amount of charge to reduce the surface was similar to the charge calculated assuming the surface being covered by monolayer. The activity of the immobilized enzyme was tested by the capbility of oxidizing a substrate, ABTS (2,2-azine-bis-(3-ethylbenzthioline-6-sulfonic acid) and it was maintained for $2\~3$ days at $4^{\circ}C$. The immobilzed laccase showed about $10\~15\%$ activity compared to that in solution. The laccase-modified electrode showed the activity of elefoocatalytic reduction of oxygen in the presence of mediator, $Fe(CN)_6^{3-}$ The addtion of azide which is an inhibitor of laccase compeletly eliminated the catalytic current.

• Journal of Mushroom
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• v.2 no.3
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• pp.127-139
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• 2004

Laccases are multicopper-containing enzymes which catalyze the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. They often occur as isoenzymes, either constitutive or inducible, that oligomerize to multilateral complexes, what allow for penetration to the woody cell wall structure. White rot basidiomycete fungi may produce a number of laccase isoenzymes, some constitutively and others after induction. Fungal laccase is commonly induced by many ions, such as $Cu^{2+}$, $Cd^{2+}$ $Ca^{2+}$, $Li^+$, $Mn^{2+}$, $Ag^+$, $Hg^{2+}$, Mn and $Fe^{3+}$, phenolic compounds, some organic compounds, such as ethanol, isopropanol, cAMP, caffeine, p-anisidine, viscosinamide and paraquat, and nitrogens and even heat shock. A combination of Cu and pHB (p-hydroxybenzoic acid) made it possible to extend the inducible laccase activities over 30-fold. But the most effective inducer of laccase in the basidiomycete and other higher fungi is 2,5-xylidine, over 160-fold stimulation of laccase activity. The laccases are frequently encoded by gene families, as e.g. in Pycnoporus cinnabarinus, from which the lcc3-1 or the allelic form lac1 and lac3-2 have been cloned and sequenced. In the case of inducible forms the post-inductional laccase formation depends upon the synthesis of mRNA and the induction is due to the synthesis of a new protein.

• Journal of the Korean Wood Science and Technology
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• v.29 no.3
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• pp.104-111
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• 2001

There are some evidence that active enzymatic proteins, e.g. fungal laccase, exist in the naturally occured soil humus. This study was performed to investigate the covalent binding of fungal laccase to the humic acid-iron complex, and to measure laccase activity of immobilized ones. Seven methods were adopted to form the covalent binding of fungal laccase with soil humic acids complexed with iron. Using these seven methods it was possible to change the dimension of spacer arm between laccase and support, and also to regulate the mode of covalent binding of this enzyme. The spacer arm was regulated from 2C to 11C. There was not observed any straight relationship between the spacer arm longitude and the laccase activity after immobilization, but the binding mode more effective than the former. Three out of the seven methods gave the high activity of immobilized laccase, and which active products of laccase immobilization was stable up to 10 days after the process. It is indicated that natural soil condition might be prevented the laccase activation by the toxic influence of some phenolic humic compounds. It was shown, for the first time, the possibilities to obtain the high activity of fungal laccase by binding to humic acids, and especially in complex with iron.

• Proceedings of the Korea Technical Association of the Pulp and Paper Industry Conference
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• 2001.04a
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• pp.18-25
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• 2001

A new wood-degrading fungus Trichophyton rubrum LKY-7 secretes a high level of laccase in a glucose-peptone liquid medium. The production of laccase by the fungus was barely induced by 2,5-xylidine. The laccase has been purified to homogeneity through three chromatography steps in an overall yield of 40%. The molecular mass of the purified laccase was about 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase had the distinct blue color and had basic spectroscopic features of a typical blue laccase: two absorption maxima at 278 and 610 nm and a shoulder at 338 nm. The N-terminus of the laccase has been sequenced, revealing high homology to laccases from wood-degrading white-rot fungi such as Ceriporiopsis subvermispora. The enzyme had a "low" redox potential (0.5 V vs normal hydrogen electrode), yet it was one of the most active laccases in oxidizing a series of representative substrates/mediators. Compared with other fungal laccases, the laccase has a very low Km value with ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid] as a substrate and a very high Km value with violuric acid as a substrate. The laccase has the isoelectric point of 4.0. The laccase had very acidic optimal pH values (pH 3-4) while it was more stable at neutral pH than at acidic pH. The laccase oxidized hydroquinone faster than catechol and pyrogallol. The oxidation of tyrosine by the laccase was not detectable under the reaction conditions. The laccase was strongly inhibited by sodium azide and sodium fluoride. fluoride.

• KSBB Journal
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• v.17 no.2
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• pp.189-194
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• 2002

Laccase produced from Trametes sp. was immobilized on CNBr-activated Sepharose-4B (CAS4B) and tested for degradation of azo dyes. Laccase was efficiently immobilized on CAS4B. Immobilization of laccase on CAS4B increased pH, thermal and proteolytic stabilities. Optimum pH and temperature of immobilized laccase were pH 3 and 40$\^{C}$, respectively as same as those of free laccase. The K$\_$m/($\mu$mol/ml) values of free and immobilized laccase for Reactive Blue 19 as the substrate were 0.34 and 2.07, respectively V$\_$max/($\mu$mol/mL$.$min) values of them were 0.12 and 0.1, respectively. In repeated batch reactions, conditions retained high stability and degradation of dye for immobilized laccase were pH 5 and 30$\^{C}$. HBT didn\\`t decrease highly activity of immobilized laccase. Immobilized laccase was very stable for degrading dyes continuously in a packed-bed reactor containing laccase immobilized on CAS4B. For continuous degradation of 100 $\mu$M Reactive Blue 19 and 50 $\mu$M Acid Red 57 in the presence of 0.1 mM HBT under optimum conditions, immobilized laccase retained 70% of degradation ability even after 30 hours.