• 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.

• 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.

• 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.

• Journal of Industrial Technology
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• v.37 no.1
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• pp.16-20
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• 2017

A new putative laccase gene (soncotA) which show 78% homology with that from Bacillus licheniformis (liccotA) was isolated from draft genome sequence of Bacillus sonorensis KCTC 13918. A 1,545 bp of PCR product corresponding 514 amino acids was cloned into NdeI-NotI site of pET21c and expressed as soluble form in E. coli. About 59 kDa size of recombinant laccase was purified into homogenity by Ni-NTA column and laccase activity was confirmed by zymography. The enzymatic properties of recombinant laccase were characterized. The specific activity of B. sonorensis laccase was 0.033 fold lower than that of Bacillus licheniformis laccase. The finding of new laccase gene broadened the enzymatic diversity of Bacillus species laccases.

• 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.

• Journal of the Korean Society of Clothing and Textiles
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• v.45 no.5
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• pp.866-880
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• 2021

This study aimed to ex situ colorize laccase-entrapped bacterial cellulose (BC) with natural phenolic dyes, namely,madder, turmeric, and cochineal, and to determine the effect of laccase entrapment on the dyeability of BC using color strength (K/S) analysis. Results showed that laccase entrapment improved the dyeability of BC and that pre-entrapment was the most effective method, compared with meta-entrapment and post-entrapment methods. In addition, surface characterizations confirmed the successful entrapment of laccase inside the BC nanostructure and retention of the cellulosic and crystalline structures of BC. The washing durability test confirmed that the K/S value of BC had improved after laccase entrapment. Furthermore, laccase-entrapped BC colorized with cochineal dye had the highest washing durability due to the high molecular weight of cochineal dyerelative to the other dyes. This study suggests a novel method for enhancing the dyeability and washing durability of BC colorized ex situ with natural phenolic dyes by laccase entrapment.

• 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.

• Journal of Life Science
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• v.25 no.6
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• pp.673-679
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• 2015

The culture conditions needed to maximize the production of laccase from Pholiota highlandensis mycelia were investigated. Among the tested media for laccase production, Coriolus versicolor medium (CVM; 2% dextrose, 0.4% peptone, 0.6% yeast extract, 0.046% KH₂PO₄, 0.1% K₂HPO₄, 0.05% MgSO₄·7H₂O) showed the highest activity for the enzyme. Then, to optimize culture conditions for laccase activity, the influences of various carbon, nitrogen, phosphorus, and inorganic salt sources in CVM were investigated. The optimum culture medium was 2% fructose, 0.4% peptone with 0.6% yeast extract, 0.05% NaH₂PO₄, and 0.05% MgSO₄·7H₂O as carbon, nitrogen, phosphorus, and inorganic salt sources, respectively. Several aromatic compounds in the medium enhanced laccase activity to varying degrees. Guaiacol induced maximum laccase production, yielding 114.1 U/ml laccase activity after cultivation for 11 days at 25℃. The optimum pH and temperature for laccase production were 8.0 and 35℃, respectively. Native polyacrylamide-gel electrophoresis (PAGE) followed by laccase-activity staining with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate was performed to identify the presence of laccase under the optimum conditions studied. Zymogram analysis of the supernatant culture showed an enzymatic band with a molecular mass of about 90 kDa.

• Journal of Mushroom
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• v.19 no.4
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• pp.285-293
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• 2021

The purpose of this study was to identify and characterize the laccase genes of Flammulina velutipes var. lupinicola. Five laccase genes (g1934, g1937, g2415, g2539, g5858) were selected based on the copper binding site and signal peptide analysis results using the laccase gene selected from the F. velutipes var. lupinicola genome. The size of the laccase genes of F. velutipes var. lupinicola were 1,488 bp~1,662 bp. As a result of cDNA sequence analysis, 14 to 17 introns were identified in the laccase genes. The cleavage site predicted as the signal peptide of the laccase gene was found to be located between 20 bp and 34 bp from the N-terminus. In addition, separation and purification were performed to characterize the F. velutipes var. lupinicola laccases, and the optimal activity of the separated and purified proteins were analyzed by pH, temperature and time. Five bands with laccase activity were found from zymogram analysis. The optimal pH of the reaction was 5.5, the optimal temperature was found to be 40℃. Therefore, characterization of the laccase genes identified in this study should help in better understanding the biomass decomposition of F. velutipes var. lupinicola.

• 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.