• Journal of the Korean Wood Science and Technology
• /
• v.34 no.4
• /
• pp.46-53
• /
• 2006

This study was to screen white-rot fungi possesing strong lignin degrading enzymes, glucose-1 oxidase (GOD), laccase (LAC) and Mn-peroxidase (MnP), based on their decolorization activity of Remazol Brilliant Blue R (RBBR). In the midst of 20 tested fungi, 9 isolates were shown 4 kinds of activities such as RBBR decolorization, GOD, LAC and MnP. Relatively high active strains were identified as Phlebia radiata, Trametes versicolor, Abortiporus biennis, Gleophyllum odoratum and Cerrena unicolor. In particular, T. versicolor, G. odoratum, and C. unicolor, which have high activities of LAC, were used to confirm the optimal temperature and pH and to evaluate the effect of inducer, 2,5-xylidine on their LAC activity. The optimum temperatures for mycelial growth were $28^{\circ}C$ for T. versicolor and G. odoratum, and $25^{\circ}C$ for C. unicolor. The optimum pH for mycelial growth was 5.5. Three strains showed the increase of LAC enzyme activity by the addition of 2,5-xylidine. T. versicolor had the highest LAC activity of $22,700nkat/{\ell}$, corresponding to 11.3 times, G. odoratum $15,400nkat/{\ell}$, 9 times and C. unicolor $17,330nkat/{\ell}$, 5.5 times higher than those of the control.

• Journal of the Korean Wood Science and Technology
• /
• v.33 no.5 s.133
• /
• pp.76-85
• /
• 2005

The degradation of pentachlorophenol (PCP) by lignin degrading fungi was performed. Several fungi, Abortiporus biennis, Cerrena unicolor and Trametes versicolor, were tested to evaluate the inhibitory effect of PCP on their growth. At the extremal concentration of PCP $(500\;{\mu}M)$, only C. unicolor showed relatively fast growth (60% within 14 days) in the comparison to the control culture. In the case of A. biennis and C. unicolor, when initial PCP concentration was $50\;{\mu}M$, about 88.2% and 79.5% of PCP degradation were achieved within 3 days, respectively. When 2,5-xylidine (0.2 mM) was added to the C. unicolor culture, as high as 98% of PCP degradation was achieved within just an hour after its addition. A. biennis removed 44% of PCP at the same condition. PCP was completely disappeared when laccase activities reached to maximum.

• Journal of the Korean Wood Science and Technology
• /
• v.35 no.4
• /
• pp.61-66
• /
• 2007

The study was performed to purify and characterize laccase in culture of Trametes versicolor. The fungus was grown in liquid culture media of PDB and added 2,5-xylidine (0.2 mM) after 5 days to enhance the production of laccase. The fungal culture was incubated at $25^{\circ}C$ on a rotary shaker (120 rpm) for 7days, and the culture broth was clarified through Glass filter (GF/C). The aqueous solution was concentrated by ultramicrofiltration (Viva flow 50, GE Healthcare Bioscience, USA) and loaded onto a Hitrap Q FF column. Laccase activity could be detected at one peak, and this enzyme has a molecular mass of approximately 53kDa as determined by SDS-PAGE The optimum pH and temperature for syringaldazine were 5.0 and $60^{\circ}C$, respectively. The specific activity of crude, concentrated and purified laccase were 32, 409, and 1,243 U/mg, respectively.

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

• Journal of Korea Technical Association of The Pulp and Paper Industry
• /
• v.29 no.4
• /
• pp.7-17
• /
• 1997

균체가 생산하는 laccase 효소가 다방면으로 이용되면서, 이 효소를 균체로부터 대량으로 생산하고 분리.정제하여야 할 필요성이 대두되고 있다. 아울러 이 효소의 활성을 유도하기 위한 inducer 로서 2,5-xylidine 이 주로 사용되고 있는바, 이 xylidine 의 유독성이 인정되면서 사람에게 독성을 주지 않는 환경친화적 inducer의 검색이 필요하게 되었다. 본 연구에서는 백색부후균 Cerrena unicolor가 분비하는 laccase 효소의 유도를 위한 inducer로서 ferulic acid를 사용하였으며, 균체로부터 생산 및 분리된 laccase 효소의 정제특성을 구명코자 하였다. 본효소(constitutive enzyme)로서 I 및 II를, ferulic acid를 inducer로 사용한 경우 inducing 효소 III을 분리.정제하였다. 본 효소 I 및 II의 Michaelis 정수는 각각 737 M, 716 M 이었고, inducing효소 III은 167 M 로서, 기질에 대한 높은 친화성을 보여주고 있다. 분자량도 각각 65 kD, 63 kD 였으며, inducing효소 III은 59 kD 였다. 두효소 모두 15-19%의 당 및 단백질분자당 4M의 동(Cu)을 함유하고 있었다. 정제효율은 효소 I 및 II가 10.1%, 9.4% 였으며, III은 3.2% 였다. 모든 효소의 최적 pH 는 5.5였으며, 최적온도는 비교적 높은 $40^{\circ}C$였다.

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

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

• Microbiology and Biotechnology Letters
• /
• v.27 no.6
• /
• pp.477-483
• /
• 1999

For Trametes sp. CJ-105, a kind of white-rot fungi which was collected from the mountain of Korea and was proven to be effective in decolorizing a wide range of structurally different synthetic dyes, the optimum conditions for mycelial growth and laccase(E.C. 1.10.3.2) production were investigated. Among various carbon sources, glucose showed the highest potential for the mycelial growth and laccase production, the optimum concentration being 2% glucose. For the nitrogen source, asparagine was good for the mycelial growth, while ammonium tartrate for laccase production(optimum concentration: 0.04%). The addition of thiamine and biotin increased both th emycelial growth and laccase production. When 2,5-xylidine was added as an inducer after the first day of culture, the production of alccase was seven-times higher than that in the absence of the inducer. The optimum pH and temperature conditions for laccase production by Trametes sp. CJ-105 were pH 5.0 and $25^{\circ}C$, respectively. In the 5L fermentation, the production of laccase reached a maximum of 340U/ml at the time when the ammonium ion was being rapidly depleted.

• Environmental Analysis Health and Toxicology
• /
• v.18 no.2
• /
• pp.121-129
• /
• 2003

The genotoxicity of pendimethalin [N-(l-ethylpropyl)-2, 6-dinitro-3, 4-xylidine, C$\_$13/H$\_$19/N$_3$O$_4$, M.W.=281.3, CAS No. 40487-42-1], one of selective herbicide, was evaluated in bacterial gene mutation system, chromosome aberration in mammalian cell system and in vivo micronucleus assay with rodent. In bacterial gene mutation assay, pendimethalin revealed dose-dependent mutagenic potential in 313 ∼ 5,000 ${\mu}$g/plate of Salmonella typhimurium TA 98 and TA 1537 both in the absence and presence of S-9 metabolic activation system, and TA 100 only in the absence of S-9 mixture. In the TA 1535, slight increase of revertant was also observed in the presence of S-9 metabolic activation system. No mutagenic potential was observed in the TA 1535 without metabolic activation system and TA l00 in the presence of S-9 mixture. In mammalian cell system using Chinese hamster lung (CHL) fibroblast, no clastogenicity of pendimethalin was observed both in the absence and presence of S-9 metabolic activation system in the concentration range of 2.32∼9.28 ${\mu}$g/ml. And also, in vivo bone marrow micronucleus assay, pendimethalin revealed no clastogenic potential in the dose range of 203∼810 mg/kg body weight after oral administration in mice. Consequently, in vitro chromosome aberration with mammalian cells and in vivo bone marrow micronucleus assay revealed no clastogenic potential of pendimethalin. However, pendimethalin revealed mutagenic potential in bacterial gene mutation assay.