• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.864-871
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• 2013

The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was $60^{\circ}C$, but it was not stable at high temperature. The enzyme could remain stable at $30^{\circ}C$ for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system ($k_{obs}$ = 0.151) than that in the buffer solution ($k_{obs}$ = 0.028).

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.228-234
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• 2006

The culture conditions were investigated to maximize the production of laccase from Fomitella fraxinea mycelia. Among the tested media, mushroom complete medium (MCM) showed the highest production of the enzyme. The optimum culture medium was 2% dextrose, 0.4% $(NH_4)_{2}HPO_4$, 0.05% $Na_{2}HPO_{4}{\cdot}7H_{2}O$, and 0.05% KCl as carbon, nitrogen, phosphorus, and inorganic salt sources respectively. SDS-PAGE followed by laccase activity staining using 2,6-djmethoxyphenol as the substrate was performed to identify the laccase activity under culture conditions studied. Zymogram analysis of the culture supernatant showed a laccase band with a molecular mass of 50 kDa. The enzyme production from F. fraxinea was reached to the highest level after the cultivation for 10 days at $25^{\circ}C$ and initial pH 8. The enzyme activity of the culture supernatant was most active at $50^{\circ}C$ and pH 5.

• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.62-68
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• 2009

The culture conditions to maximize the production of laccase (EC 1.10.3.2) from Fomitopsis pinicola mycelia were investigated. Among the tested media for the enzyme production, mushroom complete medium (MCM ; 2% dextrose, 0.2% peptone, 0.2% yeast extract, 0.05% $KH_2PO_4$, and 0.05% $MgSO_4{\cdot}7H_2O$) showed the highest activity of the enzyme. To optimize the culture condition for the laccase activity, influence of various carbon and nitrogen sources was investigated in MCM. Among various carbon and nitrogen sources, 2% glucose and 0.4% peptone showed the highest production of the enzyme, respectively. For the phosphorus and inorganic source, 0.05% $NaH_2PO_4$ and 0.05% $CaCl_2$ were best for the enzyme activity. The enzyme production was reached to highest level after the cultivation for 8 days at $25^{\circ}C$. Native polyacrylamide gel electrophoresis (PAGE) followed by the laccase activity staining using 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate was performed to identify the laccase under culture conditions studied. Zymogram analysis of the culture supernatant showed a laccase band with molecular mass of 52 kDa. The optimum pH and temperature for the enzyme activity were $80^{\circ}C$ and pH 3.0.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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• pp.250-258
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• 2019

Peniphora sp. JS17, isolated from forest old tree, produced extracellular enzymes that decolorized human hair melanin. The JS17 strain had laccase and manganese peroxidase activity while it did not has lignin peroxidase activity. Batch culture indicated that the melanin decolorization activity of JS17 strain originated from laccase. The culture conditions to maximize the production of melanin bleaching enzymes from Peniophora sp. JS17 mycelia were investigated. Among the tested media for the laccase production, minimal medium (2% glucose, 0.2% malt extract, 0.1% $KH_2PO_4$, 0.4% $MgSO_4{\cdot}7H_2O$) showed the highest activity of laccase. Then, to optimize the culture condition for the laccase activity, the influence of various carbon and nitrogen sources was investigated in minimal medium. Among various carbon and nitrogen sources, 2% xylose and 0.4% tryptone showed the highest production of laccase, respectively. The enzyme was purified using $(NH_4)_2SO_4$ precipitation and Hitrap Q sepharose column, and the purified enzyme showed two isoenzymatic bands with molecular masses of about 70 kDa by SDS-PAGE. The melanin decolorization activity was 77% and 55% within 48 h in the presence of 1-hydroxybenzotriazole (HBT) and syringaldehyde, respectively, whereas only about 9% melanin decolorized in case of no mediator.

• Journal of Microbiology
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• v.43 no.3
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• pp.301-307
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• 2005

The feasibility of laccase production by immobilization of Pleurotus ostreatus 1804 on polyurethane foam (PUF) cubes with respect to media composition was studied in both batch and reactor systems. Enhanced laccase yield was evidenced due to immobilization. A relatively high maximum laccase activity of 312.6 U was observed with immobilized mycelia in shake flasks compared to the maximum laccase activity of free mycelia (272.2 U). It is evident from this study that the culture conditions studied, i.e. biomass level, pH, substrate concentration, yeast extract concentration, $Cu^{2+}$ concentration, and alcohol nature, showed significant influence on the laccase yield. Gel electrophoretic analysis showed the molecular weight of the laccase produced by immobilized P. ostreatus to be 66 kDa. The laccase yield was significantly higher and more rapid in the packed bed reactor than in the shake flask experiments. A maximum laccase yield of 392.9 U was observed within 144 h of the fermentation period with complete glucose depletion.

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

• Journal of the Korean Wood Science and Technology
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• v.29 no.4
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• pp.60-66
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• 2001

For examine anti-degradation factors of acetylated wood, acetylated wood was incubated on Trametes versicolor. The laccase activity was examined in broth culture and solid fermentation that contain acetylated chips. The change of acetyl groups and chemical composition in the acetylated wood having massloss analysed by $^{13}C$-CP/MAS NMR. The laccase activity was detected in broth culture. When the T. versicolor contact to acetylated wood directly, the laccase activity was very low and couldn't maintain during test periods. Through the analysing of $^{13}C$-CP/MAS NMR, the acetylation took place carbohydrates as well as lignin and hydroxyl group of amorphous region was more easily substituted that of crystalline region The spectral analyses of $^{13}C$-CP/MAS NMR were shown that introduced acetyl bond was stable against fungal attack.

• Journal of Microbiology
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• v.43 no.6
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• pp.555-560
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• 2005

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of $6.2\%$ using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of $50^{\circ}C$. The $K_m$ value of the enzyme for substrate ABTS is $12.8{\mu}M$ and its corresponding $V_{max}$ value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

• Microbiology and Biotechnology Letters
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• v.48 no.4
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• pp.463-470
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• 2020

The JS18 strain was isolated from an old tree forest and produced extracellular enzymes that decolorize synthetic melanin. Phylogenetic analysis, based on the internal transcribed spacer (ITS) sequence, indicate that JS18 belongs to the Trametes velutina species. JS18 demonstrated laccase activity but no manganese peroxidase or lignin peroxidase activity. Batch culture indicated that the melanin decolorization activity of JS18 strain originated from the laccase. Syringic acid and CuSO4 induced maximum laccase production, yielding 98 U/ml laccase activity after cultivation for 7 days at 25℃. T. velutina secretes an extracellular laccase in GYP medium, and this enzyme was purified using (NH4)2SO4 precipitation, Hi-trap Q Sepharose columns and gel filtration. The molecular weight of the purified enzyme was estimated to be 67 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme produced 80% of its melanin decolorization activity within the first 24 h of evaluation in the presence of 1-hydroxybenzotriazole (HBT), while only about 4% of the melanin was decolorized in the absence of the mediator. The greatest decolorization was observed at 1.5 mM/l HBT, which decolorized 81% of the melanin within the first 24 h. The optimum pH and temperature for this decolorization were found to be 5.0 and 37℃, respectively. Our results suggest the possibility of applying HBT induced T. velutina JS18 laccase-catalyzed melanin decolorization.

• 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.