• 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.

• Journal of Microbiology
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• v.44 no.6
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• pp.617-621
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• 2006

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress ($H_2O_2$) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.249-253
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• 2016

The inky cap, Coprinellus congregatus, produces several laccase isozymes during its life cycle: both hyphal tip laccase and sclerotial laccase are involved in the fungal development. When this fungus was transferred to an acid liquid medium (pH 4.0-4.5), a new laccase was synthesized and secreted into the culture supernatant. In order to examine its regulation by external pH, green fluorescent protein gene was ligated at the downstream of the promoters having different lengths. These expression vectors having different promoter lengths were inserted into the fungal transformation vector, pBARGEM7-1. These expression vectors were introduced to the mating type a1 and a2 monokaryons, and the transformants were selected by the phosphinothricin resistance. Transformant a1 (a1TF) and transformant a2 (a2TF) were mated with each other to generate homozygotic dikaryon transformants. All these transformants were grown in neutral liquid medium for 5 days, and then the whole cell homogenates were transferred to the acidic liquid medium (pH 4.1). After 36 h incubation at $25^{\circ}C$, cells were harvested for the analysis of GFP expression. GFP expression was detected in the transformant having full-length promoter (2.0 kb), but other transformants having shorter length promoter (shorter than 1.29 kb) failed to show the fluorescence. Therefore, the acid-responsive element in the laccase promoter should be localized between -2.0 kb ~ -1.29 kb region.

• Korean Journal of Microbiology
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• v.42 no.3
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• pp.235-237
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• 2006

When fruit bodies of Coprinellus congregatus were matured, they were autolysed to form black ink. During the developmental changes, cell walls of basidia were degraded. Laccase formed melanin which was the typical black pigment of fungi, and chitinase hydrolyzed the chitin which was a component of fungal cell wall. When laccase and chitinase genes were used as the probe for the Northern analysis to confirm their expression during the fruit body development, both gene expressions were increased as the mushroom was getting matured.

• Mycobiology
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• v.43 no.3
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• pp.280-287
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• 2015

In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium. In order to investigate their biochemical characteristics, these laccases were heterologously expressed in Pichia pastoris using the pPICHOLI-2 expression vector. Expression of the laccases was facilitated by intermittent addition of methanol as an inducer and sole carbon source, in order to reduce the toxic effects associated with high methanol concentration. The highest expression was observed when the recombinant yeast cells were grown for 5 days at $15^{\circ}C$ with intermittent addition of 1% methanol at a 12-hr interval. Investigation of enzyme kinetics using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate revealed that the primordium-specific laccase Lacc12 was 5.4-fold less active than Lacc6 at low substrate concentration with respect to ABTS oxidation activity. The optimal pH and temperature of Lacc12 were 0.5 pH units and $5^{\circ}C$higher than those of Lacc6. Lacc12 showed maximal activity at pH 3.5 and $50^{\circ}C$, which may reflect the physiological conditions at the primordiation stage.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.10-13
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• 2008

Endocrine disrupting chemicals (EDCs) are hard to be degraded in nature, and are also accumulated in diverse organisms. They finally give negative effects to human through the food web. White rot fungi which have lignin-degrading enzymes have high potentials for degradation of recalcitrant compounds, and a white rot fungus, Phlebia tremellosa, isolated in Korea show good degrading activity against the endocrine disrupting phthalates. We have isolated a laccase cDNA which was involved in the degradation of EDCs, and constructed a laccase expression vector to use in the genetic transformation of P. tremellosa. The expression vector was stably integrated into the chromosomal DNAs and showed increased laccase activity in transformants. One of transformants showed not only increased degradation of several EDCs but also faster estrogenic decreasing activities generated by the EDCs.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2057-2063
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• 2018

Laccases can oxidize a variety of phenolic and non-phenolic substrates including synthetic dyes. In this research, a laccase gene Lcc9 from Laccaria bicolor was chemically synthesized and optimized to heterogeneous expression in Pichia pastoris and Arabidopsis thaliana. The properties of recombinant laccase expressed by P. pastoris were investigated. The laccase activity was optimal at 3.6 pH and $40^{\circ}C$. It exhibited $K_m$ and $V_{max}$ values of $0.565mmol\;l^{-1}$ and $1.51{\mu}mol\;l^{-1}\;min^{-1}$ for ABTS respectively. As compared with untransformed control plants, the laccase activity in crude extracts of transgenic lines exhibited a 5.4 to 12.4-fold increase. Both laccases expressed in transgenic P. pastoris or A. thaliana could decolorize crystal violet. These results indicated that L. bicolor laccase gene may be transgenically exploited in fungi or plants for dye decolorization.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.107-110
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• 2007

The putative laccase gene, yacK of Escherichia coli, K-12 is not expressed in lab culture conditions. The laccase gene was amplified by PCR and subcloned into pET28a vector. The laccase overproduced in E. coli harboring pET28a was purified by His-affinity column chromatography. The purified laccase, which has the apparent molecular weight of 55,000 on the SDS-polyacrylamide gel showed enzyme activity on the guaiacol solution and agar plate. Optimum temperature and pH were around 65$^{\circ}C$ and 5.0, respectively.

• Mycobiology
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• v.45 no.4
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• pp.379-384
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• 2017

In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons.

• 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).