Hyoung T. Choi

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57∼97 % homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.

Removal of estrogenic activity from endocrine-disrupting chemicals by purified laccase of Phlebia tremellosa

A white-rot basidiomycete, Phlebia tremellosa, produced a laccase that showed increased activity during degradation of phthalates. A laccase was purified through the ion exchange chromatography and preparative gel electrophoresis, and the estimated molecular weight was 75 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 degrees C, respectively. The K(m) value of the enzyme was 55.7 microM, and the V(max) was 0.0541 OD min(-1) U(-1) for o-tolidine. Purified laccase reduced the estrogenic activity of four different endocrine-disrupting chemicals. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was involved in the removal of estrogenic activity.

Enhanced expression of chitinase during the autolysis of mushroom in Coprinellus congregatus

Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregates, produced mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregates that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.

Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus

Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was used as the substrate. The Km value and Vmax for the substrate A, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min−1unit−1, respectively.

Degradation of endocrine disrupting chemicals by genetic transformants in Irpex lacteus with an inducible laccase gene of Phlebia tremellosa.

Irpexlacteus was genetically transformed using an laccase expression vector to get increased laccase producing strains. Stable integration of the vector was confirmed by PCR using the vector-specific primers, and the transformants showed increased laccase activities. When the transformants were grown with several endocrine disrupting chemicals, laccase activity of each transformant was induced up to six times higher than that of the wild type. They showed increased degrading activities against EDCs as well as increased removal rates of estrogenic activities generated by the EDCs than the wild type, and the laccase expression was increased during the degradations of the EDCs.

Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in Phlebia tremellosa.

A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.

Growth inhibition of the yeast transformant by the expression of a chitinase from Coprinellus congregatus

Coprinellus congregatus generates several chitinases during its entire life cycle: at the growing hyphal stage and at the mushroom autolysis stage. We have isolated a chitinase gene (chi1) from the mushroom tissue at the autolysing stage, and constructed a chitinase expression vector to get large amount of enzyme protein. Chitinase 1 (chi1) cDNA was heterologously expressed in Saccharomyces cerevisiae by gal1 promoter. The transformants showed no specific change in growth characteristics under normal growth conditions. However the expression of the gene by the gal1 promoter in the yeast transformants resulted in complete growth inhibition, while laccase expression by the gal1 promoter showed normal growth. The chitinase activities from the transformants were also more than 3 times higher than that of the recipient strain, and the chitinase expression by the real time-PCR also showed increased expression of the chi1 in the yeast transformant. Expression of a chitinase which was produced at the mushroom autolysing stage of C. congregatus resulted in yeast growth inhibition.

Tagging and localization of a phospholipase D gene in Coprinellus congregatus by restriction enzyme-mediated integration and pulsed-field gel electrophoresis

We have identified a phospholipase D gene (pld) fragment from a transformant generated by restriction enzyme-mediated integration in Coprinellus congregatus, which is a mushroom-forming basidiomycete. A fragment of pld of this fungus has been cloned from a transformant by gene tagging. The transformation vector has been inserted into pld and this has resulted in a decreased enzyme activity of the transformant compared with the wild strain. C. congregatus has seven chromosomes, the range of its genome size is 1.6-4.7 Mb and pld is located at chromosome 4 where the transformation vector has been inserted.

Antifungal chitinase against human pathogenic yeasts from Coprinellus congregatus

The inky cap, Coprinellus congregatus, produces mushrooms which become autolyzed rapidly to generate black liquid droplets, in which no cell wall is detected by microscopy. A chitinase (Chi2) which is synthesized during the autolytic phase of C. congregatus inhibits the growths of Candida albicans and Cryptococcus neoformans up to 10% at the concentration of 10 μg/ml, about 50% at concentration of 20 μg/ml, and up to 95% at the concentration of 70 μg/ml. Upon treatment these yeast cells are observed to be severely deformed, with the formation of large holes in the cell wall. The two yeast species show no growth inhibition at the concentration of 5 μg/ml, which means the minimum inhibitory concentrations for both yeast species are 10 μg/ml under these experimental conditions.

Enhanced Expression of Glucose 2-Oxidase in Phlebia tremellosa by Addition of Phthalates

Abstract Most fungi possess several hydrogen peroxide-generating enzymes, glucose oxidase and pyranose oxidase. Pyranose oxidase can use glucose as its substrate to generate hydrogen peroxide. White rot fungi, which degrade diverse recalcitrant compounds, contain lignin-degrading enzymes, and lignin peroxidase and manganese peroxidase require hydrogen peroxide for their enzymatic reactions. In this study, we isolated a cDNA fragment of pyranose oxidase from Phlebia tremellosa using PCR and examined its expression under the degradation conditions of diethylphthalate (DEP). Pyranose oxidase expression was enhanced up to 30% by the addition of DEP, and this result supports the possible involvement of pyranose oxidase in the degradation of recalcitrant compounds.