Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 2 de 2
Filtrar
Mais filtros

Base de dados
Ano de publicação
Tipo de documento
Intervalo de ano de publicação
1.
Appl Environ Microbiol ; 72(7): 5020-6, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16820501

RESUMO

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.


Assuntos
Genes Reporter , Lacase/metabolismo , Fungos Mitospóricos/enzimologia , Stachybotrys/enzimologia , Sequência de Aminoácidos , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Aspergillus niger/enzimologia , Aspergillus niger/genética , Benzotiazóis , Biotecnologia/métodos , Celulase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Indicadores e Reagentes/metabolismo , Lacase/genética , Fungos Mitospóricos/genética , Dados de Sequência Molecular , Stachybotrys/genética , Ácidos Sulfônicos/metabolismo , Trichoderma/enzimologia , Trichoderma/genética
2.
Curr Genet ; 45(6): 383-9, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15071756

RESUMO

The advent of fluorescent proteins as vital dyes had a major impact in many research fields. Different green fluorescent protein (GFP) variants were established in prokaryotic and eukaryotic organisms within the past 10 years, and other fluorescent proteins were discovered and applied. We expressed the Discosoma red fluorescent protein, DsRed (T4), the improved monomeric red fluorescent protein (mRFP1) and the blue fluorescent protein (BFP) in the filamentous fungus Aspergillus nidulans. Whereas DsRed requires tetramer formation for fluorescence, mRFP1 functions as monomer. We used sGFP, DsRed (T4), mRFP1 and BFP for nuclear and/or mitochondrial labelling. To facilitate gene tagging, we established a number of cloning vectors for the efficient, simultaneous fusion of any protein with mRFP1, BFP and sGFP or the haemagglutinin epitope, 3xHA. A PCR-amplified gene of interest can be inserted into the expression vectors without cloning but using homologous recombination in vitro (GATEWAY). The vectors contain the argB gene as a selection marker for A. nidulans and the inducible alcA promoter for control of expression. The system allows labelling of a protein with several tags in one recombination reaction. Both the nutritional marker gene and the promoter are frequently used in other fungi, suggesting that this set of expression vectors will be very useful tools for gene analysis on a genome-wide scale.


Assuntos
Aspergillus nidulans/genética , Genes Reporter/genética , Vetores Genéticos , Proteínas Luminescentes/genética , Recombinação Genética , Núcleo Celular/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Transfecção , Proteína Vermelha Fluorescente
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA