RESUMEN
A biocontrol fungus isolated from rotting wood was identified as a Trichoderma strain (named as Trichoderma sp. ZH1) by internal transcribed spacer (ITS) sequences of rRNA genes. The laccase yield of Trametes sp. AH28-2 in cocultivation with Trichoderma sp. ZH1 reached 6,210 U l(-1), approximately identical to those induced by toxic aromatic inducers. Cocultures maintained 60-70 % of their highest laccase activity obtained at 5 days after inoculation of the biocontrol fungus, at least for 20 days. Furthermore, a novel laccase isozyme (LacC) was obtained through the fungal interactions. The molecular weight of LacC is about 64 kDa, and its isoelectric point is 6.6. The temperature and pH optimum for LacC to oxidize guaiacol are 55 degrees C and 5.0, respectively. LacC is stable both at 60 degrees C and pH 4.0-8.0. Furthermore, the K (m) values of LacC for various substrates were also determined. Our work demonstrates a safe strategy for the production of industrial laccases, instead of the traditional method of chemical induction.
Asunto(s)
Lacasa/biosíntesis , Polyporales/enzimología , Trichoderma/clasificación , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/aislamiento & purificación , Estabilidad de Enzimas , Guayacol/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Cinética , Lacasa/química , Lacasa/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Oxidación-Reducción , Filogenia , Análisis de Secuencia de ADN , Temperatura , Trichoderma/genéticaRESUMEN
Three novel laccase isozyme genes, lacA, lacB, and lacC, have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE, and CreA consensus sequences were identified in the lacA, lacB, and lacC promoter regions. Different from the TATA boxes of lacA and lacB at about -100, the TATA box of lacC is located at -172. For all the isozymes, copper ion is essential for laccase synthesis in Trametes sp. AH28-2. More interestingly, different aromatic compounds can selectively induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of laccase A (LacA) while 3,5-dihydroxytoluene mainly stimulating the production of laccase B (LacB). Quantitative reverse transcriptase-polymerase chain reaction showed that the accumulation of laccase messenger RNA transcripts is accompanied by the increase of corresponding enzyme activity in cultures. The glucose-repression effect on laccase expression in Trametes sp. AH28-2 was also observed. Furthermore, lower Cu2+ concentration (lower than 0.5 mM) can induce LacA and a novel laccase (LacC), and the latter will disappear when Cu2+ concentration is increased up to 1-2 mM. Upon induction by 3,5-dihydroxytoluene, the ratio of LacA to LacB decreased in the later phase of induction.
Asunto(s)
Basidiomycota/enzimología , Basidiomycota/genética , Lacasa/biosíntesis , Lacasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Basidiomycota/metabolismo , Clonación Molecular , Cobre/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , Regulación Enzimológica de la Expresión Génica , Isoenzimas , Lacasa/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Resorcinoles/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Toluidinas/metabolismoRESUMEN
The white-rot fungus Trametes sp. AH28-2 can synthesize extracellular laccase by induction in cellobiose-based liquid culture medium. Both yields and composition of laccase isozymes, produced by Trametes sp. AH28-2, would be quite different with induction by different small-molecule aromatic compounds, o-toluidine, guaiacol and 3,5-dihydroxytoluene, which affected microbial growth and the synthesis of laccase isozymes differentially. Higher concentrations of the three inducers could considerably increase laccase isozymes yields but not change the laccase composition. Coculturing of Trametes sp. AH28-2 with either Aspergillus oryzae or Gloeophyllum trabeum showed a few effects on laccase production. Laccase isozyme, laccase B, was selectively induced by 3,5-dihydroxytoluene and purified to homogeneity by two-step chromatography. Purified laccase B appeared as blue, with a broad peak at about 600 nm and a shoulder peak at about 330 nm. The ratio of absorbance at 280 nm to that at 600 nm was 21. Every molecule of laccase B had approximately four copper atoms. Molecular mass of laccase B was estimated to be 74 kDa on SDS-PAGE, 72 kDa by FPLC and was determined to be 71 454 Da by mass spectrum. After being treated with N-glycosidase F, laccase B lost 25% of its molecular mass. The isoelectric point of laccase B was 4.0. Its optimal pH and temperature for oxidizing guaiacol were respectively 4.7 and 45 C. The half-life of the enzyme at 60 C was 14.0 min. The enzyme showed a good stability in a range of pH value of 3.5-7.5. The K(m) values of the enzyme toward substrates syringaldazine, guaiacol, ABTS, and DMOP were respectively 28.0, 1249.0, 177.0 and 109.8 µM. The corresponding V(max) are 504.0, 1910.0, 117.4 and 159.0 µM min(-1) mg(-1). In addition, activity of laccase B was inhibited strongly by sodium azide and cyanide, mildly by SDS and trifluoroacetic acid, but only weakly by dimethyl sulfoxide.
RESUMEN
A group of 140 cotton textile workers from Shanghai, Shandong, Guangxi, and Beijing have had their chest radiographs taken using similar requirements. Most had come from the preparatory departments of cotton mills and had a history of exposure to cotton dust for at least 20 years. As controls, 140 healthy individuals with no dust exposure were matched with respect to sex, age, and smoking history. All the radiographs were read according to the ILO International Pneumoconiosis Classification, and the manifestations belonging to categories 0/0 and 0/1 were grouped as "normal," whereas categories 1/0, 1/1, and 1/2 and above as "abnormal." There was no significant difference in X-ray abnormalities between cotton textile workers and controls (P greater than 0.05). But when the data on the cotton textile workers and controls were combined, an appreciable difference in the incidence of radiographic abnormalities was found between smokers and non-smokers (P less than 0.001). Cotton dust exposure may induce nonspecific interstitial pulmonary changes, but these changes were exaggerated by cigarette smoking. These two factors appeared to have an additive effect on the pulmonary X-ray findings.