RESUMEN
BACKGROUND: As the strongest antagonist of the platelet activating factor, ginkgolide B (GB) possesses anti-ischemic, anti-oxidant and anti-convulsant properties, and it is used for the treatment of thrombosis in clinical practice. Till now, GB is usually obtained from extraction of Ginkgo biloba leaves through column chromatography with an extremely low yield and high cost, which can not meet clinical requirement. Therefore, it is urgent to find a new method to prepare GB. RESULTS: In the current study, we studied the ability and mechanism to transform multi-component ginkgolide into GB by Coprinus comatus in order to enhance the GB yield. Except for ginkgolide A (GA) and GB, all the other ginkgolides in the extract were transformed by the strain. In the case of culture medium containing 20 g/L glucose, the transformation product was identified as 12% GA and 88% GB by high performance liquid chromatography-Mass spectrometry (HPLC-MS), two stage mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR). Partial GA was also transformed into GB according to the yield (76%) and the content of GA in the raw ginkgolide (28.5%). Glucose was the key factor to transform ginkgolides. When glucose concentration in medium was higher than 40 g/L, all ginkgolides were transformed into the GB. Proteomic analysis showed that C. comatus transformed ginkgolide into GB by producing 5 aldo/keto reductases and catalases, and enhancing the metabolism of glucose, including Embden-Meyerhof pathway (EMP), hexose monophophate pathway (HMP) and tricarboxylic acid (TCA). CONCLUSIONS: C. comatus could transform ginkgolides into GB when the medium contained 40 g/L glucose. When the strain transformed ginkgolides, the glucose metabolism was enhanced and the strain synthesized more aldo/keto reductases and catalases. Our current study laid the groundwork for industrial production of GB.
Asunto(s)
Coprinus/metabolismo , Ginkgo biloba/química , Ginkgólidos/química , Ginkgólidos/metabolismo , Lactonas/química , Lactonas/metabolismo , Extractos Vegetales/metabolismo , Biotransformación , Cromatografía Líquida de Alta Presión , Coprinus/química , Coprinus/enzimología , Electroforesis en Gel Bidimensional , Extractos Vegetales/química , ProteómicaRESUMEN
In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.
Asunto(s)
Coprinus/enzimología , Coprinus/genética , Endonucleasas de ADN Solapado/genética , Regulación Fúngica de la Expresión Génica , Meiosis/fisiología , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Endonucleasas de ADN Solapado/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Filogenia , Antígeno Nuclear de Célula en Proliferación/genética , Profase/genética , Profase/fisiología , ARN de Hongos/análisis , ARN Mensajero/análisis , Fase S/genética , Fase S/fisiología , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Coprinus congregatus synthesized and secreted high amounts of laccase under acidic culture condition (pH 4.1) transiently. We have cloned a genomic DNA fragment between the copper binding regions I and II of a laccase by PCR from C. congregatus. This fragment was used as a probe to clone a laccase cDNA from the acidic culture. The cDNA (clac2) consisted of 1817 bp which contains 1086 bp encoding 362 amino acids. The N-terminal sequence of the purified CLAC2 revealed that the first 16 amino acids were removed by the modification. The purified protein had two copper binding regions, although other fungal laccases had four. According to a Northern blotting analysis the clac2 was expressed not in neutral culture but in acidic culture only. The transcription of the clac2 as well as the laccase activity reached a maximum after 24 h upon transferring to an acidic culture, and then decreased very rapidly.
Asunto(s)
Coprinus/genética , Proteínas Fúngicas , Genes Fúngicos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Coprinus/enzimología , ADN Complementario , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Lacasa , Datos de Secuencia Molecular , Oxidorreductasas/química , Reacción en Cadena de la PolimerasaRESUMEN
Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.
Asunto(s)
Coprinus/enzimología , Espectroscopía de Resonancia Magnética , Peroxidasa/química , Secuencia de Aminoácidos , Asparagina/química , Sitios de Unión , Glutamina/química , Hemo/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Peroxidasa/genética , Fenilalanina/química , Proteínas Recombinantes/química , Especificidad por SustratoRESUMEN
Chitin synthase from Coprinus cinereus (Schaeff. ex Fr.) S. F. Gray (= C. lagopus sensu Buller) was used as a model for chitin synthase from insects. The effect of dimilin (difluorobenzuron), captan (trichloromethylsulfonyl fungicide), kitazin P (organophosphorus ester fungicide) and parathion (organophosphorus insecticide) on the fungal enzyme was compared with the effect of nikkomycin (nucleosidepeptide antibiotic).