RESUMEN
A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental Tm of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented KM for CMC of 11.2 mg/mL, Vmax of 0.13 µmol/min, kcat of 52 s-1, and kcat/KM of 4.7 (mg/mL)-1 s-1. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and ß-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.
Asunto(s)
Celobiosa/metabolismo , Celulasa/biosíntesis , Celulosa 1,4-beta-Celobiosidasa/biosíntesis , Celulosa 1,4-beta-Celobiosidasa/aislamiento & purificación , Proteínas Fúngicas/biosíntesis , Penicillium/enzimología , Dicroismo Circular , Estabilidad de Enzimas , Genoma Fúngico , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Filogenia , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , TemperaturaRESUMEN
Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-ß-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/química , Celulosa 1,4-beta-Celobiosidasa/aislamiento & purificación , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Trichoderma/enzimología , Secuencia de Aminoácidos , Fenómenos Biofísicos , Biofisica , Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Especificidad por Sustrato , Trichoderma/química , Trichoderma/genéticaRESUMEN
The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.