A Novel Cellobiohydrolase I (CBHI) from Penicillium digitatum: Production, Purification, and Characterization

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 T_m 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 K_M for CMC of 11.2 mg/mL, V_max of 0.13 μmol/min, k_cat of 52 s^-1, and k_cat/K_M 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 β-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.