RESUMEN

A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60 degrees C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-beta-D-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-13C6 as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.

Asunto(s)

Celulosa/metabolismo , Penicillium/enzimología , beta-Glucosidasa , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Biotecnología/métodos , Celulosa/química , Medios de Cultivo , Estabilidad de Enzimas , Biblioteca de Genes , Glucosa/química , Glucosa/farmacología , Calor , Concentración de Iones de Hidrógeno , Cinética , Penicillium/genética , Penicillium/crecimiento & desarrollo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Xilanos/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificación , beta-Glucosidasa/metabolismo