Profiling of the β-glucosidases identified in the genome ofPenicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

Abstract

Enzymatic lignocellulosic biomass conversion to bioethanol is dependent on efficient enzyme systems with β-glucosidase as a key component. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus;Penicillium funiculosumusing genomics, transcriptomics, proteomics and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in theP. funiculosumgenome, three were found to be extracellular, as evidenced by presence of signal peptides and mass spectrometry. Among the three secreted β-glucosidase, two belonged to the GH3 and one belonged to GH1 families. Modelled structures of these proteins predicted a deep and narrow active site for the GH3 β-glucosidases (PfBgl3A andPfBgl3B) and a shallow open active site for the GH1 β-glucosidase (PfBgl1A). The enzymatic assays indicated thatP. funiculosumsecretome showed high β-glucosidase activities with prominent bands on 4-methylumbelliferyl β-D-glucopyranoside (MUG) zymogram. To understand the contributory effect of each of the three secreted β-glucosidases (PfBgls), the corresponding gene was deleted separately and the effect of the deletion on β-glucosidase activity of the secretome was examined. Although not the most abundant β-glucosidase,PfBgl3A was found to be the most significant one as evidenced by a 42 % reduction in β-glucosidase activity in the ΔPfBgl3A strain. To improve the thermostability, two mutants ofPfBgl3A were designed with the help of molecular dynamics (MD) simulation and were expressed in Pichia pastoris for evaluation. ThePfBgl3A mutant (Mutant A) gave 1.4 fold increase in the half-life (T1/2) of the enzyme at 50 °C.