Abstract:
β-Glucosidases (BGL) are key members of the cellulase enzyme complex that determine efficiency of lignocellu losic biomass degradation, which have shown great functional importance to many biotechnological systems. A
previous reported BGL from Neosartorya fischeri (NfBGL) showed much higher activity than other BGLs. Screening
the important residues based on sequence alignment, analyzing a homology model, and subsequent alteration of
individually screened residues by site-directed mutagenesis were carried out to investigate the molecular deter minants of the enzyme's high catalytic efficiency. Tyr320, located in the wild-type NfBGL substrate-binding
pocket was identified as crucial to the catalytic function of NfBGL. The replacement of Tyr320 with aromatic
amino acids did not significantly alter the catalytic efficiency towards p-nitrophenyl β-D-glucopyranoside
(pNPG). However, mutants with charged and hydrophilic amino acids showed almost no activity towards
pNPG. Computational studies suggested that an aromatic acid is required at position 320 in NfBGL to stabilize
the enzyme-substrate complex formation. This knowledge on the mechanism of action of the molecular determi nants can also help rational protein engineering of BGLs.
