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Background  β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from  Chaetomella raphigera  with high substrate affinity, and is an efficient β-glucosidase supplement to  Trichoderma reesei  cellulase mixtures for the saccharification of lignocellulosic biomass.    Results  We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in  Saccharomyces cerevisiae . Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in  Pichia pastoris . Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in  Pichia pastoris  resulted in 2.7-fold higher production of recombinant protein, higher  V  max  and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in  P. pastoris . When combined with the  T. reesei  cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2.    Conclusions  D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher  V  max  than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in  P. pastoris . Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13068-021-01973-3.

Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution