Abstract
Backgroundβ-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.ResultsWe 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 Vmax 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.ConclusionsD2-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 Vmax 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.
Highlights
Lignocellulosic biomass is considered a sustainable source of value-added products such as biofuels
Expression of recombinant D2‐BGL in S. cerevisiae and P. pastoris Saccharomyces cerevisiae is commonly used for the construction of mutant libraries via random mutagenesis because of its ability to reconstitute linearized DNA fragments into plasmids by homologous recombination [21]
We tested the suitability of S. cerevisiae to express wild-type D2-BGL (WT D2)
Summary
Lignocellulosic biomass is considered a sustainable source of value-added products such as biofuels. Physical and chemical pre-treatments are required to break down lignocellulose into lignin, hemicellulose and cellulose [1]. Kao et al Biotechnol Biofuels (2021) 14:126 to release the glucose that can be transformed into biofuels and other chemicals via catalytic conversion [2]. Endo-glucanases and exo-glucanases degrade cellulose into cellooligomers and cellobiose, whereas β-glucosidases play a crucial role in releasing the final product (i.e., glucose) from cellobiose [3]. Among microbial β-glucosidases, the fungal GH3 enzymes have been widely studied for their high catalytic efficiency towards cellobiose and cello-oligomers, and they are considered to be the most suitable β-glucosidase supplement for cellulase mixtures used in biomass saccharification [11,12,13]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
