Characterisation and expression of recombinant beta-glucosidase 2 from Trichoderma asperellum UPM1

Trichoderma sp. is a fungus capable of producing three categories of cellulase for cellulose degradation into glucose; endoglucanase, cellobiohydrolase and β- glucosidase. However, native production of β-glucosidase from fungi is often at low concentrations and takes a longer time. Furthermore, product inhibition caused by glucose on β-glucosidase reduces resulting yields, making it the rate limiting enzyme and represents an obstacle for commercial cellulose hydrolysis. Studies on β-glucosidases produced by Trichoderma sp. have elucidated two variants, which have been classified into glycosyl hydrolase (GH) families 1 and 3, with attention given to GH family 1 (Bgl2), owing to its relatively lower sensitivity to glucose inhibition, a desirable character for bioprocess development for efficient lignocellulosic biomass saccharification. As such, using locally isolated Trichoderma asperellum UPM1, this study has sought to characterise the bgl2 gene isolated and following heterologous expression in Escherichia coli, characterise the recombinant enzyme for enzyme activity and glucose tolerance. Trichoderma asperellum bgl2 (Tabgl2) gene sequence isolated was found to be 1398 nucleotides in length, encoding a protein of 465 amino acids in length, with an estimated molecular weight of 52798.31 Daltons. The identity of Trichoderma sp. glycosyl hydrolase family 1 β-glucosidase was affirmed by the presence of N-terminal signature of 15 amino acids in length, cis-peptide bonds at A180-P181 and W417-S418, conserved active site motifs with glutamate (E) residues (‘TFNEP’ and ‘VTENG’), 17 corresponding substrate binding and a lone conserved stabilising tryptophan (W) residue. Automated protein structure homology-modelling revealed the common triosephosphate isomerase (TIM) barrel fold, functioning as a monomer while protein phylogeny analyses positioned the isolated protein to a clade with known Trichoderma sp. β- glucosidases. Intracellular protein localisation was confirmed by the absence of a signal sequence. Suggestive glucose tolerance was inferred from the presence of 14 of 22 consensus residues from known glucose tolerant amino acid residues as well as the presence of corresponding residues L167 and P172, crucial in the retention of the active site’s narrow cavity, found in glucose tolerant Bgl2 from Trichoderma reesei. Characterisation thus proceeded by codon optimisation of the gene followed by transformation and heterologous expression in Escherichia coli using plasmid vector pET-20b(+), targeted for periplasmic expression of recombinant T. asperellum Bgl2 (TaBgl2). Protein expression analysis using SDS-PAGE showed the presence of a ~52 kDA protein in size while the crude enzyme extracts showed a specific activity of 0.0081 U/mg in the periplasmic fraction, 11.6-fold higher than in the periplasmic fraction of the E. coli host without IPTG induction. Glucose tolerance was affirmed with 40% of relative activity retained in a concentration of 0.2 M glucose. Thus, the relatively low sensitivity of TaBgl2 to inhibition by glucose makes this enzyme a potential candidate for further analyses in cellulose hydrolysis.

Text FBSB 2019 31 IR.pdf Download (1MB)

Actions (login required)

View Item