A novel thermostable and glucose-tolerant chimeric cellulase (AfCel6A-L-AfBgl1.3) combining cellobiohydrolase (AfCel6A) and β-glucosidase (AfBgl1.3) activity for efficient biomass conversion.

The cellulolytic chimera AfCel6A-L-AfBgl1.3 was constructed by fusing genes encoding cellobiohydrolase (AfCel6A) and β-glucosidase (AfBgl1.3) via a pentapeptide flexible linker (L). The genes were cloned into the pPICZαA vector and Pichia pastoris X-33 cells expressed them as a soluble protein. The chimera was purified after expression for six days. SDS-PAGE analysis revealed a single homogeneous protein band. The individual enzymes had optimal temperatures of 60 °C (AfCel6A) and 40 °C (AfBgl1.3), but fusion shifted the optimal temperature of the AfCel6A domain in the chimera to 40 °C. At 50 °C, both individual AfCel6A and chimeric AfCel6A retained ∼70% activity after 24 h, whereas chimeric AfBgl1.3 maintained 17% activity; individual AfBgl1.3 was inactive at this temperature. The catalytic efficiency ( k cat / K M ) of AfCel6A and AfBgl1.3 domains in the chimera was 1.8 and 1.5-fold higher than the catalytic activity of the individual enzymes, respectively. AfCel6A-L-AfBgl1.3 tolerated glucose more than the individual enzymes and retained over 120% activity under high glucose concentrations (1000 mM). Mn 2+ strongly activated both domains (AfCel6A, 237.0%; AfBgl1.3, 167.7%), whereas Co 2+ moderately enhanced their activity (AfCel6A,176.0%; AfBgl1.3, 128.0%). Thin-layer chromatography and high-performance liquid chromatography analyses of carboxymethyl cellulose (CMC) hydrolysis by the chimeric enzyme AfCel6A-L-AfBgl1.3 revealed glucose release, thereby confirming its bifunctional and synergistic activity. Adding the chimera to Celluclast® 1.5L boosted glucose yields by 16% (CMC), 22% (corncob), 46% (rice straw) and 101% (steam-exploded bagasse). This synergy mitigated product inhibition, enhanced saccharification, and makes chimeras valuable components of lignocellulosic biofuel cocktails. • AfCel6A-L-AfBgl1.3 was constructed by fusing AfCel6A and AfBgl1.3 via a flexible (GGGGS) 3 linker; • The chimera was 120% more tolerant to high glucose concentrations than the individual enzymes; • The AfCel6A and AfBgl1.3 domains retained ∼73% and 17% activity after 12 h at 50 °C; • Compared to the individual enzymes, the AfCel6A and AfBgl1.3 domains in the chimera exhibited 1.8 and 1.5-fold higher catalytic efficiency, respectively; • Supplementing an enzyme cocktail with the chimera enhanced rice straw and steam-exploded sugar bagasse hydrolysis by 46% and 101%