Artículo

One strategy to decrease both the consumption of crude oil andenvironmental damage is through the production of bioethanol from biomass.Cellulolytic enzyme stability and enzymatic hydrolysis play important roles in thebioethanol process. However, the gradually increased ethanol concentration oftenreduces enzyme activity and leads to inactivation, thereby limiting the final ethanolyield. Herein, we employed an optimized Two-Gene Recombination Process(2GenReP) approach to evolve the exemplary cellulase CBHI for practical bioethanolfermentation. Two all-round CBHI variants (named as R2 and R4) were obtained withsimultaneously improved ethanol resistance, organic solvent inhibitor tolerance, andenzymolysis stability in simultaneous saccharification and fermentation (SSF). Notably,CBHI R4 had a 7.0- to 34.5-fold enhanced catalytic efficiency (kcat/KM) in thepresence/absence of ethanol. Employing the evolved CBHI R2 and R4 in the 1Gbioethanol process resulted in up to 10.27 percent (6.7 g/L) improved ethanol yield (ethanolconcentration) than non-cellulase, which was far more beyond than other optimization strategies. Besides bioenergy fields, thistransferable protein engineering routine holds the potential to generate all-round enzymes that meet the requirement inbiotransformation and bioenergy fields.