For the purpose of a industrial process of cellulosic ethanol production, an efficient β-glucosidase was evolved by L-Shuffling starting from three parental genes (i.e., Chaetomium globosum glucosidase putative gene, Trichoderma reesei bgl1 gene, and Neurospora crassa glucosidase putative gene, named genes A, B, and C, respectively) originating from microbial biodiversity and showing 70% of identity at the amino acid level. Enzyme B (encoded by bgl1 gene) was chosen as a reference so that the backbone of the evolved enzymes would be based on this enzyme. Two rounds of L-Shuffling and colonies screening (20,000 colonies per round) on chromogenic glucose substrate were performed. Compared with native β-glucosidase, the most evolved enzyme has a 242-fold increased kcat for the pNPGlc substrate. After expression of this improved β-glucosidase in T. reesei, a new efficient enzymatic cocktail was secreted by the strain allowing for a 4-fold decrease in cellulase loading without any loss in hydrolysis performanc...