To improve the Spent mushroom substrate (SMS) saccharification, cloning, recombinant expression in Escherichia coli and characterization of two new GH5 family cellulases (Cel1 and Cel2) were performed. Based on enzymes properties, Cel2 was selected for the generation of 30,000 random mutants by directed evolution in order to develop improved biocatalysts. Error-prone Polymerase Chain Reaction was used for diversity generation in cel2 gene and the screening for activity of mutants allowed selection of 63 improved variants that were subjected to a scale up production. Among these, 13 clones exhibited two-fold higher activity than Cel2 and a higher thermoresistance after 72h. The performances of these mutants in the hydrolysis of pretreated SMS/ wheat straw (40/60) were compared to the wild type Cel2 in conjunction with a commercial enzymatic mixture (MetZyme® SUNO™ BOOSTER 144). All the mutants exhibited a glucose yield two-fold or four fold higher than wild-type Cel2 after 72h of incubation.