Cellulases have great potential to be widely used for industrial applications. In general, naturally occurring cellulases are not optimized and limited to meet the industrial needs. These limitations lead to demand for novel cellulases with enhanced enzymatic properties. Here, we describe the enzymatic and structural properties of two novel enzymes, EG3_S1 and EG3_S2, obtained through the single-gene shuffling approach of Cel12A(EG3) gene from Trichoderma reseei. EG3_S1 and EG3_S2 shuffled enzymes display 59 and 75% identity in protein sequence with respect to native, respectively. Toward 4-MUC, the minimum activity of EG3_S1 was reported as 5.9-fold decrease in native at 35 degrees C, whereas the maximum activity of EG3_S2 was reported as 15.4-fold increase in native activity at 40 degrees C. Also, the diminished enzyme activity of EG3_S1 was reported within range of 0.6-to 0.8-fold of native and within range of 0.5-to 0.7-fold of native toward CMC and Na-CMC, respectively. For EG3_S2 enzyme, the improved enzymatic activities within range of 1.1- to 1.4-fold of native and within range of 1.1-to 1.6-fold of nativewere reported toward CMC and Na-CMC, respectively. Moreover, we have reported 6.5-fold increase in the kcat/Km ratio of EG3_S2 with respect to native and suggested EG3_S2 enzyme as more efficient catalysis for hydrolysis reactions than its native counterpart.