Design and characterizations of two novel cellulases through single-gene shuffling of Cel12A (EG3) gene from Trichoderma reseei

Abstract

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 singlegene 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°C, whereas the maximum activity of EG3-S2 was reported as 15.4-fold increase in native activity at 40°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.