Abstract：Low enzymatic activity and high cost are the two main problems that limit the industrial applications of cellulose. In order to enhance the enzymatic activity of neutral endoglucanase activity, error-prone PCR was conducted to engineer the Bacillus subtilis C-36 endoglucanase gene. Two optimum mutants, b-15 and b-28 were obtained, with an endoglucanase activity 2.1 folds and 3.6 folds increased, respectively. The sequence of b-15 endoglucanase gene showed six nucleotide substitutions leading to four mutated amino acids; and b-28 endoglucanase gene showed one nucleotide substitution leading to one mutated amino acid. According to the 3D structure of endoglucanase mimicked by SWISS-MODEL, the four mutated amino acids of b-15 were either located at the corner between the fourth and fifth α-helix in the catalytic domain or at the fifth β-fold or the corner between the ninth and tenth β-fold in the carbohydrate-binding domain. And the mutation of b-28 was located at the fourth β-fold in the carbohydrate-binding domain. Following the orthogonal experiment, the mutant b-15 and b-28 could reach to an endoglucanase activity of 4.542 U/mL and 5.136 U/mL through fermentation, respectively, both of which were much higher than the wild-type control. These results have provided a base for further research of endoglucanase.
