Improving Endoglucanase Activity from Bacillus subtilis by Error-Prone PCR

Abstract:

Endoglucanase cooperates with cellobihydrolase and β-1,4-glucosidase to translate cellulose to glucose completely.Now lots of researchers found that low enzymatic activity and high cost are the two main problems that restrict the industrial applications for cellulase.In order to enhance the enzymatic activity of the neutral endoglucanase activity,error-prone PCR strategy was conducted on Bacillus subtilis C-36 endoglucanase gene.The mutant F-10 with endoglucanase activity improved 4.2 folds respectively were obtained.The sequence of F-10 showed five nucleotides substitutions to two mutated amino acids.SDS-PAGE showed genes are correctly expression.Enzymatic properties of mutant F-10 showed that:The optimal temperature and pH value changed compared with the original enzyme,it’s optimal temperature and pH is 45 ℃ and 5.6,respectively.The enzyme maintained over 80% of the original enzymatic activity after incubated from pH 4.5 to 10.0 at 50 ℃ for 30 min and the enzyme maintained over 80% of the original enzymatic activity in 60~75℃,but when temperature upon 75 ℃,then enzymatic activity decreased sharply.