Enzyme and Microbial Technology, Vol.86, 52-58, 2016
Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR
Endoglucanase is a key enzyme involved in cellulose hydrolysis and can be used in multiple industrial fields. In this study, we used error-prone PCR to engineer the endoglucanase, Cel8M, from Escherichia coli. The Cel8M belongs to the glycoside hydrolase family 8 and shows 99% identity with the reported endoglucanase from E. coli K12. Through screening of approximately 10,000Cel8M variants, two variants, Cel8M(E15) and Cel8M(E18), respectively showing 1.42 fold and 1.61 fold increased activities, were obtained. Through sequence analysis, it was found that Cel8M(E15) had two mutations, with the residues Ala(9) and Glu(353) respectively substituting the residues Val(9) and Lys(353) of Cel8M; while Cel8M(E18) had one mutation with the residue Ser(117) replacing the residue Gly(117) of Cel8M. Based on the analysis of the predicted 3D structure of Cel8M, it was suggested that changes of K353E and G117S might directly affect the substrate binding affinity and therefore contribute to the improved activities of Cel8M(E15) and Cel8M(E18). Based on all the results we had, it is believed that this study should provide a useful reference for the future engineering of other endoglucanases from glycoside hydrolase family 8. (C) 2016 Elsevier Inc. All rights reserved.