title: Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR.
abstract: 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, Cel8ME15 and Cel8ME18, respectively showing 1.42 fold and 1.61 fold increased activities, were obtained. Through sequence analysis, it was found that Cel8ME15 had two mutations, with the residues Ala9 and Glu353 respectively substituting the residues Val9 and Lys353 of Cel8M; while Cel8ME18 had one mutation with the residue Ser117 replacing the residue Gly117 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 Cel8ME15 and Cel8ME18. 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.
authors.affiliation: Provincial Key Laboratory of the Conservation and Exploitation Research of Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu, Anhui, People's Republic of China. Electronic address: linling8@mail.ahnu.edu.cn.
authors.forename: Ling
authors.initials: L
authors.lastname: Lin
authors.affiliation: Provincial Key Laboratory of the Conservation and Exploitation Research of Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu, Anhui, People's Republic of China.
authors.forename: Chenggen
authors.initials: C
authors.lastname: Fu
authors.affiliation: Guangdong Rongda Bioengineering Company Limited, Qingyuan, Guangdong, People's Republic of China.
authors.forename: Weiqian
authors.initials: W
authors.lastname: Huang
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D002480:Cellulase
keywords: 3D-Structural modelling; Congo red; Endoglucanase; Error-prone PCR