Cloning,Expression, Enzymatic Properties And Synergistic Interaction Of Glucanase CEL8A And CEL48B

In this study,endoglucanase gene cel8A from Enterobacter cloacae (E. cloacae) and exoglucanase gene cel48B from Bacillus licheniformis(B. licheniformis) were studied.Firstly, Endoglucanase gene cel8A from E. cloacae was cloned and expressed,and the enzymatic properties was studied,including the optimum pH,the optimum temperature、Km and specific activity. More over, exoglucanase gene cel48B from B. licheniformis was cloned and expressed,and the enzymatic properties were studied.Then,three site-directed mutagenesis E38Q、E49Q、D228N were performed, three mutant enzyme were compared with the wild-type enzyme. Finally,the synergistic interaction of endoglucanase and exoglucanase was studied.The endoglucanase gene sequence were obtained from NCBI (National Center of Biotechnology Information),and this gene with size of1.0kb was amplified using PCR technology and cloned into expression vector pQE30for construction of plasmid pQE30-cel8A. The recombinant plasmid was expressed in Escherichia coli XL1-blue (E. coli XL1-blue). The recombinant protein was purified by nickel affinity chromatography, which showed a clear single band on SDS-PAGE having a molecular weight of37kD. It was consistent with the expected prediction. Hydrolysis halo was formed in the CMC-Na substrate plate by E. coli XL1-blue/pQE30-ce18A.The enzymatic properties of purified enzyme were determined. Its optimum temperature、optimum pH、Km、Vmax值and specific activity were40℃、7.0、46.95mg/mL、0.38μmol/min and3.56U/mg, respectively.In the same way, the exoglucanase gene sequence were obtained from NCBI,and this gene with size of2.1kb was amplified using PCR technology and cloned into expression vector pQE30for construction of plasmid pQE30-cel48B.The recombinant plasmid was expressed in E. coli XL1-blue.The recombinant protein was purified by nickel affinity chromatography,which showed a clear single band on SDS-PAGE having a molecular weight of77kD.It was consistent with the expected prediction.The enzymatic properties of purified enzyme were determined.The exoglucanase cel48B showed activity with substrate cellotetrose and amorphous cellulose, respectively. The thin layer chromatography(TLC) analysis showed that the products from substrate catalyzed by the enzyme were mixtures contained glucose and cellobiose.The result of TLC demonstrated that cel48B have enzymatic activity of exoglucanase.Then,the site-directed mutagenesis of cel48B were performed,three site (E38Q、E49Q and D228N,which near to the active site) were mutated.Compared with the wild-type enzyme,the mutant enzyme E38Q and D228N had not enzymatic activity,while the mutant E49Q remained its enzymatic activity.In the study of synergistic interaction of endoglucanase and exoglucanase, their synergistic interaction was less than the sum of them.The synergistic interaction of endoglucanase and exoglucanase need to make further efforts.