Improvement of extracellular secretion efficiency of Bacillus naganoensis pullulanase from recombinant Escherichia coli: Peptide fusion and cell wall modification.


School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China; Suqian Industrial Technology Research Institute of Jiangnan University, Suqian, 223814, China. Electronic address: [Email]


Pullulanases are well-known starch-debranching enzymes that are widely used for hydrolysis of a-1,6-glycosidic linkages in starch, pullulan, amylopectin, and other oligosaccharides. Escherichia coli is a popular heterologous expression host for generating target enzymes. However, cells have to be disrupted to obtain the target enzyme due to the weak extracellular secretion of E. coli. In order to facilitate subsequent characterization and application of pullulanase, improving its secretion efficiency from E. coli is a major challenge that must first be solved. Considering the transport mechanism involving signal peptide as well as permeability of the cell wall, we adopted two strategies to improve the extracellular secretion of pullulanase from E. coli: (1) fusion of a negatively charged peptide at the N-terminal of the target enzyme to guide it out of the membrane of E. coli and (2) modification of the composition of the cell wall to increase its permeability. In this study, both strategies showed positive influence on the extracellular secretion of pullulanase. After fusing a negatively charged peptide at the N-terminal, the extracellular enzymatic activity increased by more than 4 times compared to the pullulanase without peptide fusion. By modifying the permeability of the cell wall, the extracellular enzymatic activity increased by 12 times. In addition, the two strategies were also used to improve the secretion efficiency of the truncated pullulanase ΔN106, which has higher enzymatic activity than the full-length protein. The strategies employed have valuable implications for increasing the extracellular secretion efficiency of target proteins from recombinant E. coli.


Amino acid tag,D,D-carboxypeptidase,Extracellular secretion,Negatively charged peptide,Pullulanase,