Wang Xinye, Chen Yinqi, Nie Yao, Xu Yan
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
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.
Protein Expr Purif. 2019 Mar;155:72-77. doi: 10.1016/j.pep.2018.09.011. Epub 2018 Sep 21.
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.
普鲁兰酶是众所周知的淀粉脱支酶，广泛用于水解淀粉、普鲁兰、支链淀粉和其他寡糖中的α-1,6-糖苷键。大肠杆菌是用于产生目标酶的常用异源表达宿主。然而，由于大肠杆菌细胞外分泌能力较弱，必须对细胞进行破碎才能获得目标酶。为了便于后续对普鲁兰酶进行表征和应用，提高其在大肠杆菌中的分泌效率是首先必须解决的主要挑战。考虑到涉及信号肽的转运机制以及细胞壁的通透性，我们采用了两种策略来提高大肠杆菌中普鲁兰酶的细胞外分泌：（1）在目标酶的N端融合带负电荷的肽以引导其穿出大肠杆菌细胞膜；（2）修饰细胞壁的组成以增加其通透性。在本研究中，这两种策略均对普鲁兰酶的细胞外分泌产生了积极影响。在N端融合带负电荷的肽后，与未融合肽的普鲁兰酶相比，细胞外酶活性提高了4倍以上。通过修饰细胞壁的通透性，细胞外酶活性提高了12倍。此外，这两种策略还用于提高截短型普鲁兰酶ΔN106的分泌效率，该酶比全长蛋白具有更高的酶活性。所采用的策略对于提高重组大肠杆菌中目标蛋白的细胞外分泌效率具有重要意义。
