School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Collaborative Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu 214122, China.
Int J Biol Macromol. 2019 Jul 1;132:759-765. doi: 10.1016/j.ijbiomac.2019.04.002. Epub 2019 Apr 3.
1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl β-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.
1,4-α-葡聚糖分支酶（GBE，EC.2.4.1.18）在淀粉和糖原的合成中起着关键作用，许多研究人员已将其在大肠杆菌中作为胞内酶过表达。在这项研究中，发现来自嗜热葡萄糖芽孢杆菌和嗜热盐单胞菌的 GBE 在大肠杆菌中单独表达时会分泌到培养基中，尽管没有任何信号肽。事实上，尽管生物信息学工具预测这两种蛋白质都将定位于细胞质，但发现无需添加诱导剂异丙基β-D-硫代半乳糖苷即可实现高水平表达和非经典分泌。进一步的实验表明，分泌是一个两步过程，通过周质空间发生。结果排除了 Sec 途径或 TAT 途径的参与。相反，研究结果表明细胞膜完整性与两种 GBE 的分泌之间存在关系，并表明它们的 N 端在其表达和分泌中起重要作用。
