School of Bioengineering, Dalian University of Technology, Dalian 116024, China.
School of Bioengineering, Dalian University of Technology, Dalian 116024, China; School of Ocean Science and Technology, Dalian University of Technology, Panjin 124221, China.
Int J Biol Macromol. 2021 Jul 1;182:1473-1483. doi: 10.1016/j.ijbiomac.2021.05.110. Epub 2021 May 19.
κ-Carrageenan oligosaccharides with many excellent biological properties could be produced by κ-carrageenases selectively. In this study, based on the encoding gene of full length κ-carrageenase obtained from Pseudoalteromonas sp. ZDY3 and the reported mature secreted κ-carrageenase composed of 275 amino acid residues (N26-T300), CgkPZ_GH16 was expressed in E. coli, but no soluble active protein could be detected. Fortunately, the signal peptide of wild-type κ-carrageenase was recognized, and cleaved in the soluble and folding form in E. coli, the K and k values of CgkPZ_SP_GH16 was 1.007 mg/mL and 362.8 s. By molecular dynamics simulations, it was showed that YjdB domain might affect the activity of κ-carrageenase. Due to the absence of mature processing modification system in E. coli, YjdB was remained in recombinant full length κ-carrageenase, and the lost catalytic efficiency of CgkPZ was compensated by expression level and thermal stability. Interestingly, CgkPZ_GH16_YjdB was expressed soluble without the signal peptide, which indicated that YjdB could contribute to the expression and folding of κ-carrageenase. These results provide new insight into the effects of different modules of κ-carrageenase on the expression and properties of enzyme.
κ-卡拉胶寡糖具有许多优异的生物学特性，可以通过κ-卡拉胶酶有选择地生产。在本研究中，基于从假交替单胞菌 ZDY3 获得的全长 κ-卡拉胶酶的编码基因和报道的由 275 个氨基酸残基组成的成熟分泌型 κ-卡拉胶酶（N26-T300），在大肠杆菌中表达了 CgkPZ_GH16，但未检测到可溶的活性蛋白。幸运的是，野生型 κ-卡拉胶酶的信号肽被识别，并以可溶性和折叠形式在大肠杆菌中被切割，CgkPZ_SP_GH16 的 K 和 k 值分别为 1.007 mg/mL 和 362.8 s。通过分子动力学模拟表明，YjdB 结构域可能影响 κ-卡拉胶酶的活性。由于大肠杆菌中缺乏成熟的加工修饰系统，YjdB 残留在重组全长 κ-卡拉胶酶中，而 CgkPZ 的催化效率损失则通过表达水平和热稳定性得到补偿。有趣的是，CgkPZ_GH16_YjdB 没有信号肽也可以可溶性表达，这表明 YjdB 可以有助于 κ-卡拉胶酶的表达和折叠。这些结果为不同模块的 κ-卡拉胶酶对酶的表达和性质的影响提供了新的见解。
