本研究探讨了(His)6-tag位置和N/C-末端截短对重组副溶血弧菌磷脂酶D（VpPLD）酶学特性的影响。研究发现：(His)6-tag位置对于VpPLD的活性有较大影响，其中(His)6-tag位于N-末端时测得酶活力最高，而位于C-末端时最低，且活性差异不依赖于水解底物。此外，(His)6-tag位于C-末端导致VpPLD的最适反应pH从7.0变为8.0；删除VpPLD成熟蛋白N-末端前34个氨基酸能显著增强VpPLD对1,2-二肉豆蔻酰基-sn-甘油-3-磷脂酰-L-丝氨酸（DMPS）的水解活性，但对于其他磷脂的水解活性并没有明显改变；删除VpPLD成熟蛋白C-末端肽段（469~487）会显著降低其对磷脂底物的催化活性；与(His)6-VpPLD-WT和(His)6-VpPLD-Δ469-487相比，(His)6-VpPLD-Δ451-487对1,2-二棕榈酰基-sn-甘油-3-磷脂酰胆碱（DPPC）、1,2-二肉豆蔻酰基-sn-甘油-3-磷脂酰-(l'-rac-甘油)（DMPG）和1,2-二肉豆蔻酰基-sn-甘油-3-磷脂酰乙醇胺（DMPE）的选择性均显著降低，该结果表明C-末端肽段（451-469）对VpPLD的底物选择性发挥重要影响作用。以上结果表明，(His)6-tag的位置以及N/C-末端肽段对于重组副溶血弧菌磷脂酶D的催化活性和底物选择性具有重要影响作用。

In the present study, the effects of (His)6-tag’s location and N/C-terminal truncation on the enzymatic characteristics of recombinant Vibrio parahaemolyticus phospholipase D (VpPLD) were investigated. It was found that (His)6-tag’s location had a great influence on the activity of VpPLD. When (His)6-tag was located at the N-terminal, VpPLD exhibited the highest activity. The lowest VpPLD activity was detected when (His)6-tag was located at the C-terminal of VpPLD, with the differences in activity independent on the hydrolysis substrate. In addition, the optimum pH of VpPLD was changed from 7.0 to 8.0 due to the location of (His)6-tag at its C-terminal. When the first 34 amino acids at the N-terminus of mature VpPLD were deleted, the hydrolytic activity of VpPLD towards 1,2-Dimyristoyl-sn-glycero-3-phospho-L- serine (DMPS) was significantly enhanced, although no change was found in the hydrolytic activity towards other phospholipids. When the C-terminal amino acids (469~487) of VpPLD were deleted, its catalytic activities towards all the tested phospholipids decreased significantly. Compared with (His)6-VpPLD-WT and (His)6-VpPLD-Δ469-487, the selectivity of (His)6-VpPLD-Δ451-487 for1,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (DMPG) and 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE) decreased significantly, indicating that the C-terminal amino acids (451~469) had a great impact on the substrate selectivity of VpPLD. The above results showed that the position of (His)6-tag and the N/C-terminal peptide had an important effect on the catalytic activity and substrate selectivity of the VpPLD.

国家重点研发计划项目（2018YFC0311100）；国家自然科学基金项目（31972014）