Abstract: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.
