A Study On The Enzymatic Properties Of Vibrio Parahaemolyticus Phospholipase D And The Function Of C,N-terminal Peptides On Its Substrate Selectivity

Phospholipase D?PLD;EC 3.1.4.4?is a class of enzymes that catalyze the hydrolysis of dimethyl phosphate bonds,which can be used as a variety of phospholipid and phospholipid derivatives,the action point is phosphoric acid to replace the ester bond between the base,the product is glycerides phosphate and hydroxyl compounds.As an important industrial enzyme,it has great application in food and pharmaceutical industry.Phospholipase D has a wide distribution,among which the microbial source of phospholipase D has caused the main concern.At present,the research and development of microbial sources of PLD is mainly concentrated in terrestrial sources,the report of marine microbial source of PLD is still in a blank.The abuntant microbial resources existed in the ocean provide a broad development space for us.It is of great value and significance to study and develop novel PLDs from marine microorganism.Taking the phospholipase D-VpPLD?GenBank:EXJ48329.1?from Vibrio parahaemolyticus as the research object of present study.To explore the effect of?His?₆-Tag on the adsorption properties and hydrolysis properties of recombinant proteins,three kinds of?His?₆-Tag recombinant VpPLDs?VpPLD-WT-?His?₆,?his?₆-VpPLD-WT,?His?₆-VpPLD-WT-?His?₆?were constructed at the same time by Escherichia coli recombinant expression system.It was found that VpPLD with?His?₆-Tag at N-terminus tend to retain on the interface of air-water compared to?His?₆-Tag at C-terminus.VpPLD with?His?₆-tag at N-terminus had a stronger insertion capacity into DMPS and DMPE monolayers.However,the VpPLD with?His?₆-Tag at C-terminus is stronger in the binding ability on DMPS and DMPC monolayers.When?His?₆-Tag is located at C-terminus,the selectivity of VpPLD to hydrolyze the phospholipids with different acyl chain length was changed and prefer to hydrolysis DMPC,DSPC and DOPC,When the?His?₆-Tag is located at N-terminus or both N-terminus and C-terminus of VpPLD,the selectivity of VpPLD to different polar head phospholipid substrates was also changed,making it more prefer to hydrolysis DMPC,DPME and DPMG.Protein sequence analysis of the VpPLD indicated that the C,N-terminal peptide segment of VpPLD is different from other PLDs that has been reported.In order to investigate the effect of C,N-terminal peptides on enzymatic activity of VpPLD,VpPLD mutants with C,N-terminal peptides truncation were constructed.Based on the monolayer technique,the adsorption properties of these mutants on different phospholipid substrates were characterized.Results indicated that the absence of C-terminal peptides of VpPLD didn't change on its binding selectivity to different phospholipid monolayers.While removing beta-sheet structure?aa434-469?and helix structure?aa469-487?of VpPLD,the binding affinity of VpPLD was enhanced to each phospholipid substrate.In addition,removing either the loop structure?aa1-25?or helix structure?aa26-34?of VpPLD changed the binding preference of VpPLD from DMPS/DMPC to DMPE.Moreover,based on the method of detecting enzymatic activity with ironsalicylic acid,the hydrolysis properties of truncated mutants to different phospholipid substrates were characterized.Result indicated that missing C-terminal peptides of VpPLD changed its selectivity to phospholipid substrates,and mutants prefer to hydrolyze DOPC,DMPS and DMPC.Deleting the N-terminal peptides of VpPLD made the enzyme prefer to hydrolyze DMPS.