Enzymatic Properties Of Vibrio Harveyi Phospholipase D And Effects Of Terminal Peptide Chains On Enzymatic Properties

Vibrio harveyi belongs to the genus Vibrio in the Vibrioaceae family.It is mainly infected with shrimps and fish.It is widely distributed in the marine environment and is a common pathogen in seawater.It is mainly found in free water bodies and floating animals and plants,and seabed sediments.The PLD in this study was derived from the pathogenic bacteria Vibrio harveyi,and its homology modeling analysis revealed that it has a unique structure different from other PLDs—the C-terminal highlights a peptide chain,and the C-terminal and N-terminal peptide chains are related to their catalytic activity.Therefore,characterizing the enzymatic properties of Vibrio harveyi phospholipase D?VhPLD?and revealing that the functions of the C-terminal and N-terminal peptide chains in the development of enzyme activity not only help to understand the structural and functional relationship of the enzyme,but also lays a foundation for future molecular transformation and industrial application of phospholipase.It is of great guiding significance for the prevention and control of diseases caused by Vibrio harveyi for this enzyme.In this paper,the phospholipase VhPLD was used as the research object,and the recombinant expression of VhPLD was obtained by recombinant E.coli expression.Based on this,the enzymatic properties were analyzed by phosphatidyl p-nitrophenol?PpNP?method and monolayer technique.By designing and constructing C-terminal and N-Terminal peptide deletion mutants,comparative analysis of the effects of C-terminal and N-terminal peptides on the enzymatic properties of phospholipase VhPLD before and after deletion.The specific research content and results are as follows:?1?Construction and enzymatic properties of wild-type VhPLD recombinant expression strains.Firstly,the phospholipase VhPLD E.coli recombinant expression strain pET21a-VhPLD SHuffle T7 was constructed,and the target protein was obtained by Ni2+affinity chromatography,G25 and Q column anion exchange chromatography.Secondly,the optimum reaction conditions were determined using PpNP as a substrate.The results showed that the optimal reaction temperature of VhPLD phospholipase activity was 45°C and pH was8.0;benzene had an activation effect on the enzyme activity of VhPLD-WT.Finally,the interface adsorption parameters were determined by monolayer technique.The catalytically active central histidine mutant?VhPLD-H157A?was designed by site-directed mutagenesis to avoid the influence of hydrolysis on the adsorption process.When DMPS was used as the substrate,the maximum insertion pressure?MIP?value was the largest.The results showed that VhPLD-H157A had the strongest binding ability to DMPS monolayer;The synergy factor a of the four substrates used to study the enzyme protein?dimyristoyl phosphatidylcholine?DMPC?,dimyristoyl phosphatidylethanolamine?DMPE?,dimyristoyl phosphatidylglycerol?DMPG?and DMPS?is greater than 0,indicating the positive effect of the four substrates and the enzyme protein;the??₀ of the four substrates is greater than their equilibrium surface pressure,further confirming that the enzyme protein migrates more easily from the underwater phase to the interface and binds to the phospholipid.?2?Effect of C-terminal peptide chain on the enzymatic properties of VhPLD.Firstly,The C-terminal truncated E.coli recombinant expression strain pET21a-VhPLD-??472-483?SHuffle T7 and pET21a-VhPLD-??437-483?SHuffle T7 were successfully constructed,and the pure protein was obtained.Secondly,the optimum reaction conditions were determined using PpNP as a substrate.The results showed that the optimum temperature condition of VhPLD-??472-483?was 50°C,and the optimum temperature condition of VhPLD-??437-483?enzyme activity was 60°C;VhPLD-??472-483?and VhPLD-??437-483?of the optimum pH was 8.0;ethyl acetate and diethyl ether significantly promoted the enzyme activity of the C-terminal truncation mutants.Finally,monolayer technology experiments show that:Comparing the MIP values,the deletion of the peptide chain 437-472 has a positive effect on the interfacial binding of VhPLD to DMPE;comparing the synergy factor a,it was found that the peptide chain 437-472 promoted the binding of VhPLD to DMPE;comparing??₀ showed that the deletion of the peptide chains 472-483 and 437-472 were favorable for their binding to DMPG.?3?Effect of N-terminal peptide chain on the enzymatic properties of VhPLD.Firstly,The N-terminal truncated E.coli recombinant expression strain pET21a-VhPLD-??25-39?SHuffle T7 was successfully constructed,and a purer target protein was obtained.Secondly,the optimum reaction conditions were determined using PpNP as a substrate.The results showed that the optimum temperature condition of VhPLD-??25-39?was 45°C,and the optimum pH of VhPLD-??25-39?was 8.0;n-hexane promoted the enzyme activity of N-terminal truncation mutants.Finally,monolayer technology experiments show that:Comparing the MIP values,the deletion of the N-terminal peptide chain facilitates the interface of VhPLD with DMPC and DMPG;comparing the synergistic factor a,the deletion of the N-terminal peptide chain is more favorable for its binding to DMPE;comparing??₀,Deletion of the N-terminal peptide chain is more advantageous for its binding to DMPC.