Expression,purification And Biochemical Properties Of A Novel Glycerophosphodiester Phosphodiesterase

Glycerophosphodiesterase phosphodiesterases(GDPDs;EC 3.1.4.46)are ubiquitous among prokaryotic and eukaryotic organisms.The primary function of these enzymes is the degradation of various glycerophosphodiesters to glycerol-3-phosphate(G3P)and corresponding alcohol moiety(e.g.,choline,serine,ethanolamine,inositol).It is a key enzyme in the phospholipid metabolism pathway which maintains G3 P concentrations,for phospholipid remodelling and synthesis.In this study,a novel GDPD from Pyrococcus furiosus DSM 3638(pfGDPD)was successfully expressed and we extensively characterized the pfGDPD protein,including its phylogenetic relationship and enzyme characteristics.The results were showed as follows: 1.Bioinformatics Analysis and Construction of Expression Strain of GDPD-PF2003Analysis of sequence alignment and phylogenetic tree by GDPD with other sources found that GDPD-PF2003 has a conserved sequence shared by the GDPD superfamily.That means GDPD-PF2003 is a member of the GDPD superfamily.The predicted molecular weight of the protein was 29.14 KDa and the isoelectric point pI was 5.19.The GDPD-PF2003 homologous source derived from Thermococcus kodakarensis KOD1 GDPD was selected as template for homology modeling,and the GDPD-PF2003 three-dimensional structure model was successfully constructed.The plasmid pET28a-GPDP-PF2003/DH5? was extracted and successfully transformed into E.coli Shuffle T7 to construct the expression strain of GDPD-PF2003.2.Expression and Purification of GDPD-PF2003The fermentation conditions of GDPD-PF2003 were optimized,and the optimized fermentation scheme and protein purification program were optimized.The optimal expression conditions of the recombinant plasmid pET28a-PF2003/E.coli Shuffle T7 in the 100 ml fermentation system(placed in the 500 mL Erlenmeyer flask)were as follows: After adding 0.1 mM IPTG in the logarithmic growth phase,temperature of 28?,200 rpm culture 4 h after the bacteria.Under these conditions,the optimal protein expression was obtained.The protein purification protocol of GDPD-PF2003 was established by observing the corresponding purification conditions.The expressed protein was purified by Ni2 +-NTA column affinity adsorption chromatography,Sephadex G25 gel chromatography and tangential flow ultrafiltration system To obtain a purity of more than 95% of the GDPD-PF2003 protein.3.Enzymatic Properties of GDPD-PF2003The enzymatic properties of GDPD-PF2003 were studied,and the enzymatic properties of the enzyme were obtained.The optimum reaction temperature of GDPD-PF2003 is 55?,which is lower than or higher than this temperature.When the temperature is higher than 80?,the enzyme activity is lost.The half-lives of the enzyme at 50?,55? and 60? were 17.6 h,17.8 h and 0.93 h,respectively.The optimum pH for GDPD-PF2003 is 8.5,and the enzyme activity is lost when the pH is less than 7.0.The experimental results show that GDPD-PF2003 is a metal ion-dependent enzyme,and it can not show the catalytic effect on BpNPP in the absence of metal ions.The activation of Mn2 + in several ions was the most significant,and the optimum activity was at 5 mM.In this study,the effect of ether on the activity of GDPD-PF2003 was significantly inhibited in the organic solvent.In addition,the enzyme was more resistant to organic solvents.The circular dichroism spectroscopy of GDPD-PF2003 showed that the protein was stable at 55? and the calculated Tm was 59.6?.4.Hydrolysis Characteristics of Different Phospholipid SubstratesThe effects of GDPD-PF2003 on the hydrolysis of PC,GPC,1-LPC and 2-LPC were investigated.The effects of GDPD-PF2003 on different substrates were discussed.Among the four different substrates,GDPD-PF2003 had the highest activity of GPC hydrolysis,followed by 1-LPC,and it was inactive for 12 hours under experimental conditions.Indicating that the enzyme has the activity of lysophospholipase D in the same enzyme.By observing the molecular docking of the substrate with the GPDP-PF2003 model,GPC showed a higher activity because the substrate molecule was more stable with the enzyme-catalyzed pocket.