Enzymatic Preparation Of Glycerylphosphorylcholine In Aqueous Medium And Acyl Migration Mechanism

L-alpha glycerylphosphorylcholine （L-α-GPC）, comprised of choline, glycerol, and phosphate, is wellknown as the precursor for producing acetylcholine and phosphatidylcholine （PC） in the body, and forhaving important medical applications in neurological and psychiatric disorders of the human brain, such asalzheimer’s disease, cerebellar ataxia, schizophrenia, and bipolar affective disorder. These potential,valuable uses for L-α-GPC have prompted exploration into its possible role in the brain. Unfortunately,L-α-GPC is scarce in natural sources. The present work described a simple and cost-effective method forproducing L-α-GPC by the green enzymatic technology and the resins-silica gel column chromatographyassociated purification technology, which solved the problem of the low conversion rate, low purity, andenvironmental pollution. Meantime, the amino acid sequence of phospholipase A₁was determined, and thecatalytic mechanism of activity center, auto-acyl migration mechanism, and the adsorption kinetics andthermodynamics of silica gel were also studied.An environmental friendliness enzymatic preparation of L-α-GPC was established in this study.L-α-GPC was first produced by phospholipase A₁hydrolysis of phosphatidylcholine （PC） in an aqueousmedium. PC and L-α-GPC were quantitative analyzed by HPLC-ELSD. The effects of substrateconcentrations, temperature, enzyme loading, and dosage of CaCl₂on the PC conversion and L-α-GPCyield were discussed. Based on the single-factor experiments, next four process variables significantlyaffected the L-a-GPC yield and were used to develop the experimental design: substrate concentrations（mg/mL, w of soy lecithin powder/v of deionized water）, reaction temperature （oC）, enzyme loading （U/mL,U/v of total reaction solution）, and dosage of CaCl₂（mg/mL, w/v of total reaction solution）. Experimentswere designed by Design Expert7.0software for modeling and optimization of the effects of theexperimental factors on L-a-GPC yield. The optimal condition was confirmed as follows: reaction time3.5h, temperature53oC, enzyme loading28.2U/mL, substrate concentration51.5mg/mL, and dosage ofCaCl₂1.9mg/mL; the L-a-GPC yield increased by96.8%, which was close to the amount predicted by themodel. Meanwhile, kinetic model was established. On the base of Arrhenius empirical equation, theactivation energy of phospholipase A₁hydrolysis was5.96kJ/mol. Briggs-Haldane steady enzymatic modelwas fitted for enzymatic hydrolysis process. Kinetic parameters K_m, V_maxand kIwas4.02×10^-2mol/L,10.05mol/（L·min） and1.33×10²mol/L, respectively.However, the purity of enzymatic reaction product is not high and requires further purification.Therefore, resin-silica gel column chromatography combinations for purifying L-α-GPC were established.The optimal condition for eliminating Ca²⁺: the ability processing enzymatic reaction solution of001×7resin,25.1mL/g; the concentration of solution,720μg/mL; and adsorption flow rate,1mL/min. Theoptimal condition for eliminating Cl^-: the ability processing enzyme reaction solution of D311resin,29.6mL/g; the concentration of solution,997μg/mL; and adsorption flow rate,1mL/min. The optimalcondition for L-α-GPC purification by silica gel: eluent,80%aqueous methanol （methanol: water, v:v）;loading amount,24.4mg/g; loading concentration,16.6mg/mL; and the mobile phase flow rate,2mL/min.The final L-α-GPC was decolorized with activated carbon at60oCfor1.5h. The resin and silica gel showedremarkable ability for L-α-GPC isolation after10uses. Finally, colorless L-α-GPC was obtained at99.8%purity,69.8%recovery, a specific rotation of-2.5°, Ca²⁺residue5.8ppm, and Cl^-residue8.7ppm via afour-step procedure. The resulted L-α-GPC was charactered by ultra performance liquidchromatography-electrospray ionization-quadrupole-time of flight-mass mass spectrometry （UPLC-Q-TOFMS/MS）, attenuated total reflection fourier-transform infrared spectroscopy （ATR-FTIR）, and nuclearmagnetic resonance （NMR）. The results indicated that it was in good agreement with the standard.The successful determination of the amino acid sequence of the phospholipase A₁was carried out byMALDI-TOF/TOF MS and TOF-MS/MS. The spatial structure of3D model of phospholipase A₁wassimulated by the3D software and homology modeling. Four space model was obtained by the3D software, including Ball, Stick, Ribbons1, and Ribbons2. However, this technique cannot continue to explore thespatial structure of the enzyme active center. Therefore, the spatial structure of phospholipase A₁was thensimulated by the homology modeling using1G6T （PDB） as a template. The spatial structure of the enzymeactivity center was further simulated by molecular docking techniques. The results showed that the activitycenter contained three amino acid residues, including Asp202, His280and Ser141. The catalyticmechanism of the phospholipase A₁was inferred through the spatial structure of the molecules and theamino acid residues that it was more similar to the “Ser-His-Asp” triad catalytic mechanism of lipase.Meantime, the hydrolysis process of soybean lecithin powder was analyzed and studied bythree-dimensional microscope from the microscopic point of view.The specifity of phospholipase A₁was testified by quantitative analysis of¹³C NMR. PhospholipaseA₁catalytic hydrolysis progress was monitored by HPLC-RID. The result showed that Sn-1fatty acid ofPC was hydrolyzed by phospholipase A₁to produce Sn-2-LPC, then spontaneous acyl migration occurredin which the Sn-2acyl moved to the Sn-1position to form Sn-1-LPC, and finally the enzyme hydrolyzedthe remaining acyl group to produce L-α-GPC. The effect of some reaction factors, such as the solutionpolarity, pH, reaction temperature, substrate concentration and reaction time on acyl migration regulationand inhibitory mechanism were investigated.The adsorption kinetics and thermodynamics of L-α-GPC on silica gel column chromatography wereresearched. The result showed that the adsorption rate of L-α-GPC was governed by interface diffusioncontrol rate model and adsorption time t was in the good linear relationship with1-3（1-X）2/3+2（1-X）. Theadsorption isotherm of L-α-GPC could be described with the equation of Langmiur （R2>0.99）. When theloading concentration was constant, the adsorption ability of silica gel was decreased with the increasing oftemperature, that is to say, the adsorption of silica gel for L-α-GPC was exothermic reaction. Theadsorption thermodynamics analysis on the equation of Clausius-Clapeyron showed that, under differentadsorption capacity of80mg/g,160mg/g and240mg/g, the adsorption enthalpy change （△HAm） was24.85kJ/mol,23.82kJ/mol,22.72kJ/mol, respectively.