水相体系酶法制备甘油磷脂酰胆碱及酰基转移机理研究
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摘要
甘油磷脂酰胆碱（L-alpha glycerylphosphorylcholine, L-α-GPC）由胆碱、甘油和磷酸盐组成，是乙酰胆碱和磷脂酰胆碱合成的胆碱源。在治疗人体大脑的精神混乱和神经混乱方面具有重要的医药应用价值，如治疗阿尔茨海默氏症、小脑性共济失调、精神分裂症和双相情感障碍等，但是就作为药用原料及相关研究而言需要较高纯度的L-α-GPC。然而遗憾的是天然L-α-GPC的含量较少。本研究针对制备高纯L-α-GPC中存在的转化率低、纯度低及环境污染等方面的问题，创建了绿色生物制取技术和树脂-硅胶柱色谱联用纯化技术，进行了磷脂酶A_1氨基酸序列测定及活性中心催化机理、自动酰基转移机理、硅胶吸附动力学和热力学等方面的研究，开发出了高纯度的L-α-GPC产品。
首先，建立了一种水相体系磷脂酶A_1水解大豆粉末磷脂制备L-α-GPC的方法。通过研究pH、底物浓度、温度、磷脂酶A_1添加量和CaCl_2添加量等酶反应条件对磷脂酰胆碱（PC）转化率和L-α-GPC得率的影响，确定了磷脂酶A_1水解反应的显著影响因素，利用响应面对反应体系进行优化。得到最佳酶反应条件：底物浓度51.5mg/mL，反应温度53℃，磷脂酶A_1添加量28.2U/mL，CaCl_2添加量1.9mg/mL，反应时间3.5h，最终L-α-GPC得率为96.8%。同时，根据Arrhenius经验方程，计算出磷脂酶A_1水解反应的活化能为5.96kJ/mol；利用Briggs-Haldane稳态法酶催化模型对酶催化水解过程进行模拟，并计算出反应的动力学参数K_m，V_(max)和kI分别为4.02×10~(-2)mol/L、10.05mol/(L·min)和1.33×10~2mol/L。
采用树脂-硅胶柱色谱联用纯化技术对酶水解产物进行纯化，依次进行了树脂吸附Ca~(2+)和Cl~-、硅胶纯化、活性炭脱色。通过树脂吸附实验，确定了去除Ca~(2+)的最优条件：处理酶反应液的能力25.1mL/g，Ca~(2+)的初始浓度720μg/mL，吸附流速1mL/min。吸附Cl~-的最优条件：处理酶反应液的能力29.6mL/g，Cl~-的初始浓度997μg/mL，吸附流速1mL/min。阳离子交换树脂和阴离子交换树脂分别用HCl和NaOH再生。通过硅胶纯化实验，确定了去除其它磷脂和杂质的最优条件：洗脱剂80%甲醇、上样量24.4mg/g、上样浓度16.6mg/mL、洗脱流速2mL/min。硅胶在150℃的烘箱中，烘烤3h就可以使吸附饱和的硅胶再生。脱色条件：活性炭作脱色剂，60℃脱色90min。经过树脂-硅胶柱色谱联用四步纯化，最终L-α-GPC产品的纯度为99.8%、回收率为69.8%、比旋光度为-2.5o，Ca~(2+)残留5.8ppm，Cl~-残留8.7ppm。通过LC-MS/MS、ATR-FTIR、~1H NMR和~(13)C NMR等检测技术对L-α-GPC产品进行表征，结果与L-α-GPC标准品一致。
采用MALDI-TOF/TOF MS和TOF-MS/MS测定磷脂酶A_1的氨基酸序列，利用3D软件和同源模建技术分别对磷脂酶A_1的氨基酸序列进行空间结构模拟。通过3D软件模拟得到Space fillin、BallStick、Ribbons1和Ribbons2等四种空间模型。同时，利用同源模建技术成功设计出磷脂酶A_1的3D空间构型；在此基础上，进一步利用分子对接技术研究了磷脂酶A_1的活性中心，得到了Asp202、His280和Ser141三个氨基酸残基组成的活性中心及其空间结构。经过氨基酸残基组成和空间结构分析，推导出磷脂酶A_1水解PC的“Ser-His-Asp”三联体催化机理；同时，本研究还利用三维显微镜从微观的角度对磷脂酶A_1水解PC的过程进行分析和研究，证实了酶的水解过程是界面反应。
对L-α-GPC的制备机理进行了研究，通过定量~(13)C NMR证明了磷脂酶A_1是Sn-1位专一性水解酶，结合借助HPLC-RID的磷脂酶A_1水解PC全过程分析，推测酶水解制备L-α-GPC的关键步骤是Sn-2-LPC自动酰基转移。进一步对溶液极性、pH、温度、底物浓度和反应时间等因素对酰基转移的影响进行了探讨，推导出Sn-2-LPC自动酰基转移机理和抑制机理。
对自制L-α-GPC样品进行硅胶吸附动力学和热力学研究，发现界面扩散控制速率模型1-3(1-X)~(2/3)+2(1-X)能很好的拟合吸附过程。同时，用Langmiur方程对硅胶吸附L-α-GPC的吸附等温线进行拟合，计算出不同温度条件下的回归方程，相关系数均在0.99以上，并且证实了硅胶的吸附为放热反应。最后，利用Clausius-Clapeyron方程计算出80mg/g、160mg/g和240mg/g三个吸附量的硅胶吸附热力学参数，焓变(△HAm)分别为24.85kJ/mol、23.82kJ/mol、22.72kJ/mol。
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 A_1was 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 A_1hydrolysis 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 CaCl_2on 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 CaCl_2(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 ofCaCl_21.9mg/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 A_1hydrolysis was5.96kJ/mol. Briggs-Haldane steady enzymatic modelwas fitted for enzymatic hydrolysis process. Kinetic parameters K_m, V_(max)and kIwas4.02×10~(-2)mol/L,10.05mol/(L·min) and1.33×10~2mol/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 Ca~(2+): 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°, Ca~(2+)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 A_1was carried out byMALDI-TOF/TOF MS and TOF-MS/MS. The spatial structure of3D model of phospholipase A_1wassimulated 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 A_1was 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 A_1was 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 A_1was testified by quantitative analysis of~(13)C NMR. PhospholipaseA_1catalytic hydrolysis progress was monitored by HPLC-RID. The result showed that Sn-1fatty acid ofPC was hydrolyzed by phospholipase A_1to 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.
首先，建立了一种水相体系磷脂酶A_1水解大豆粉末磷脂制备L-α-GPC的方法。通过研究pH、底物浓度、温度、磷脂酶A_1添加量和CaCl_2添加量等酶反应条件对磷脂酰胆碱（PC）转化率和L-α-GPC得率的影响，确定了磷脂酶A_1水解反应的显著影响因素，利用响应面对反应体系进行优化。得到最佳酶反应条件：底物浓度51.5mg/mL，反应温度53℃，磷脂酶A_1添加量28.2U/mL，CaCl_2添加量1.9mg/mL，反应时间3.5h，最终L-α-GPC得率为96.8%。同时，根据Arrhenius经验方程，计算出磷脂酶A_1水解反应的活化能为5.96kJ/mol；利用Briggs-Haldane稳态法酶催化模型对酶催化水解过程进行模拟，并计算出反应的动力学参数K_m，V_(max)和kI分别为4.02×10~(-2)mol/L、10.05mol/(L·min)和1.33×10~2mol/L。
采用树脂-硅胶柱色谱联用纯化技术对酶水解产物进行纯化，依次进行了树脂吸附Ca~(2+)和Cl~-、硅胶纯化、活性炭脱色。通过树脂吸附实验，确定了去除Ca~(2+)的最优条件：处理酶反应液的能力25.1mL/g，Ca~(2+)的初始浓度720μg/mL，吸附流速1mL/min。吸附Cl~-的最优条件：处理酶反应液的能力29.6mL/g，Cl~-的初始浓度997μg/mL，吸附流速1mL/min。阳离子交换树脂和阴离子交换树脂分别用HCl和NaOH再生。通过硅胶纯化实验，确定了去除其它磷脂和杂质的最优条件：洗脱剂80%甲醇、上样量24.4mg/g、上样浓度16.6mg/mL、洗脱流速2mL/min。硅胶在150℃的烘箱中，烘烤3h就可以使吸附饱和的硅胶再生。脱色条件：活性炭作脱色剂，60℃脱色90min。经过树脂-硅胶柱色谱联用四步纯化，最终L-α-GPC产品的纯度为99.8%、回收率为69.8%、比旋光度为-2.5o，Ca~(2+)残留5.8ppm，Cl~-残留8.7ppm。通过LC-MS/MS、ATR-FTIR、~1H NMR和~(13)C NMR等检测技术对L-α-GPC产品进行表征，结果与L-α-GPC标准品一致。
采用MALDI-TOF/TOF MS和TOF-MS/MS测定磷脂酶A_1的氨基酸序列，利用3D软件和同源模建技术分别对磷脂酶A_1的氨基酸序列进行空间结构模拟。通过3D软件模拟得到Space fillin、BallStick、Ribbons1和Ribbons2等四种空间模型。同时，利用同源模建技术成功设计出磷脂酶A_1的3D空间构型；在此基础上，进一步利用分子对接技术研究了磷脂酶A_1的活性中心，得到了Asp202、His280和Ser141三个氨基酸残基组成的活性中心及其空间结构。经过氨基酸残基组成和空间结构分析，推导出磷脂酶A_1水解PC的“Ser-His-Asp”三联体催化机理；同时，本研究还利用三维显微镜从微观的角度对磷脂酶A_1水解PC的过程进行分析和研究，证实了酶的水解过程是界面反应。
对L-α-GPC的制备机理进行了研究，通过定量~(13)C NMR证明了磷脂酶A_1是Sn-1位专一性水解酶，结合借助HPLC-RID的磷脂酶A_1水解PC全过程分析，推测酶水解制备L-α-GPC的关键步骤是Sn-2-LPC自动酰基转移。进一步对溶液极性、pH、温度、底物浓度和反应时间等因素对酰基转移的影响进行了探讨，推导出Sn-2-LPC自动酰基转移机理和抑制机理。
对自制L-α-GPC样品进行硅胶吸附动力学和热力学研究，发现界面扩散控制速率模型1-3(1-X)~(2/3)+2(1-X)能很好的拟合吸附过程。同时，用Langmiur方程对硅胶吸附L-α-GPC的吸附等温线进行拟合，计算出不同温度条件下的回归方程，相关系数均在0.99以上，并且证实了硅胶的吸附为放热反应。最后，利用Clausius-Clapeyron方程计算出80mg/g、160mg/g和240mg/g三个吸附量的硅胶吸附热力学参数，焓变(△HAm)分别为24.85kJ/mol、23.82kJ/mol、22.72kJ/mol。
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 A_1was 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 A_1hydrolysis 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 CaCl_2on 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 CaCl_2(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 ofCaCl_21.9mg/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 A_1hydrolysis was5.96kJ/mol. Briggs-Haldane steady enzymatic modelwas fitted for enzymatic hydrolysis process. Kinetic parameters K_m, V_(max)and kIwas4.02×10~(-2)mol/L,10.05mol/(L·min) and1.33×10~2mol/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 Ca~(2+): 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°, Ca~(2+)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 A_1was carried out byMALDI-TOF/TOF MS and TOF-MS/MS. The spatial structure of3D model of phospholipase A_1wassimulated 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 A_1was 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 A_1was 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 A_1was testified by quantitative analysis of~(13)C NMR. PhospholipaseA_1catalytic hydrolysis progress was monitored by HPLC-RID. The result showed that Sn-1fatty acid ofPC was hydrolyzed by phospholipase A_1to 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.
引文
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