利用溶血磷脂酰乙醇胺的酰基转移调控酶促制备甘油磷脂酰乙醇胺的机理研究

L-alpha glycerylphosphorylethanolamine (L-α-GPE) is well known for having important medical applications in neurological and psychiatric disorders of the human brain. Unfortunately, L-α-GPE is scarce in natural sources. A green bio-preparation of L-α-GPE is considered to be a attractive alternative strategy for high efficiency, economic, and environmental friendliness. Phospholipase A1 (Lecitase Ultra) as one of the industry scale enzyme, which hydrolyzes the Sn-1 fatty acyl-ester bonds of phospholipids, has potential application for enzymatic preparation of Sn-2-lysophosphatidyl ethanolamine (Sn-2-LPE) on scale-up. The Sn-1-lysophosphatidyl ethanolamine (Sn-1-LPE) and L-α-GPE were occasionally obtained during the preparation of Sn-2-LPE from LPE, which might be attributing to acyl migration phenomenon. It is the key problom that the mechanism of acyl migration of lysophospholipids on the enzymatic selectively preparation of L-α-GPE. The intermolecular rearrangement transformation of Sn-2-LPE to Sn-1-LPE was accomplished through the five-membered ring of the acyl migration. Based on the previous researches, the molecular conformation and configuration of the five-membered ring of the acyl migration will be captured and determined with advanced technique. The mechanism of acyl migration will be revealed. The enzymatic hydrolysis preparation of L-α-GPE will be selected as reaction model. The effect of enzymatic reaction system and amphoteric hydroxide on the acyl migration will be deplored. The effect of acyl donor on the selectivity of acyl migration and the rearrangement of five-membered ring will be investigated on the basis of analysis of configuration and kinetics. The mechanism and regulation of acyl migration on the enzymatic selectively hydrolysis preparation of L-α-GPE will be proved. The result of this research will provide the fundamental data, methodology, and scientific references for the novel preparation of functional lipids, and for the novel application of biotechnology in lipid modification.

甘油磷脂酰乙醇胺（L-α-GPE）在治疗神经混乱方面具有重要药用价值，但其天然含量较少，它的绿色生物制取技术逐渐成为未来的研究方向。磷脂酶A1是专一水解磷脂Sn-1位酰基制备溶血磷脂（Sn-2-LPE）的工业用酶。在制备Sn-2-LPE的研究中发现Sn-1-溶血磷脂酰乙醇胺（Sn-1-LPE）和L-α-GPE生成，推测其原因可能发生了酰基转移现象。酰基转移过程的关键在于Sn-2-LPE经五元环中间体分子内重排产生Sn-1-LPE。本项目以Sn-2-LPE的酰基转移调控酶促制备L-α-GPE为研究模型，利用现代分析技术捕捉、鉴定过渡态五元环中间体；探明反应体系和两性氢氧化物对酰基转移的影响及调控；利用构型和动力学分析研究酰基供体对酰基转移选择性及五元环中间体分子内重排的影响，揭示酰基转移对酶促制备L-α-GPE的调控机理，为生物技术在磷脂改性中的应用提供理论指导及功能性脂质的开发奠定基础。

甘油磷脂酰乙醇胺（L-alpHa glycerylpHospHorylethanolamine，L-α-GPE）能治疗人体的人体大脑的精神混乱和神经混乱方面的疾病，具有重要的医药价值，但其天然含量较少，它的绿色生物制取技术逐渐成为未来的研究方向。磷脂酶A1是专一水解磷脂Sn-1位酰基制备溶血磷脂（Sn-2-LPE）的工业用酶。针对生物酶解制备L-α-GPE过程中Sn-2-LPE转化Sn-1-溶血磷脂酰乙醇胺（Sn-1-LPE）的酰基转移现象，本研究通过13C NMR技术进行过渡态五元环中间体的捕捉和鉴定，确定了反应体系和两性氢氧化物对酰基转移的影响及调控规律，并利用构型和动力学分析研究酰基供体对酰基转移选择性及五元环中间体分子内重排的影响，揭示酰基转移对酶促制备L-α-GPE的调控机理，为生物技术在磷脂改性中的应用提供理论指导及功能性脂质的开发奠定基础。.首先，通过13C NMR技术进行过渡态五元环中间体的捕捉和鉴定，证明了酰基转移是磷脂酶A1水解PE制备L-α-GPE的关键步骤，证实了Sn-2-LPE具有向Sn-1-LPE的自动酰基转移，而Sn-1-LPE的酰基转移基本不发生。.其次，进行了反应体系（溶液极性、pH、温度、底物浓度和反应时间等因素）对酰基转移率影响的研究。发现水、甲醇、异丙醇等极性比较大的体系能溶解Sn-2-LPE，并且随着溶剂极性的增加酰基转移率也增加。酸性条件下随着溶液pH的升高酰基转移率逐渐下降；在碱性条件下，随着pH升高酰基转移率也升高；中性条件下酰基转移率最低为22.6%。在0-35℃，酰基转移率比较低；在35-45℃，酰基转移率随温度的升高快速升高；当温度超过55℃，酰基转移率保持一个较高值。浓度对酰基转移率的影响是随浓度的增加先增加后减少，在底物浓度5 mg/mL，有最大的酰基转移速率为32%。50 min之内，酰基转移率随时间延长快速增加；随后缓慢增加，当超过180 min时，酰基转移达到平衡。 .在酰基转移抑制机理的研究过程中，发现Al(OH)3、Zn(OH)2、B(OH)3等物质对Sn-2-LPE的酰基转移能很好的抑制。以硼酸为例，推测酰基转移的抑制机理可能是Sn-1位上羟基氧的孤对电子可以和硼原子的空轨道组成配位键，抑制了氧负离子的形成，切断了其进攻羰基碳正离子形成五元环中间体的途径，从而实现对酰基转移的抑制。