手性微环境中脂肪酶催化拆分手性醇的研究

Cationic surfactant CTAB and cetyl pyridyl bromide, anionic surfactant AS, AOT and sodium oleate, nonionic surfactant Triton X-100 and LS45, natural surfactant APG, and Gemini surfactant Dynol-604 were chosen to form amphihilic associated systems such as liquid crystal, microemulsion, vesicle and microemulsion based organogels, which were all thought to be nice pseudo-biological micro-enviorment. Composition of the systems was verified systematically, and polarity of micro region transformed regularly while structure of the system changed. Light scattering method has been used to determine the size of microemulsion, polar microscope and 2H NMR method were used to find the structure of liquid crystal, and lamellar distance was determined through small angle x-ray diffraction method. Freeze etching TEM, 1H NMR, Raman spectroscope, viscosity method and electric conductance determination were used to test variation of micro structure of the system. Stereo selective esterification reaction of racemic 2-octanol and octylic acid catalyzed by lipase PSL, CCL, CRL, HPL was studied in these systems. HPLC and NMR method were used to determine the time course of esterification reaction, and give out activity and stereo selectivity of lipase molecule. UV and fluorescence spectroscopy study prompted that strengthen of molecule conformation rigidity of enzyme entrapped in microemulsions corresponded to increase of enzyme activity, and electric interaction between enzyme molecule and surfactant interface layer initiated the conformation variation of enzyme. Effect of organic solvent, hydro ratio w0, pH value of buffer solution, lamellar distance of liquid crystal and micro structure of system on activity and stereo selectivity of lipase were studied, and optimum surfactant associated system in which the lipase molecule could exhibit super activity were got. It was found that PSL exhibited super-activity and high stereo electivity in CTAB reversed microemulsion, ee(S) value of residual 2-octanol after reacted 48 hour could be 95%; V0R/V0S was 18.09, which is twice higher than that in organic medium without surfactant. CCL and CRL had high activity in OAS microemulsion and liquid crystal. HPL showed super activity in OAS reversed microemulsion and Triton X-100+OAS composite system. The kinetics of esterification reaction of lauric and 1-pentanol catalyzed by CCL lipase was studied in AOT microemulsion and it was showed that the reaction followed a Ping-Pong Bi-Bi mechanism. Lipase has also been immobilized in gelatin-containing AOT microemulsion-based organogels, the lipase-containing MBGs prove to be a novel solid-phase catalysts for the use in polar organic solvents and have a good physical stability. These lipase-containing MBGs retained its higher activity after 10 runs esterification reactions. NMR self-diffusion study showed that diffusion of substrate molecule in associated system play a great role on the reaction rate. Variation of composition and change of micro structure coursed difference of self-diffusion coefficient of molecules in the system and then affected the rate of esterification reaction and activity of enzyme. MBPM method was used to study dynamic diffuse behavior and partition arrange of surfactant in solution.It was determined that chosen surfactant associated system, such as microemulsion or liquid crystal could be nice micro enviourment for lipase catalyzed esterification reaction, organic substrates and aqueous soluble enzyme molecule could be both solubilized efficiently, and not only activity of enzyme, but also stereo selectivity could be super high in optimum surfactant associated system, then optic active alcohol could be got by stereo selective esterification reaction catalyzed by lipase entrapped in surfactant associated system.

以手性两亲分子形成的胶束、微乳液、液晶、囊泡、表面活性剂凝胶等有序缔合体为微反应器，研究脂肪酶催化酯合成与酯交换反应的动力学，寻找酶呈现超活性的最佳微环境体系及条件，探讨反应机理，建立理论模型，并利用脂肪酶催化酯合成和酯交换反应拆分手性醇，研究微环境对脂肪酶构象和立体选择性的影响，得到高光学纯度的手性醇。