Stabilization of Pickering Emulsion by Terpolymer Microspheres and Application of Immobilized Enzyme
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摘要: 以苯乙烯（St）、马来酸酐（MA）、甲基丙烯酸缩水甘油酯（GMA）为单体，通过自稳定沉淀聚合反应制备了带有环氧基团的形貌规整、粒径均一的纳米粒子（PMG），该纳米粒子可作为乳化剂稳定皮克林乳液，并利用环氧基团来固定化脂肪酶。系统研究了单体投料比、溶剂对纳米粒子形貌和组成的影响，以及纳米粒子添加量、脂肪酶质量浓度、溶剂对皮克林乳液粒径和酶催化反应的影响。结果表明，当n(GMA)∶n(St)∶n(MA)为2∶1∶1、纳米粒子质量分数为0.5%、脂肪酶初始质量浓度为3 mg/mL、正庚烷与去离子水体积比为5∶5时，固定化脂肪酶的催化比活性最高，是游离酶催化比活性的7.5倍，且具有优良的重复使用性能。Abstract: Through self-stable precipitation polymerization, styrene (St), maleic anhydride (MA), and glycidyl methacrylate (GMA) were copolymerized to obtain nanoparticles with regular morphology and uniform particle size. This one-pot method has no stabilizer and emulsifier, and requires no stirring. The post-treatment is simple, and the product can be obtained only by centrifugation. The effects of monomer feed ratio and solvent on the morphology and chemical composition of nanoparticles were studied by Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The prepared nanoparticles can be used as emulsifiers to stabilize Pickering emulsion and immobilize lipase through epoxy groups. It was verified that lipase was anchored on the oil-water interface by immobilizing on the particles. The influence of the contact angle of particles and the solvent volume ratio of n-Heptane to water (RO/W) on the emulsion size was observed by fluorescence microscope. With the increase of contact angle and RO/W, the emulsion size decreases, which is also beneficial to the enzymatic reaction. The effects of nanoparticle mass content, lipase concentration, Ro/w on the size of Pickering emulsion and subsequent enzyme catalyzed reaction were investigated. Results showed that when n(GMA)∶n(St)∶n(MA) is 2∶1∶1, the mass content of nanoparticles was 0.5%, the mass concentration of lipase was 3 mg/mL, and RO/W was 5∶5, the specific activity of immobilized enzyme was the highest, up to 7.5 times that of free enzyme, and it showed excellent reusability.
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图 3 PMG的扫描电镜照片
Figure 3. SEM images of PMG
(a, d)—IPA; (b, e) — IPA/DMB (8/2, Volume ratio); (c, f) —DMB
图 5 Pickering乳液的光学显微镜照片
Figure 5. Optical microscope pictures of Pickering emulsion
(a) Contact angle of 37°, (b) Contact angle of 56°; RO/W(c) 3∶7, (d) 4∶6, (e) 5∶5
图 6 PMG纳米粒子稳定的Pickering乳液固定化脂肪酶的荧光显微镜照片
Figure 6. Fluorescence microscope images of lipase immobilized with Pickering emulsion stabilized by PMG nanoparticles
图 7 （a）PMG粒子质量分数，（b）脂肪酶质量浓度，（c）单体摩尔比，（d）溶剂体积比对固定化脂肪酶的催化比活性的影响
Figure 7. Relative enzyme activity of immobilized enzymes with (a) particle mass fraction, (b) lipase concentration, (c) molar ratio of GMA to St, (d) RO/W
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