【作者】 王翠；

【导师】 高静；

【作者基本信息】 河北工业大学 ， 生物化工， 2011， 硕士

【摘要】 仿生硅化过程用于固定化酶,克服了传统溶胶凝胶法的缺点,在温和条件下(室温,中性pH)更好保持酶的活性,提高包埋率和包埋能力；创造更适宜的微环境,显著提高酶的稳定性。不仅为固定化酶技术提供了新的方法,而且进一步拓展了仿生矿化的应用。本论文主要研究仿生制备纳米氧化硅载体用于固定化酶,主要研究内容如下：首先,以聚乙烯亚胺(PEI)为诱导剂诱导纳米氧化硅形成,同时固定化辣根过氧化物酶(HRP)。FT-IR表征结果证实载体为氧化硅,且HRP被成功固定于载体中。SEM分析显示,生成的含HRP的氧化硅粒子为200-500 nm的球形颗粒。固定化HRP的包埋率为65.3%,酶活回收率为70.8%,稳定性得到显著提高。将固定化HRP用于处理含酚废水,苯酚去除率最高可达73.1%。固定化HRP重复使用5次,仍保留初始催化活性的51.7%。其次,研究了木瓜蛋白酶(papain)自诱导仿生合成氧化硅的同时将其自身包埋固定化的过程。考察了不同因素对固定化效果的影响,实验结果表明,固定化papain的包埋率最高达82.6%,酶活回收率达83.1%。与游离papain相比,固定化papain显示出良好的温度、pH、储存和重复使用稳定性。初步探讨了木瓜蛋白酶自诱导仿生硅化过程的机理。对动力学常数的研究表明,papain经自诱导仿生固定化后其底物亲和性并没有受到严重破坏。最后,将papain交联固定在氨基修饰过的磁性粒子上,通过papain诱导使磁性粒子(MNPS)表面包覆一层氧化硅,并同时将papain包埋于其中,实现温和固定化和高效分离的有效结合。FT-IR显示MNPS表面成功修饰了氨基功能基团,且磁性粒子交联酶(EMNPS)表面包覆有仿生硅化形成的氧化硅。SEM显示经氧化硅包覆后样品的平均粒径增大,分散性得到改善。研究发现磁载体最大载酶量为0.24 mg papain-mg-1 MNPS,此时EMNPS和交联酶后包覆氧化硅(EMNPS/SiO2)的活性分别3.72和3.66 u·mg-1 MNPS。EMNPS/SiO2的各项稳定性比EMNPS有了进一步提升。与游离papain相比,EMNPS与底物的亲和力略有升高,而EMNPS/SiO2与底物的亲和力降低。更多还原

【Abstract】 Encapsulation is demonstrated to be an effective method for enzyme immobilization. Compared to traditional encapsulation method, the biomimetic approach, which occurs under mild conditions, can create benign microenvironment for the immobilized enzyme and improve the stability of enzyme. The method offers a new program for the immobilized enzyme technology, and further expands the application of biomimetic mineralization. The study discusses biomimetic method for the preparation of nanosilica for enzyme encapsulation. The details in this study were summarized as follows:Firstly, polyethyleneimine-mediated silica formation was used to immobilize horseradish peroxidase (HRP) under room temperature and neutral pH for the removal of phenol. SEM image showed that the immobilized HRP particles were near-spherical and homogeneous ranging from 200-500 nm. FT-IR spectra indicated that HRP was successfully encapsulated within the silica matrix. The results showed that 65.3% of HRP was immobilized and 70.8% of its initial activity could be retained. As well, the immobilized HRP exhibited improved thermal, pH and storage stabilities. The resulting silica-encapsulated HRP was used into the reaction of phenol removal. The phenol removal rate could reach 73.1%. After 5 repeated cycles, almost 51.7% of its initial activity was retained.Secondly, papain was immobilizated through the biomimetic silicification process. This method made use of the silicification activity of papain itself. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly, and papain was encapsulated simultaneously. The results showed that the encapsulation efficiency and the recovery activity were 82.6% and 83.1%, respectively. The mechanism of papain encapsulation was tentatively proposed. Furthermore, the encapsulated papain exhibited enhanced thermal, pH, recycling and storage stabilities. Kinetic analysis showed that the biomimetic silica matrix did not significantly hinder the mass transport of substrate or the release of product.Finally, an approach combining biomimetic mineralization with Fe3O4 magnetic particles (MNPS) was proposed to prepare immobilized papain that was easily separated from reaction mixture. FT-IR spectra showed that aminosilane-modified MNPS was synthesized, and EMNPS was coated with biosilica. SEM suggested that the silica layer increased the average size and disperasion of the sample. The study indicated the loading of papain was 0.24 mg-mg-1 MNPS and the activity of EMNPS and EMNPS/SiO2 were 3.72 and 3.66 u-mg-1 MNPS, respectively. Furthermore, the stability of EMNPS/SiO2 was better than EMNPS. Compared with free papain, EMNPS has slightly higher affinity, and EMNPS/SiO2 has lower affinity with substrate.更多还原