【作者】 魏炜；

【导师】 胡祖明；

【作者基本信息】 东华大学 ， 材料科学与工程， 2013， 博士

【摘要】 酶是一类特殊且高效的生物催化剂。为了将其优势充分发挥,在过去的一百多年中,有关酶的固定化方法的研究和固定化体系的开发,受到人们的广泛关注。种类丰富、形式多样的固定化酶体系被开发出来,改善了游离酶容易失活和难以回收利用的不足,但却引入固定化过程中酶活性的过度损失和固定化酶容易从载体上脱落等问题。因此,迫切需要设计和开发稳定且高效的新型固定化酶体系。基于此研究背景,为制备应用性更好和整体性能更完善的固定化酶体系,本论文基于单蛋白纳米胶囊化技术平台,设计和制备了几种新型的固定化酶体系：1.选取有机磷水解酶,采用两步法制备了有机磷水解酶纳米胶囊(nOPH)。通过动态光散射(DLS)、透射电镜(TEM)、(?)胶电泳、红外光谱(FTIR)对其形貌和结构进行表征。结果显示,制备的nOPH是单蛋白纳米胶囊结构,成功实现了对有机磷水解酶的纳米胶囊化固定。通过对其催化活性、热稳定性、有机溶剂稳定性和储存稳定性进行测评,结果显示,nOPH比固定前具有更好的催化活性和稳定性。通过体外细胞毒性测试和体内动物预实验,对将其用于制备有机磷化合物解毒制剂的潜力进行评估,结果显示,nOPH具有低细胞毒性和明显的体内安全性和有效性,可以进一步开发制备有机磷水解化合物的解毒制剂。2.在成功制备nOPH的基础上,把单蛋白纳米胶囊固定化酶技术和有机载体固定化酶技术相结合,设计制备了一种新型复合固定化酶体系,nOPH／细菌纤维素复合体系(nOPH/BC)。采用荧光素标记的方法证实了nOPH和BC膜间通过稳定的共价键连接实现固定。对nOPH/BC的催化活性、热稳定性、循环使用稳定性和储存稳定性进行测评。结果显示,在nOPH/BC复合体系中,经过双重固定化的有机磷水解酶,其热稳定性得到明显的提高,比仅采用纳米胶囊固定化酶的稳定性更高；可以有效地被多次回收和循环使用；在室温条件下,可以以湿润或者干燥的状态,长时间稳定的储存。这种基于单蛋白纳米胶囊技术,与BC载体相结合,成功制备了综合性能增强、重复循环使用性好的nOPH/BC复合体系,为开发敏感度高、稳定性好、实用性强的有机磷化合物体外检测和防护体系提供了新的可能。3.把单蛋白纳米胶囊固定化酶技术和无机载体固定化酶技术相结合,通过两步固定的方法,成功设计和制备了一种新型复合固定化酶体系,nOPH/介孔硅复合材料应用体系(nOPH/MS)。通过对游离酶和固定化酶进行DLS、TEM、红外光谱、以及氮气吸附-脱附等温测试(BET)和脱附孔径测试(BJH),对nOPH/MS复合体系的形貌和结构进行了表征。还对游离酶、nOPH以及nOPH/MS复合体系中酶的活性、热稳定性、有机溶剂稳定性和循环使用稳定性进行了评价。结果显示,以单蛋白纳米胶囊为构建单元,成功制备了具有生物活性的双重固定化酶体系。在该体系中,OPH的动力学参数Km和Kcat均升高,催化活性得到提升；其热稳定性和有机溶剂稳定性得到显著提高,比仅采用纳米胶囊固定化酶的稳定性更高；nOPH/MS复合体系可被有效地回收和循环使用。这种具有生物活性的双重固定化酶体系的成功制备,不仅增强了OPH的应用性,还为其他蛋白质和酶的固定化研究提供了新平台。4.在单蛋白纳米胶囊技术的平台上,引入原位沉淀聚合的方法,设计并制备了一类具有温度响应性能的蛋白纳米胶囊(nBSA)和辣根过氧化物酶纳米胶囊(nHRP)。采用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)、TEM和DLS对BSA的修饰度,nBSA的形貌和结构,及其温度响应性能进行了表征。并用HeLa细胞对nBSA的体外安全性进行了初步评价。结果表明,通过改变NAS/BSA的比例,可在一定范围内实现对蛋白丙烯酰化程度的控制；通过调节NIPAM/BSA的比例,成功制备了粒径大小在7.4～17nm之间的nBSA,实现了对nBSA粒径的调控；制备了响应温度在33～41℃之间的nBSA;当环境温度高于响应温度时,nBSA出现粒径剧增的响应现象,其粒径大小较响应前增大16～33倍；细胞毒性实验结果显示,nBSA具有较低的细胞毒性。对nHRP进行表征结果显示：粒径大小为13.7nm单分散的nHRP;其在环境温度高于33℃时出现响应变化,其粒径可从13.7nm剧增到250nm左右,且具有可逆的响应性能；nHRP与游离HRP相比,其热稳定性明显提高；50℃时,将nHRP高速离心后,固定化酶可被多次有效地分离和回收。这种基于温度响应型蛋白纳米胶囊平台,整合酶的精细化固定、高效分离和回收于一体的新技术,为制备更经济的固定化酶体系提供了新思路。综合以上,本论文采用单蛋白纳米胶囊技术,成功设计和制备了几种新型固定化酶体系,并对各体系的综合性能以及其在不同领域的应用潜力进行了评估和探讨,为其他固定化酶体系的研究提供了新参考。更多还原

【Abstract】 Enzyme is a class of special and highly efficient biocatalysts. To better utilize its specialty, in the past century, great effects were made to develop approaches and methods to immobilize enzymes. Immobilized systems of many different types of enzymes in versatile forms were invented to improve enzymatic activity and stability performance. However, new problems came along as well, such as excessive activity loss during immobilization process and enzyme linkage from the substrate.Herein, in order to prepare robust immobilized enzyme systems with enhanced capability, the design, preparation and evaluation of several enzyme immobilization systems based on the single-protein nanocapsule platform were conducted as follows.1. Organophosphorus hydrolase was chosen as the target enzyme in this study. Organophosphorus hydrolase nanocapsules (denoted as nOPH) were synthesized by using a simple two-step process. The formation of nOPH was confirmed with various characterization techniques. The morphology and structure of nOPH were characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), agarose gel electrophoresis, and infrared spectroscopy. The results show a successful formation of nOPH. Proposed polymer shell is indeed constructed around each enzyme molecule forming single-protein nanocapsules, a new immobilized OPH system. The catalytic performance and durability study show nOPH with enhanced acidity and significantly improved stability against various denaturation factors, including elevated temperature, freeze-thaw cycles, organic solvents and long-term storage. The nOPH cytotoxicity study clearly suggests that nOPH exhibits low cytotoxicity. Besides, a preliminary in-vivo study demonstrates the great potentials of using nOPH as therapeutic and prophylactic agents against OP intoxication. The capability to fabricate nOPH with significantly enhanced activity and stability provide a novel platform for various applications, exemplified in the study by the development of organophosphate detoxification agents, decontamination agents, and native building blocks toward the fabrication of OPH nanocomposites.2. A bioactive composite was designed and fabricated by using nOPH as building blocks, a combination of protein nanocapsule technique and existing organic carrier. A novel robust nOPH/bacterial cellulose composite (denoted as nOPH/BC) was successfully synthesized and characterized. The formation of nOPH/BC was confirmed with fluorescein-isothiocyanate-labelling test, indicating there is a covalent linkage between nOPH and BC foam. The catalytic activity, thermal stability, storage stability, and recyclability of the prepared nOPH/BC were tested and evaluated. Results show that in the nOPH/BC system, double immobilized enzyme not only demonstrates good residual activity, but also displays improved thermal stability over nOPH. Moreover, the nOPH/BC composite performs good storage at room temperature in both dry foam form and swelling pad form and presents excellent recyclability. The successful combination of single-protein nanocapsulation and bacterial cellulose carrier, not only results a robust and recyclable nOPH/BC composite, but also demonstrates its great potential to develop sensitive and stable organophosphate decontamination, detoxification and protection systems.3. Another bioactive composite was designed and fabricated by using nOPH as building blocks. This bioactive nOPH/mesoporous silica (denoted as nOPH/MS) composite is a double immobilized enzyme system, which was prepared according to above simple two-step approach. The successful construction of nOPH was confirmed with DLS, TEM and FTIR, while the fabrication of nOPH/MS composite was confirmed by above characterizations and additional BET and BJH analysis. The catalyst activity overall durability of native OPH, nOPH and nOPH/MS composite were tested and compared. The study shows a successful construction of nOPH/MS composite. Enzyme immobilization in nOPH/MS composite leads to an increase value of both Michaelis constant (Km) and turnover number (Kcat), indicating an enhancement of enzymatic performance. Besides, strengthened thermal and solvent stability were also observed in nOPH/MS over nOPH. Furthermore, an excellent recyclability and reusability of the composite with well retained enzymatic activity was also proved by tests. This robust nOPH/MS composite built up from nOPH demonstrates an overall enhanced capability and applicability. Studies not only demonstrate an effective route towards the synthesis of bioactive composites that are highly active, stable and recyclable, but also provide an approach to design and fabricate double immobilized systems for other enzymes.4. A new class of thermal-responsive protein nanocapsules, thermo-responsive BSA nanocapsules (denoted as nBSA) and horseradish peroxidase nanocapsules (denoted as nHRP) were prepared separately via in-situ precipitation polymerization. The modification degree, morphology, structure, and thermo-responsive property of prepared nanocapsules were characterized by MALDI-TOF MS, TEM and DLS, while resazurin-based cytotoxicity assay was applied to study its cytotoxicity. The results show that a series of uniformed monodispersed nBSA were prepared. By adjusting the NIPAM/BSA ratio, different sized nBSA were synthesized, which have reversible thermo-responsive properties and response to different temperatures. With the increase of the NIPAM/BSA ratio from2to6, the particle size of nBSA increases from7.4to17nm, and at the same time, the responsive temperature of nBSA decreases between41and33℃. For each sized nBSA, when the environmental temperature is above the responsive one, its particle size increases dramatically (between16to33times) comparing to its size at lower temperature. The low cytotoxicity of nBSA, indicating its potential to be safely applied in vivo. Similarly, the modification degree, morphology and structure, thermo-responsive property study of prepared nHRP was conducted. Besides, its catalytic activity and thermal stability results show a mono-dispersed nHRP was prepared successfully, with a responsive temperature at33℃and a reversible response property. It also exhibits significantly improved thermal stability, which accordingly can be applied to separate and recycle nHRP at50℃with a well-retained activity, indicating a new delicate way to immobilize, separate and recycle enzymes. This is of great interest to build economical immobilization systems.In summary, novel enzyme immobilization systems were designed and fabricated base on single-protein nanocapsulation technique. Comprehensive performance of each system was evaluated, potential applications in various fields were proposed accordingly. This work presents itself as a new reference to the study of effective immobilized enzyme systems.更多还原