Synthesis And Properties Of Single Protein Nanocapsules And Nanocomposites

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.