Study On The Improvement Of Protein Stability By Biomineralization

Biomineralization refers to the mineral deposition processes by living organisms. From bacteria, fungus in microcosm to plant, animal in macrocosm, biomineral participate in biotic activities of all organisms. Living organisms selectively adsorb elements from environment and synthesis biomineral with highly ordered hierarchical structures and specific functions under rigid biological regulation. These biomineral exerts supporting, protecting, optical and magnetic functions in living organisms. In addition, it has been proved to play an important role in improving organisms’ resistant ability in harsh environment.Inspired by these biomineralization phenomena, inorganic materials has been introduced to organisms which have no biomineralization ability in a biomimetic way. It can not only improve their performance under extreme environment, but also endow them with special abilities. Protein, one of the most important compositions in organisms, plays an irreplaceable role in people’s life because of its good catalytic capability and specificity as medicine. However, the practical applications of protein are always limited by their intrinsic drawbacks such as poor stability, low operational stability, difficult to recycle and so on. Therefore, it is of great significance to find an simple and effective way to improve protein stability. In this thesis, we improved protein stability by introducing an inorganic exterior shell on protein surface and then studied the protection mechanism. Furthermore, by introducing functional nanomaterials, we explored the application of protein-material nanocomposites in biosensor. This thesis contained five chapters:In chapter 1, we briefly introduced the basic concepts of biomineralization, including the variety and function of biomineral and biomineralization processes in living organisms. Then we described biomimetic principle, the function of organic substrate in biomineralization and the applications of organism biomimetic modification. Subsequently, we summarized the basic knowledge of protein, its drawbacks in application and approaches to stable protein. Based on above understanding, it inspired us to improve protein stability by biomimetic mineralization.In chapter 2, we incorporated the protein into amorphous calcium phosphate by in situ biomineralization and found a thermal protective effect of amorphous calcium phosphate on enzyme. Via the study of protein structure, H-D exchange rate and water content of amorphous minerals, we found that the amorphous mineral tightly bonds abundant water molecules around the entrapped proteins to stabilize the conformation of the enclosed protein by decreasing the hydrogen bond exchange during the heat treatment.In chapter 3, by combining in situ biomineralization technique with functional nanomaterials, we introduced amorphous silica shell to protein surface. After functionalization of protein with PEI by chimeical modification, the PEI-functionalized enzymes were concentrated by magnetic nanoparticles and then incorporated within the silica layer by in situ biosilification. The results showed that the functional material incorporated protein composites had a high immobilization rate, significant enhancement in protein thermostability and organic resistance, as well as great catalytic ability in a board range of temperatures. This enzyme-material hybrid, biomineralization-based method could be considered as a feasible biomolecule immobilization strategy in future.In chapter 4, considering the peroxidase-like activity of magnetic nanoparticle and the improvement of protein stability under acidic environment by biosilification, we used the synthetic Fe3O4@C-glucose oxidase-silica nanocomposites for glucose determination in one step. This method showed good linearity, high sensitivity, high selectivity and excellent reusability due to the magnetic characteristic from the Fe3O4 core. It inspired us that the combination of biomineralization and functional nanomaterial could make good use of the particular characteristics of material and biomolecule. It showed great potential in the application of analysis and biosensor.In chapter 5, we concluded the studies in this dissertation. Our studies clearly demonstrated that the biomimetic mineralization strategy could be successfully applied in improving protein stability and explained the key role of structural water in amorphous phase in stable protein conformation. We further proposed that it held great promise in the development of biosensor by the combination of biomieralization technique and functional nanomaterials. Moreover, according to these conclusions, we also analyzed the shortages of this work and the problems needed to be solved in future study.