| The adsorption behavior of protein and enzyme on solid surface and the relationship between the adsorption and protein conformation, bioactivity have become an interesting and hot topic in the fields of biomedical and biomaterials research. With the development of biotechnology especially the development of genetic technology and biopharmaceutical industry, the possibility of extraneous bioactive protein releasing into the environment become greater. On the other hand, biological methods including enzyme technology have been widely applied in environmental remediation process. It should be important to study the behavior of protein and enzyme on various solid surfaces.In the part one of this thesis, the significance and progress on the researches related with the adsorption of biomolecule on solid surface and their bioactivity change was reviewed. The relationship between the protein conformation and bioactivity, as well as the factors affecting the protein bioactivity and stability was also reviewed. The effect of adsorption on the protein conformation, bioactivity and stability was mainly discussed. Various method and models for studying the protein adsorption on solid surface or solid-liquid interface were summarized. Meanwhile, adsorption of biomolecules on typical environmental solid surface and their bioactivity or stability change was also discussed.In part two, the methods of extracting and purification of crylAb toxin expressed from transgenic plants were established. It was the first time that toxin purified from transgenic plants instead of from bacterium was used in the studies of conformational analysis, environmental behavior and mechanism of bioactivity change upon the adsorption of the protein on the solid surface.In the chapter one of part two, a protein purification strategy was proposed based on bioinformatics analysis. The differences in surface charge density and hydrophobicity between target protein and coexist protein were analyzed by protein structure prediction software. Based on the prediction results, a sequential ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC) were employed for the purification of crylAb toxin, which was only expressed in low quantity in the transgenic plants. The purified protein could meet the demands of followed study. Since the protein activity was significantly related with its conformation, the method for detecting the protein conformation change must be developed. In the chapter 2, enzyme-linked immunoassay method was confirmed to have the ability for probing the CrylAb conformational change. The method was based on the change of binding capacity of the antibody with antigen resulting from antigen conformation transition. Conformational stability of CryIAb protein in different temperature, solvent composition, pH and different organic solvents was measured. Results showed that the antibody on the micro-well plate of ELISA kits could sensitively and selectively recognized conformation transition of CryIAb protein without need of highly purified protein. The ELISA values had highly correlation with the protein conformation and larvicidal activity, and the results were accordance with fluorescence spectroscopy results reported previously. Moreover, it was found that humic acids from soil could remarkably stabilize the conformation of CryIAb protein even in acidic condition by ELISA method.In chapter 3, sorption and desorption behaviour of CrylAb protein expressed by transgenic rice (Bt-r) on different kinds of clay minerals were studied. Larvicidal activity and stability change upon the protein adsorbed on various homo-ion saturated montmorillonite and kaolinite were also investigated. The batch experimental results showed that Bt-r could be both adsorbed strongly on montmorillonite and kaolinite, which might involve in different kinds of adsorption mechanism. The protein might be adsorbed on montmorillonite through ion exchange mechanism, while in the case of kaolinite, the protein might be adsorbed mainly by the way of hydrophobic interaction according to the elution behaviour of adsorbed protein. Among all the clay tested, the kaolinite showed highest adsorption capacity for the Bt-r. In the other three homo-ion saturated clay, the adsorption capacity for the Bt-r follows the order:Fe3+-montmorillonite>Al3+-montmorillonite>Na+-montmorillonite. Br-r adsorbed on clay minerals could be effectively extracted by the complex extracting buffer solution (Na2CO3-NaHCO3+sodium pyrophosphate+ DTT+glycerol+ Triton X-100) and the recovery could reach above 60%. The data obtained from enzyme linked immunology assay (ELISA) and larvicidal bioassay showed that the Bt-r was less stable and larvicidal activity was also lower in acidic montmorillonite than it was in basic montmorillonite such as Na+-montmorillonite. It was noticeable that the Bt-r showed high stability and larvicidal activity when it was adsorbed on kaolinite.In chapter 4, the mechanisms of adsorption of crylAb protein on various solid surface affecting its bioactivity were studied. XRD analysis showed that parts of peptide chains in the CrylAb protein could intercalate into the inner layer of montmorillonite (Mont). The layer of the Na-Mont expanded more significant than Fe3+-Mont and Al3+-Mont. The results indicated that the portion of the protein penetrated into the Na-Mont layer was larger than in the case of Fe+ -Mont and Al3+-Mont, which might lead to inhibiting its bioactivity. FTIR and Lorentzian curve fitting method testified that no obvious conformation change was found upon the toxin was adsorbed onto the Na-Mont layer. It could be deduced that the decreased larvicidal activity of crylAb toxin on Na-Mont might result from the penetrating of domain II region into clay layer and preventing it form being bound with its acceptor on the larval cell. Although the extent of intercalation into the Fe3+ -Mont and Al3+-Mont layer was much smaller, the larvicidal activity of the toxin almost disappeared after it was complexed with these acidic clay minerals. FTIR results showed that amide I absorption band of the adsorbed toxin remarkably changed, which might result from its protein conformational change in acidic condition.In chapter 5, the stability of CrylAb toxin in the present of humic substance was investigated. ELISA and larvicidal experiment found that humic acid could decrease the bio-activity of the toxin, while the fulvic acid could effectively protect the conformation stability. Gel permeation chromatography (GPC) experiments confirmed that the toxin could form a tight complex with both humic acid and fulvic acid. But the mechanism of toxin stabilized by fulvic acid still leave to be unknown.In chapter 6, FTIR spectra signal derivative method and multi-curve fitting method were employed to investigate the conformation change of the toxin on the surface with different hydrophobicity. Results showed that the surface with strong hydrophobicity (C-18) could induce the protein conformation change significantly, while the conformation of the adsorbed protein on weak hydrophobic surface (Kaolinite) was stable.Since the importance of enzyme in environmental catalysis, adsorption behaviours of catalase on different kinds of solid surface as well as its bioactivity change were also investigated.In the chapter 1 of Part 3, adsorption dynamic property on several kinds of surface was studied. Two adsorption stage could be found including fast rate adsorption by outer surface and slow rate adsorption by inner surface of clay minerals. On the other hand, orientation adjustment of the adsorbed protein might also contribute the slow adsorption process. The reversibility of enzyme adsorption on solid surface was different on the surfaces with different interaction mechanism. The adsorbed enzyme could be easily desorbed from the surface through ion-exchange interaction by salt solution. With the increase of surface hydrophobicity, the adsorption became more irreversible. The tendency of enzyme activity change caused by its adsorption on solid surface could be explained and predicted based on protein structure information such as the exposure rate of enzyme active site, hydrophillicity and hydrophobicity of peptide around the active site. Experimental result showed that extent of activity and stability change of the adsorbed enzyme variated with the property of solid surface. Strong hydrophobic surface could decrease the catalytic activity and destabilized the enzyme. Enzyme adsorbed through static electric forces was more stable. When the surface was loaded with Fe3+, the enzyme could interact with solid surface through coordination force. The conformation of the catalase might slightly shift to adaptation the multi point attachment (MPA) mode. The initial activity of catalase was decreased on the Fe3+ loaded surface, but its stability was increased.In chapter 2 of part 3, a silicate adsorbent with mesoporous surface structure was prepared by molecular imprinting method. The structure of the mesoporous materials was characterized by FTIR, XRD and SEM respectively. The material could effectively adsorb catalase with high capacity. Meanwhile, the adsorbed enzyme on the porous surface was more stable and had higher catalytic activity compared with the free catalase. Experimental result also indicated that not only the chemical and physical structure on the surface but also the porous structure could have profound effect on the stability and activity of the adsorbed enzyme. The result was obviously significant to the application of immobilized enzyme in environmental catalysis.
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