| With the success of the human genome project, converting gene into their corresponding proteins is a significant advantage for protein science and protein engineering. The expresstion of foreign recombinant proteins become an efficient solution for the biography medicine production and special chemicals. Escherichia coli is often used to be a host cellof the recombinant DNA, because of its rapid growth, great rate of protein production. In order to abtain the active protein, inclusion bodies should be dissolved, but meanwhile, the active structures are destroyed and the activities are dispared. Thus, it is necessary to accurately refold the structures and renature the activities of proteins. Protein refolding, as a unit operation during the manufacture of recombinant protein via the inclusion body process route, becomes critical. In this thesis, lysozyme is used as model protein, which is unfolded, refolded, and determined by several new methods. The creative works of this thesis are summarized as follows:1. The interaction between urea and guanidinium chloride (GuHCl) on lysozyme refolding was investigated in this work. Live micrococcus lysodeikticus was successfully introduced into refolding system. Lysozyme can be refolded from the GuHCl-denatured, DTT-reduced state in good yield of 96.54% at final protein concentration as high as 0.2 mg·mL-1. A model could be employed to elucidate refolding kinetics behavior and the kinetics constants were studied. In the coexistence of GuHCl and urea, the aggregation rate decreased by increasing urea concentration to a proper value. The cooperation of GuHCl and urea not only suppressed the competition of the aggregation reaction, but also increased the yield of refolding efficiently.2. The "phase diagram" of fluorescence was used to detect the intermediate of lysozyme unfolding and describe the unfolding pathway. At 4℃, unfolding intermediate of lysozyme could be visible, which is consistent with three-state model. For the higher temperature disturbance, the intermediate was unfolded slowly and the unfolding process seems to be a two-state mechanism at 25℃and 37℃. At 4℃, the intermediate was visible in presence of 5 M urea. However, the unfolding process was seemed to be a simple transition (Native state→Unfolding state) in presence of 4 M or 6 M urea, as the rate constants of the two transitions are different.3. A novel monitoring technique series piezoelectric quartz crystal (SPQC) sensing technique is used to monitor conductivity in unfolding and refolding processes of lysozyme in real time. The lysozyme is unfolded by sodium dodecylbenzene sulfonate (SDBS) and then refolded by cetyltrimethylammonium bromide (CTAB). During the unfolding and refolding process, the conductivity of the solution changed accordingly. The change in conductivity caused a sensitive response of the SPQC sensor. The obtained experimental results indicate that the SPQC sensing technique would be a useful and convenient tool for monitoring the interaction between ionic surfactants and protein.4. The treatment of phenol is studied by using UV (365 nm)/H2O2 system. A HPLC method for qualitative and quantitative analysis of phenol and its photo-catalyzed degradation intermediates, benzoquinone, hydroquinone and catechol is described. The compositions of moblile phase, the flow rate of mobile phase and the influence of H2O2 on the intermediate products were investigated. It is shown that UV (365 nm)/H2O2 system has a great degradation effect on high concentration of phenol.
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