The molecular interaction of a temperature stimuli-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), with lysozyme of different status was studied with an emphasis on the application of PNIPAAm for protein refolding. The refolding of lysozyme was performed by directly diluting denatured lysozyme into a refolding buffer containing PNIPAAm, in which PNIPAAm with the weight average molecular weight of 22,000, denoted as M-PNI, gave the best refolding yield in terms of the recovery of lysozyme activity. The interaction between M-PNI and lysozyme was investigated using non-reductive SDS–PAGE, circular dichroism (CD), fluorescence emission spectroscopy, and reverse phase HPLC. It was shown that the use of M-PNI increased the secondary structures of lysozyme and reduced the formation of protein aggregate. The correctly folded lysozyme has a weaker hydrophobicity compared to the denatured lysozyme. The PNIPAAm-lysozyme complex dissociates once lysozyme is correctly folded. The increase in the operational temperature leads to increases in both the refolding yield and the apparent rate of refolding. Based on above experimental results, a kinetic model of the refolding, both with and without PNIPAAm, was determined and a molecular view of lysozyme refolding using PNIPAAm was presented.

Removal of hydrophobic organic contaminants (HOCs) from soil of low permeability by electroremedia-tion was investigated by using phenanthrene and kaolinite as a model system. Tween 80 was added into the purging solution in order to enhance the solubility of phenanthrene. The effects of pH on the assorption of phenanthrene and Tween 80 on kaolinite and he magnitude of S-potential of kaolinite were examined. respectively. The effects of electric field strength indicated by electric current on the electroremediation investigated. respectively Incase of an electric field of 25mA applied for 72 hours, overs 90% of phenanthrene was removed from 424g (dry mass) of kaolinite at an energy consumption of 0.148 kW.h. The experimental results described in prcsent study show that the addition of surfactant into purging solution greatlky enhances the removal of HOCs by electroremediation.

A temperature stimuli responsive polymer, β-CD-grafted-PNIPAAm (β-CD-g-PNIPAAm), was prepared by radical polymerization using cerium ammonium nitrate as the initiator. The use of β-CD-g-PNIPAAm as the stripper and cetyltrimethylammonium bromide (CTAB) as the capturer for protein refolding was tested using lysozyme as the model protein. The stripping of CTAB from the denatured lysozyme-CTAB complex was accomplished by the β-CD segment of β-CD-g-PNIPAAm. The interactions between lysozyme in different states with CTAB and β-CD-g-PNIPAAm were characterized by fluorescence emission spectroscopy. Based on these results, a temperature stimuli responsive "artificial chaperone" composed of CTAB and β-CD-g-PNIPAAm was proposed for protein refolding, in which stripper, β-CD-g-PNIPAAm, displayed dual functions in terms of stripping CTAB via β-CD segment and inhibiting the formation of protein aggregate. The latter was accomplished by the PNIPAAm segment. This leads to an improved refolding yield particularly at high temperatures, as compared to that obtained by using β-CD as the stripper.

A novel temperature-sensitive polymer-trypsin conjugate was prepared by the covalent linking of monodispersed carboxyl-terminated poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) (PDMAPS) to trypsin. The molar ratio of the polymer to trypsin was 2.4 and its upper critical solution temperature was 14◦C. Fluroscence emission spectroscopy and circular dichroic spectroscopy showed that the conjugated trypsin retained its native conformation. The hydrolysis ofN-R-benzoyl-d,l-arginine p-nitroanilidewas carried out at different pHs and temperatures using native trypsin and the conjugate, respectively. The optimal pH was 7.8 for native trypsin and 8.0 for the conjugate. Michaelis-Menten kinetics analysis showed that, as compared to the native trypsin, the conjugated trypsin has a smaller Km that decreases with temperature, while the Vm of the conjugate was the same. When casein was used to study the catalytic effect of the conjugated trypsin on a high molecular weight substrate, an increase in temperature from 40 to 60◦C gave a 2.6-fold increase in the enzyme activity of the conjugate. The half-life for the enzyme activity at 60◦C was 4.8 min for native trypsin and 315.9 min for the conjugate. The conjugate retained 85% of the initial enzyme activity after 10 cycles of temperature swinging from 4 to 40◦C.

The concept of generating an oscillatory electroosmotic flux inside the porous particle to enhance the intra-particle mass transport was presented and a new kind of electrochromatography carried out in a five-compartment electrolyzer were developed. The adsorbent was packed in the central compartment, while the neighboring compartments were used as the elution compartments and the electrode compartments, respectively. Chromatographic separations of human serum albumin on Blue Sepharose Fast Flow, bovine serum albumin (BSA) on DEAE–Sepharose Fast Flow, and BSA on ydroxyapatite were carried out, respectively. The adsorption isotherms were shown to be independent of electric field, while the increase in the electric field strength resulted in a linear increase in the magnitude of electroosmotic flux and the improvement of the breakthrough behavior in all cases. The experiment results have demonstrated the effectiveness of the oscillatory electroosmosis in enhancing intra- and inter-particle mass transport and its high potential to large-scale hromatography.

A molecularly imprinted polymeric monolith (MIPM) was prepared by in situ polymerization using styrene, glycidyl methacrylate and methacrylic acid as monomers, divinylbenzene and triallyl isocyanurate as cross-linking agents, and ceramide III as print molecule. The texture, pore size distribution, mobile phase flow characteristic, and chromatographic performance of the MIPM and a control monolith synthesized without the print molecule were examined, respectively. The results showed that using ceramide III as print molecule significantly affected the pore structure and pore distribution of the monolith, and greatly improved the retention of ceramide III and its analogues used in cosmetics as well. The retention of ceramide III on the MIPM could be reduced by increasing the ratio of chloroform to hexane in eluting buffer. The workability of the MIPM was firstly demonstrated through the separation of a model lipid mixture containing ceramide III and ergosterol, the main sterol impurity in yeast lipid extracts. The application of the ceramide III imprinted monolith to the isolation of ceramides from yeast lipid extracts was attempted and resulted in a considerable enrichment of ceramides, as shown by FTIR analysis. This indicates the potential of ceramide III imprinted monolith synthesized in the present study in the on-line solid-phase extraction of ceramides from yeast.

Introducing an electric field into chromatography on hydroxyapatite (HAP) was attempted in order to enhance mass transfer and separation performance. A membrane spaced multicompartment electrolyzer was developed for electrochromatography on HAP. The high performance of liquid transport by electroosmotic flux was identified and described in terms of dynamic electroosmotic pressure. The application of the electric field resulted in an improved adsorption of bovine serum albumin as shown by the breakthrough curve as function of the electric field. An improved elution was also obtained in the presence of the electric field. The results show that electroosmosis is a powerful tool of liquid transport and dispersion in a packed bed of fine particles and has potential in the large-scale chromatography of biological molecules.

Knowledge of the adsorption mechanism of biological molecules on hydroxyapatite (HAP) is essential to the application of HAP chromatography and to the development of HAP-based biomedical materials. Centered on the surface charge of HAP and its impacts on adsorption of proteins, the present study started with the characterization of-potential of HAP as a function of the chemical properties of solution in terms of concentration of ions of different types, ionic strength and pH. Then the adsorption of bovine serum albumin (BSA) on HAP was carried out at the same conditions to elucidate the effects of-potential on BSA adsorption and the replacement of BSA with PO4 3−in acidic buffer. A Langmuir isotherm was obtained, indicating a single layer adsorption of BSA on HAP. Finally, the apparent activation energy and the adsorption heat were interpreted from the adsorption at different temperatures. The low magnitude of both the apparent activation energy and the adsorption heat indicated that the fast adsorption of BSA on HAP was a physical adsorption process.

Refolding of native and recombinant lysozyme was accomplished by dilution with refolding buffer containing low-concentration cetyltrimethylammonium bromide (CTAB). In both cases, the use of-cyclodextrin (β-CD) was unnecessary. Centered on the interaction between CTAB and the protein being refolded, experimental studies were conducted to investigate the effect of CTAB on the refolding yield, product distribution and the refolding kinetics. A comparative study of the artificial chaperone-assisted refolding of native and recombinant lysozyme was included to generate a more comprehensive understanding of the function of CTAB in assisting protein refolding. It was shown that the formation of CTAB-denatured lysozyme complex occurred at the beginning stage of refolding effectively inhibited the formation of aggregate, leading to an improved refolding. The dissociation of this complex occurred, when lysozyme started to fold into its native conformation catalyzed by GSSG/GSH. The use of β-CD facilitated the dissociation the CTAB-protein complex and thus increased the overall rate of lysozyme refolding. The results described by present study are also helpful for the design of surfactant and the optimization of refolding process for different proteins.

The effects of a weak electric field on the growth and metabolic behavior of Enterobacter dissolvens were investigated using glucose as the sole carbon source. A direct current (DC) was applied using salt bridge electrodes and platinum electrodes. The best stimulating effects in terms of cell growth and the dehydrogenase activity (DHA) were obtained when a DC of 10mA was applied for 12 h via the platinum electrodes. In this case, the electrolysis of water was the major electrode reaction, as determined by cyclic voltammetry. The presence of the hydrogen generated a strong reductive environment and led to a reduction of NAD/NADH ratio from 7 to 3. The specific activity of dehydrogenase and glucose consumption increased 2- and 1.5-fold, respectively. The application of the DC via the platinum electrodes also led to accelerated cell death during the later stationary phase. This is possibly due to the presence of anodic intermediates including H2O2, OH• and O2•. These results provide more details for understanding the effect of a DC on E. dissolvens, a strain with potential application in the electro-remediation of PAHs contaminated soil.