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.