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.

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.

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.

A temperature stimuli-responsive polymer, dextran-grafted-PNIPAAm (DGP), was prepared by radical polymerization using cerium nitrate as the initiator. The structure and the grafting ratio of DGP were determined by FT-IR and elemental analysis. The temperature-stimuli responsive behavior of DGP and the size of the DGP self-assemblies at different temperatures were determined by transmittance and dynamic light scattering, respectively. The use of DGP as an artificial chaperone to assist protein refolding in vitro was demonstrated using hen egg white lysozyme (lysozyme) and carbonic anhydrase bovine (CAB) as model proteins. It was shown that the hydrophobic interaction between DGP and the protein being refolded gave a significant reduction in the rate of protein aggregation and a slight reduction in the refolding rate, and consequently gave an improved refolding yield. Moreover, the refolding of lysozyme and CAB at a temperature gradient starting above the LCST of DGP and ending at a lower temperature, which make DGP change from hydrophobic to hydrophilic, gave a significant increase in the refolding yield compared to that carried out at a constant temperature. The process mechanism of DGP assisted protein refolding was presented and the importance of controlling the hydrophobicity of the solution environment for protein refolding was discussed.