Bioseparation最新文献

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed.

In this review article the function of the binding site monomers in the molecular imprinting procedure is discussed. Especially, new developments towards stoichiometric noncovalent interactions are highlighted. In stoichiometric noncovalent interactions template and binding site monomer in an 1:1 molar ratio are nearly completely bound to each other. This is only possible if the association constants are considerably high (Kass > 900 M(-1)). Using this type of interaction in molecular imprinting no excess of binding sites is necessary and binding sites are only located inside the imprinted cavity. Since all cavities can be reloaded these polymers show high capacity (e.g., for preparative application) and are especially suited for the synthesis of catalytically active imprinted polymers. Discussed are binding site interactions based on amidines (and guanidines), multiple hydrogen bonding, charge-transfer interactions, and host-guest inclusion. The systematic investigation of the underlying binding reaction is described in detail. With low-molecular weight model substances the thermodynamics of the association can be conveniently investigated, e.g., by NMR spectroscopy.

A highly selective polymer has been prepared for the selective separation of nucleotides by the surface imprinting polymerization. A dialkyl quaternary ammonium chloride was effective as the functional molecule for recognizing the difference in the structure of nucleotides. Adsorptive behavior of the ionic species of the structural analogues, inosine-5'-monophosphoric acid (IMP) and guanosine-5'-monophosphoric acid (GMP), could be controlled by changing the pH condition. Surface imprinting polymers were prepared under different pH conditions; pH 9.0 and pH 8.5. The IMP-imprinted polymers exhibited higher template effect for IMP than for a structural analogue, GMP. A reference polymer prepared without the imprint molecule neither exhibit any selectivity to IMP nor to GMP. The adsorption behavior was quantitatively evaluated by the binding constants for the IMP-imprinted polymer. The imprinting polymer was found to recognize a small structural difference in nucleotides.

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin.

This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether. Nucleic acids purified from E. coli were injected separately in the above HIC column and eluted with 1.5 M (NH4)2SO4 in the buffer. The column was able to separate single-stranded from double-stranded nucleic acids. RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands. Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough.

A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L)-phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60 degrees C. The monolith obtained was crushed, sieved to 30-90 microm and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase. From the measurement of the relative selectivity it was found that only the simultaneous presence of the carboxyl, the phenolic hydroxyl and certain peculiar substructures such as the chlorine atom assures the whole recognition of the template.