Enzyme and protein immobilization on non-porous polystyrene- based microspheres

• Main Authors: Wu, Cheng-We, 吳政誼
• Other Authors: Lee Wen-Chien
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/39409829393230260369

碩士 === 國立中正大學 === 化學工程研究所 === 84 === The major disadvantages of porous carries is that most of the surface available for protein immobilization is internal surface . Diffusional limitation can be serious and great amount of immobilized enzyme within the micro-pores of the porous beads may never be fully utilized . In a chromatographic column packed with porous adsorbents , mass transfer effects due to pore diffusion of solutes into and out of the matrix may result in a longer elution time and a lower chromatographic efficiency . The diffusion contraints with respect to substrate and product can be eliminated when the enzyme is immobilized on non-porous carriers. In an affinity chromatography system, non-porous beads can present advantages for very rapid analytical and micropreparative chromatography of bioproducts. In this work, we synthesized non-porous beads of polystyrene as the support and developed a procedure of protein and enzyme immobilization on these beads . Due to its good mechanical property , polystyrene can remain resistance against a strong acid or base under operating condictions .However , the hydrophobic benzene groups on the side-chain of polystyrene should be chemically modified (nitration and hydrogenation) and converted to yield amino groups for enzyme and protein immobilization .Three chemical coupling methods were successfully applied to carried out immobilization of β- lactamase . When penicillin-G was used as the substrate , the immobilized enzyme prepared by the method of glutaraldehyde crosslinking yields the hightest activity under the optimal conditions (pH 7.0 , temperature 30℃). The immobilized enzyme also obey the Michaelis-Menten kinetics with a value of 2.85 mM ,which is slightly larger than that (0.57 mM) of the free enzyme .It was observed that heat stability of the immobilized enzyme can be increased by the addition of sorbitol in the buffer. The technique of immobilization and the experimental results obtained in this work provide a basic for the design of a mini-enzyme-reactor consisting of a thermistor .In the affinity chromatography system , concanavalin A was immobilized onto the surface of the polystyrene support via a crosslinker, glutaraldehyde . These prepared affinity adsorbents were then packed into two columns (5×0.46 cm ID. and 5×0.2 cm ID. ). Because the specific surface area of the polystyrene beads is small , the immobilized quantity of Con A and consequ-ently the binding capacity of chromatographic columns is relatively small . the capacity factor ( ) is only at the level of 0.65 . This small value of capacity factor resulted in a slight overlap of the chromatographic peaks whenever the samples containing a sugar of affinity and a sugar of non-affinity were applied. The experimental results showed that the sample loading and the flow-rate significantly influence the capacity factor and the chromatographic efficiency . However, the data indicate that these affinity columns are suitable for the chromatographic determination of Con A-specific sugar, p-nitrophenyl-α-D- mannopyranoside and other derivations of α-D-mannopyranoside. The capacity factor is decreased by increasing sample loading and the plate height becomes larger whenever a larger sample is applied .