| Summary: | 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 .
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