Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry - Advances in Biochemical Engineering Biotechnology - page 92

optimal recovery of the enzyme depends upon the lectin concentration on the
support [44].
A method based on the FMC determination of catalytic activity of IMB in the
FMC column was used to monitor the process of lectin affinity chromatography
of invertase on Con A-bead cellulose [45]. Adsorption and desorption in the
chromatography column were examined by FMC in small samples withdrawn
from the column.Attention was given to the operating parameters and the stor-
age stability of the affinity sorbent. The binding ability of the affinity matrix
decreased with the number of consecutive chromatographic runs, although its
storage stability was satisfactory.
Binding of Con A to glycoproteins could be prevented/reversed by several
sugars which are known to interact with the lectin,such as a -methyl- D - gluco-

pyranoside, D -mannose or -methyl- D - manno pyranoside [46]. Glycosylated

enzymes, in this case invertase and glucose oxidase, could be used as competi-
tive markers for the simple and rapid determination of the relative carbohydrate
specificity of lectins for mono- and oligosaccharides. They were used as the
competitive saccharides in homogeneous and heterogeneous systems: (lectin +
enzyme + saccharide) and (lectin immobilized on support + enzyme + saccha-
ride), respectively, under static or flow conditions. The activity of the enzyme
was determined indirectly by spectrophotometry in the supernatant liquid,after
the precipitated lectin-enzyme complex was separated by centrifugation
(homogeneous system) or in a Con A-sorbent with specifically bound enzyme
(heterogeneous system at static conditions). A new, alternative method using
enzyme flow microcalorimetry was applied in flow conditions for the direct
determination of the biospecifically adsorbed enzyme. The relative carbo-
hydrate specificity of lectins (Con A) was estimated as the concentration of
the saccharide ( D -mannose,

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-methyl- D - manno pyranoside) that inhibited the
interaction to 50%. The results obtained by a precipitation-inhibition assay
in solution and a sorption-inhibition assay in a batch system intercorrelated
significantly with those obtained with a sorption-inhibition assay in the FMC
flow system [46].
Among generally appropriate ligands, specific antienzyme antibodies are
clearly the most versatile, being applicable - at least theoretically - to virtually
every enzyme.Moreover,anti-Con A antibody may also be employed,at least in
an analytical determination of freely accessible Con A pre-linked to affinity
sorbent.The amounts of accessible Con A pre-linked to bead cellulose could be
evaluated: (1) by adsorption of invertase,and (2) by means of ELISA,using both
mouse anti-Con A antibody and pig anti-mouse antibody linked with peroxi-
dase [35]. Reactions under conditions 1 and 2 were monitored in two ways:
thermometrically, by FMC, as well as by conventional spectrophotometry. The
results obtained by spectrophotometry and thermometry correlated signi-
ficantly (p = 0.028 and p = 0.0001,for ELISA and the invertase).The amounts of
immobilized Con A were directly proportional to the ELISA signal of the pero-
xidase reaction as detected by FMC (r = 0.983, p = 0.0005).A slightly less tight
correlation was found between the amounts of immobilized Con A and the
activity of invertase in conjugates when they were assayed by means of thermo-
metry (r = 0.933, p = 0.0065) and spectrophotometry (r = 0.920, p = 0.0094).

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