Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 209

2.5
Behaviour of the Immunoaffinity Immobilized Enzyme
High immobilization yields,expression of high activity of the bound enzyme
and stabilization against inactivation/ denaturation are the principal yardsticks
for the measurement of the success of any enzyme immobilization procedure
and have received considerable attention in case of immunoaffinity immobi-
lized enzymes as well. As has been already pointed out,the antibody molecule
acts as large spacer holding the enzyme at a distance from the support matrix
thereby minimizing steric hindrance and facilitating remarkable freedom to act
even on high molecular weight substrates [18]. Expression of nearly full activi-
ty by the enzyme complexed directly with antibody [22,24] or when bound to
support matrix-coupled antibody have been observed by several investigators
[26]. In addition,the K m values of several immunoaffinity immobilized enzymes
were either unaltered,exhibited minor alterations as compared those of their
respective soluble enzymes. This has been observed in the case of trypsin [26],
transglutaminase [16],NADase and urease [27] and carboxypeptidase [17,18].
The last study also described relatively unaltered K i values of the enzyme for
small molecular weight inhibitors. Table 2 lists the impressive enhancement
in the stabilities of the immunoaffinity immobilized enzymes against high
temperature,chemical denaturants,proteolysis oxidative stress,etc. Antibody
mediated stabilization may not however be a general phenomenon as reports of
labilizing antibodies are also available.
Where applicable the stability enhancement may arise out of crosslinking like
effect caused by the antibody binding on enzyme. Antibody binding appears to
involve reasonably large areas of protein antigen that in turn may comprise
more than a single oligopeptide [86,87]. Based on their studies on the interac-
tion on the influenza virus protein and the Fab' fragment of the antibody,Davis
et al. [88] suggested that a large number of non-covalent interactions are in-
volved and water molecules are eliminated from the contact areas. Shami et al.
[22] argued that reduction in the free energy of the antigen resulting from the
binding of even a moderate affinity antibody [89] may be sufficient to confer
stability as free energy changes between the folded and the unfolded states of
protein lie in the same range [90]. Furthermore although due to the large size of
the antibody a single matrix bound antibody may not bind more than one
molecule of enzyme,lateral interactions with more than one antibody molecule
may contribute significantly to the stability of the enzyme protein [91]. It is
interesting to note that some monoclonal antibodies exhibit chaperon like activity
and assist in antigen refolding [92,93] and inhibit enzyme aggregation [94].
In instances where soluble antibody is used for the enzyme immobilization,a
single enzyme molecule may interact with more than one antibody molecules
resulting in high degree of stabilization [24] like an enzyme attached via multiple
covalent [45] or non-covalent linkages [46]. Indeed the degree of stabilization
achieved with such complexes is relatively very high [22,28]. While a correlation
may exist between the thermal stability of protein and its susceptibility to
proteolysis [95],the exact mechanism by which antibodies enhance stability
against other forms of inactivation needs to be further examined.