Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 212

sions and may necessitate additional immobilization procedures [60]. As Con
A-glycoenzyme complex formation does not require high purity of either the gly-
coenzyme or lectin,possibility of cutting down of the cost of immobilization has
been demonstrated using crude lectin preparation in place of pure Con A [111].
The biological activity of Con A seems to remain unaffected by chemical
modification of the amino groups [112]. Coupling to supports has therefore been
achieved mostly via amino groups. A large number of support matrices have
been used for the coupling of Con A for the preparation of the affinity supports
[103]. More recently Fadda et al. [113] have shown that Con A can be coupled in
high yield to commercial aluminium oxide after activation by means of 3-ami-
nopropyltriethoxysilane and subsequent reaction with cyanogen bromide.
Con A has been shown to bind very strongly to copper (II) iminodiacetic acid
functions of various supports due to the presence of six histidine residues in
each subunit [114]. Such binding has also been shown to favourably orient Con
A on the support facilitating very high degree of binding to glycoproteins [115].
A simple strategy for remarkably raising the amount of Con A and glyco-
enzyme associated with solid supports has been recently developed by us [116].
It involves building up layers of Con A and glycoenzymes on Sepharose matrix
precoupled with Con A (Fig. 2). Bioaffinity layered preparation of glucose
oxidase and invertase exhibited high activity,good mechanical stability and a
layer-by-layer increase in thermal stability. The technique was applicable to all
the glycoenzymes investigated namely-glucose oxidase,invertase, -galactosi-

dase and amyloglucosidase. A somewhat similar approach was earlier used by
Gemeiner et al. [117] in the preparation of superactive immobilized invertase.
Enzymes immobilized on Con A supports show impressive gains in resistance
to inactivation induced by heat,chemical denaturation,proteolysis,storage [103,
118] and long term continuous operation for several weeks [119]. Taking into
consideration the stabilization that accompanies immobilization of glycoenzy-
mes via their carbohydrate chains using lectins [103],glycosyl-specific anti-
bodies [29,30] or by chemical reaction [120,121],it appears likely that regions
close the glycosylation sites of enzymes may be important in the unfolding of
several glycoenzymes. It is now recognized that unfolding of protein molecules
may begin at specific region of the molecule and improvement in stabiliza-
tion may be remarkable if such regions are blocked by attachment to suitable
supports [122,123].
Binding of Con A to glycoproteins can be prevented/reversed by several
sugars like methyl-

b

- D -mannopyranoside or even D -man-
nose or D -glucose which are known to interact with the lectin [124,125]. The
strength of association between Con A and glycoprotein has however been
shown to be dependent not only on the nature of glycosylation but also on the
nature of glycoprotein [126]. Some glycoenzymes like invertase exhibit extra-
ordinary strong binding and necessitate extra long incubation with the eluting
sugar [127]. It was also shown that ease of elution of the enzyme depends upon
the lectin concentration on the support. Nevertheless several lectin-based
analytical devices with reloadable glycoenzymes have been described.
Mattiasson and Borreback [128] employed glucose oxidase and peroxidase
in a thermistor devices containing immobilized Con A for the measurement of

a - D -glucopyranoside, a