Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 211

a - D -Glucopyranosyl a - D -Mannopyranosyl unit

Fig 1. Carbohydrate units recognised by concanavalin A

addition to the saccharide binding site [104]. Early studies on the lectin indicat-
ed that Con A recognizes -D.mannose with free hydroxyl at

3-,4-and 6- positions [105] indicating the structural requirements as in Fig. 1.
Glycoproteins comprising of glycosyl chains with Glc

a - D -glucose and a

a 1 Æ ,Man a 1 Æ and

GlcNAc a 1 Æ residues which are located at the non-reducing termini of the sugar

chains and 1-residues located within the sugar chain

can bind to this lectin [106]. Based on the binding of several oligosaccharides
and glycopeptides,Ogata et al. [107] suggested that high mannose type of sugar
chains bind more strongly than those of complex type and presence of at least
two binding residues is essential for a oligosaccharide to be retained by a column
of immobilized Con A.
Con A retains its affinity for carbohydrates between pH 5.0 or lower and
above pH 9.0 [108] making it suitable for the immobilization of enzymes acting
over a wide pH range. A recent study has however suggested that inspite of the
comparable affinities of the tetrameric/dimeric forms for simple carbohydrate
ligands the affinity towards more complex oligosaccharides is considerably
higher in case of the tetrameric Con A [109]. This suggested that the Con A
supports may be relatively more effective for the immobilization of enzymes
acting optimally above pH 7.0.

Æ 2Glc a 1- and Æ 2Man a

3.2
Immobilization of Glycoenzymes Using Concanavalin A

The principal strategies of enzyme immobilization with the help of Con A are
very similar to those that employ antibodies,i. e. formation of insoluble lectin-
glycoenzyme flocculate and binding to insoluble supports precoupled with lec-
tins [103]. Due to the multivalent nature of Con A and the presence of several
oligosaccharides on most glycoenzymes,Con A readily interacts with glyco-
enzymes in solution to form large insoluble lectin-glycoenzyme complexes.
Since enzyme glycosyls,through which the enzymes are associated with lec-
tins are not usually involved in catalytic process [40],the Con A enzyme com-
plexes retain high catalytic activity [110]. Remarkable stability is also exhibited
by enzymes complexed with lectins [60] presumably due to the fixation of the
enzyme molecules in the native state by several lectin molecules in the complex
[103]. The Con A-glycoenzyme complexes however have small particle dimen-