Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by...

Ficin fully immobilized on Asp-agarose beads at pH 7 but not on an aminated support. This made enzyme adsorption plus glutaraldehyde modification non-viable for this enzyme. Modifying glyoxyl-agarose beads with mixtures of Asp and 1,6-hexamethylenediamine (HA) at different ratios, mixed anion/cation exchanger supports were built. Only if HA greatly exceed Asp in the support, immobilization did not work. While only using the Asp-agarose support immobilized enzyme molecules were only ionically adsorbed after glutaraldehyde treatment (visualized in SDS-PAGE analysis), the mixed supports gave covalent immobilization. The glutaraldehyde modification of these biocatalysts permitted to establish covalent bonds with the support, and this was more effective when using higher amounts of HA in the support. When around 60 % of the groups in the support were HA, the treatment with glutaraldehyde fully suppressed enzyme release from the support after boiling in SDS. The glutaraldehyde treated biocatalysts were more stable than just the adsorbed enzymes or the enzyme adsorbed only on Asp supports and then treated with glutaraldehyde (the optimal biocatalyst retained 90 % of the initial activity while the just adsorbed ficin retained 50 % of the initial activity). This strategy can be utilized to immobilize other proteins with high isoelectric points following this immobilization strategy.