Enzyme loading in the support and medium composition during immobilization alter activity, specificity and stability of octyl agarose-immobilized Eversa Transform.

Eversa Transform (ETL) was immobilized on octyl agarose beads at two different enzymes loadings (1 mg/g and 15 mg/g) under 18 different conditions, including different pH values, buffers, additives (different solvents, Ca, NaCl). Their activity was analyzed at pH 5 and 7 with p-nitrophenyl butyrate and at pH 5 with triacetin, determining also its stability at pH 5 and 7 (in different media). Ca stabilized ETL biocatalysts while phosphate destabilized them.

Optimizing the activation of agarose beads with divinyl sulfone for enzyme immobilization and stabilization.

The focus of the present work is to find the optimal conditions for the activation of agarose beads with divinyl sulfone (DVS). The reactivity of the vinyl sulfone groups in the support was checked by the support capacity to react with ethylamine; via elemental analysis. In addition, trypsin was used as a model enzyme to test the immobilization and stabilization capabilities of the different supports.

Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by enzyme adsorption and support-enzyme glutaraldehyde crosslinking.

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.

Changes in ficin specificity by different substrate proteins promoted by enzyme immobilization.

Ficin extract has been immobilized using different supports: glyoxyl and Aspartic/1,6 hexamethylenediamine (Asp/HA) agarose beads. The latter was later submitted to glutaraldehyde modification to get covalent immobilization. The activities of these 3 kinds of biocatalysts were compared utilizing 4 different substrates, casein, hemoglobin and bovine serum albumin and benzoyl-arginine-p-nitroanilide at pH 7 and 5.

Designing tailor-made steric matters to improve the immobilized ficin specificity for small versus large proteins.

The development of strategies that can permit to adjust the size specificity of immobilized proteases by the generation of steric hindrances may enlarge its applicability. Using as a model ficin immobilized on glyoxyl agarose, two strategies were assayed to generate tailor made steric hindrances. First, ficin has been coimmobilized on supports coated with large proteins (hemoglobin or bovine serum albumin (BSA)).