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

Area of Science:

• Biochemistry
• Enzyme immobilization
• Protein chemistry

Background:

• Enzyme immobilization is crucial for biocatalyst reusability and stability.
• Different immobilization supports and methods can significantly impact enzyme activity and specificity.
• Ficin, a plant protease, has diverse applications but requires optimized immobilization strategies.

Purpose of the Study:

• To compare the activity of ficin immobilized on glyoxyl-agarose and Aspartic/1,6 hexamethylenediamine (Asp/HA)-agarose beads.
• To investigate the effect of glutaraldehyde modification on immobilized ficin activity.
• To evaluate the influence of immobilization on ficin's specificity towards various protein and peptide substrates.

Main Methods:

• Ficin was immobilized onto glyoxyl-agarose and Asp/HA-agarose supports.
• Asp/HA-immobilized ficin was further modified using glutaraldehyde.
• Enzyme activity was assayed using casein, hemoglobin, bovine serum albumin, and benzoyl-arginine-p-nitroanilide at pH 7 and 5.

Main Results:

• Immobilization on glyoxyl-agarose did not decrease ficin activity, even with extended reaction times.
• Asp/HA supports induced significant changes in ficin activity, with some substrates showing increased activity and others decreased.
• Glutaraldehyde treatment further enhanced these activity alterations, indicating significant conformational changes and altered substrate specificity.

Conclusions:

• Immobilization strategies, particularly the choice of support and chemical modification, profoundly influence ficin's enzymatic properties.
• Conformational changes induced by immobilization can lead to altered substrate specificity, impacting enzyme performance.
• These findings have implications for enzyme inactivation studies and the development of tailored biocatalysts.