Covalent coupling of calf brain prolidase

Area of Science:

• Biochemistry
• Enzyme immobilization
• Protein chemistry

Background:

• Prolidase is a crucial enzyme involved in peptide bond hydrolysis.
• Enzyme immobilization is a technique used to enhance enzyme stability and reusability.
• Understanding the properties of immobilized enzymes is essential for biotechnological applications.

Purpose of the Study:

• To investigate the biochemical properties of calf brain prolidase after covalent immobilization.
• To compare the stability, activity, and substrate specificity of free and immobilized prolidase.
• To evaluate the effects of metal ions and pH on the enzymatic activity of both preparations.

Main Methods:

• Covalent immobilization of calf brain prolidase onto CNBr-Sepharose 4B.
• Enzyme activity assays at various pH values and temperatures.
• Determination of enzyme stability under different storage conditions.
• Assessment of the effects of various metal ions and compounds on enzyme activity.

Main Results:

• Immobilized prolidase retained 32% of the free enzyme's activity.
• Free prolidase demonstrated greater stability at 20°C and 0°C, and superior thermal stability between 25°C-60°C.
• Both enzyme forms showed optimal activity at pH 4.0, but free prolidase exhibited additional maxima at pH 9.0 and 6.5.
• Heavy metal ions (Ag+, Cu+, Hg+2, Zn+2) were strong inhibitors of free prolidase, with less inhibition observed for the bound enzyme (except Zn+2).
• Co+3, Mg+2, and Mn+2 stimulated free prolidase activity, unlike the immobilized enzyme.

Conclusions:

• Covalent immobilization of calf brain prolidase on CNBr-Sepharose 4B alters its stability and kinetic properties.
• The immobilized enzyme shows reduced, but still significant, activity and altered responses to pH and metal ions.
• These findings provide insights into the behavior of immobilized prolidase, relevant for biocatalysis and enzyme engineering.