Preparation and characterization of a dextran-trypsin conjugate

Area of Science:

• Biochemistry
• Enzyme immobilization
• Protein chemistry

Background:

• Bovine pancreatic trypsin is a widely used serine protease.
• Enzyme immobilization is crucial for enhancing enzyme stability and reusability.
• Dextran, a polysaccharide, can be used as a carrier for enzyme conjugation.

Purpose of the Study:

• To conjugate bovine trypsin to dextran using cyanogen bromide activation.
• To characterize the enzymatic activity, stability, and inhibitor resistance of the resulting dextran-trypsin conjugate.
• To investigate the role of polysaccharide cross-linking in enzyme stabilization.

Main Methods:

• Activation of dextran with cyanogen bromide.
• Coupling of bovine pancreatic trypsin to activated dextran.
• Purification of the soluble dextran-trypsin conjugate using molecular sieve chromatography.
• Assays for esterase and caseinolytic activity.
• Evaluation of stability against heat, autodigestion, and denaturing agents.
• Assessment of resistance to trypsin inhibitors (e.g., ovomucoid).
• Treatment with dextranase to assess the role of the polysaccharide.

Main Results:

• The dextran-trypsin conjugate retained 53% of esterase activity but only 7% of caseinolytic activity compared to native trypsin.
• The conjugate exhibited significantly enhanced resistance to heat inactivation, autodigestion, and denaturing agents.
• The modified trypsin demonstrated increased resistance to inhibition by trypsin inhibitors, especially ovomucoid.
• Treatment with dextranase partially reversed the enhanced stability and inhibitor resistance.
• The preparation consisted of heterogeneous macromolecular aggregates of trypsin and dextran.

Conclusions:

• Conjugation of bovine trypsin to dextran via cyanogen bromide activation yields a stable enzyme preparation.
• Intramolecular cross-linking by polysaccharide chains is responsible for the stabilization of the enzyme's tertiary structure.
• The dextran-trypsin conjugate offers improved stability and resistance to inhibitors, making it potentially useful for various applications.