Deviation of Trypsin Activity Using Peptide Conformational Imprints

Area of Science:

• Biochemistry
• Enzyme immobilization
• Materials Science

Background:

• Enzyme immobilization is crucial for biocatalysis and enzyme reusability.
• Developing specific and stable immobilization methods remains a challenge.
• Porcine pancreatic alpha-trypsin (PPT) is a widely studied enzyme with significant biotechnological applications.

Purpose of the Study:

• To develop a novel methodology for site-specific immobilization of porcine pancreatic alpha-trypsin (PPT).
• To utilize peptide conformational imprints (PCIs) on magnetic particles (PCIMPs) for targeted enzyme capture.
• To evaluate the catalytic activity, kinetic parameters, and stability of immobilized PPT.

Main Methods:

• Fabrication of segment-mediated PCIs on magnetic particles (PCIMPs) to create cavities complementary to PPT structure.
• Immobilization of PPT using template-derived PCIMPs based on specific PPT sequences.
• Assessment of hydrolytic activity using N-benzoyl-L-arginine ethyl ester (BAEE) and high-performance liquid chromatography (HPLC).
• Kinetic analysis using Lineweaver-Burk plots to determine inhibition type and kinetic parameters.

Main Results:

• PCIMPs successfully created specific cavities for PPT immobilization.
• The immobilized PPT exhibited non-competitive inhibition towards the substrate BAEE.
• Optimized kinetic parameters for PPT/PCIMPs 233-245+G were determined (V = 1.47 × 10^-3 mM s^-1, K = 0.42 mM, k = 1.16 s^-1, k/K = 2.79 mM^-1 s^-1).
• Immobilized PPT maintained stable catalytic activity over four successive cycles.

Conclusions:

• Peptide conformational imprints provide an effective strategy for site-specific enzyme immobilization.
• The PCIMP method enhances the stability and reusability of porcine pancreatic alpha-trypsin.
• This approach offers a promising platform for developing robust biocatalytic systems.