Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities

Area of Science:

• Biochemistry
• Food Science
• Analytical Chemistry

Background:

• Legume seeds contain protease inhibitors, primarily Kunitz and Bowman-Birk types.
• Trypsin inhibitor activity has historically been the focus, but chymotrypsin inhibitor activity (CIA) is increasingly important for plant-based food/feed.
• Existing methods for CIA assay lack standardization and are often incompletely described.

Purpose of the Study:

• To develop a robust and accurate method for measuring chymotrypsin inhibitor activity (CIA).
• To optimize assay conditions for reliable CIA quantification in various protein products.

Main Methods:

• Investigated factors including organic solvents, substrate/enzyme concentrations, and reagent addition sequences.
• Utilized N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate in a water-miscible organic solvent (dimethylformamide).
• Developed an enzyme-last protocol with a 5 ml assay volume, calculating activity based on 40% chymotrypsin inhibition.

Main Results:

• An optimized method for CIA measurement was successfully developed, featuring dimethylformamide and an enzyme-last addition sequence.
• The method demonstrated robust performance when validated on 11 diverse protein products.
• The assay is sensitive and can be adapted for measuring chymotrypsin activity.

Conclusions:

• The developed method provides a reliable basis for the standardization of CIA measurement in plant protein products.
• Accurate CIA quantification is essential given the growing use of plant proteins in food and feed.
• This standardized assay will enhance quality control and safety in the plant protein industry.