Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities

Authors: Liu, Keshun

Submitted to: Journal of Food Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2022
Publication Date: 5/3/2022
Citation: Liu, K. 2022. Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities. Journal of Food Science. 87(5):2018–2033. https://doi.org/10.1111/1750-3841.16141.
DOI: https://doi.org/10.1111/1750-3841.16141

Interpretive Summary: Protease inhibitors of protein nature are ubiquitously distributed in the plant, animal, and microbial kingdoms, playing key regulatory roles in many biological processes. In seeds of legume crops, such as soybeans and pulses, there are two major types of protease inhibitors: trypsin inhibitors and trypsin and chymotrypsin inhibitors. Since trypsin and chymotrypsin are the two major proteolytic enzymes in mammalian digestive tracts, their inhibitors are considered antinutritional. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement. However, as plant proteins are increasingly used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity in these products as well. The problem is that at the present reported methods for measuring chymotrypsin inhibitor activity vary greatly and lack details in description. There is no standardized or official method available. Thus, results cannot be compared among studies. Furthermore, since a popular substrate is not water soluble, an organic solvent must be present. This adds complexity for the assay method development. In addressing these problems, the present study was conducted. The objective was to develop a reliable method to measure chymotrypsin inhibitor activity in various protein products with accuracy and precision. After investigating the effects of several factors, such as absorption spectra, organic solvent type and concentration, substrate and enzyme concentrations, inhibitor levels, the sequence of adding reagents, extractant and extraction time, etc., an optimized method for measurement of chymotrypsin inhibitor activity was finally developed. The method can also be used for measuring chymotrypsin activity. The robust performance of the proposed method was verified by measuring 11 assorted protein products, paving a way for standardization.

Technical Abstract: Protease inhibitors of protein nature are rich in seeds of legume crops. There are two common types: Kunitz inhibitor, which mainly inhibits trypsin, and Bowman–Birk inhibitor, which inhibits both trypsin and chymotrypsin. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement. However, as plant proteins are increasingly used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Reported methods for CIA assay vary greatly and are incompletely described. No standardized or official method is available. The present study focused on developing a robust method for accurately measuringCIA, usingN-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Since BTpNA is not water soluble, a water-miscible organic solvent must be present. After investigating the effects of several factors, such as absorption spectra, organic solvent type and concentration, substrate and enzyme concentrations, inhibitor levels (which affected % chymotrypsin inhibition), the sequence of adding reagents, extractant and extraction time, and so forth, an optimized method for CIA measurement was finally developed. It features dimethylformamide as the organic solvent, the enzyme-last sequence, 5 ml total assay volume, and calculation of the inhibitor activity based on 40% chymotrypsin inhibition. The method can also be slightly modified for measuring chymotrypsin activity. The robust performance of the method was verified by measuring 11 assorted protein products, paving a way for standardization.