Enzymatic and algebraic methodology to determine the contents of Kunitz and Bowman-Birk inhibitors and their contributions to total trypsin or chymotrypsin inhibition in soybeans

Authors: Liu, Keshun

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/2023
Publication Date: 5/8/2024
Citation: Liu, K. 2024. Enzymatic and algebraic methodology to determine the contents of Kunitz and Bowman-Birk inhibitors and their contributions to total trypsin or chymotrypsin inhibition in soybeans. Journal of Agricultural and Food Chemistry. 72(20):11782-11793. https://doi.org/10.1021/acs.jafc.3c06389.
DOI: https://doi.org/10.1021/acs.jafc.3c06389

Interpretive Summary: Soybean is a major oilseed crop and the number one source of plant protein for food and feed. However, the natural presence of protein proteinase inhibitors exerts antinutritional effects (such as reducing protein digestion, enlarging pancreas, and depressing growth), which limits soybean use. There are two types of proteinase inhibitors in soybeans: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Together, they contribute up to 6% of the total protein in the seed. Because of their difference in molecular structures, the two inhibitors show different genetic constitutions, physicochemical properties, and nutritional and health implications. Historically, trypsin inhibitor activity in soybeans has been measured enzymatically, but these measurements were not able to differentiate KTI from BBI in any way since both inhibit trypsin. Consequently, in the past three decades, several non-enzymatic methods, including immunoassays, polyacrylamide gel electrophoresis, and liquid chromatography, have been developed to quantitate KTI and/or BBI in soybeans. Yet, these methods have pros and cons. Furthermore, even with these methods, it is still hard to determine the contribution of each inhibitor toward total trypsin inhibition or total chymotrypsin inhibition. Recently, USDA-ARS researchers at Aberdeen, Idaho, have developed a brand-new methodology that has a capacity to differentiate and measure KTI and BBI, simultaneously, not only for their mass composition but also for their contribution toward total trypsin or chymotrypsin inhibition in soybeans. This so-called enzymatic and algebraic methodology serves as a much needed analytical tool for food/feed chemists and plant scientists to determine inhibitory activities and contents of KTI and BBI in soybeans simultaneously. This greatly facilitates their efforts to modify protease inhibitor content and activity through genetic manipulation and/or processing, and thus, achieve optimal nutrition and health benefits of soybeans as food and feed.

Technical Abstract: Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.