Identification of a New Soybean Kunitz Trypsin Inhibitor Mutation and Its Effect on Bowman−Birk Protease Inhibitor Content in Soybean Seed

Soybean seed contains antinutritional compounds that inactivate digestive proteases, principally corresponding to two families: Kunitz trypsin inhibitors (KTi) and Bowman–Birk inhibitors (BBI). High levels of raw soybean/soybean meal in feed mixtures can cause poor weight gain and pancreatic abnormalities via inactivation of trypsin/chymotrypsin enzymes. Soybean protein meal is routinely heat-treated to inactivate inhibitors, a practice that is energy-intensive and costly and can degrade certain essential amino acids. In this work, we screened seed from 520 soybean accessions, using a combination of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblots with anti-Kunitz trypsin inhibitor antibodies. A soybean germplasm accession was identified with a mutation affecting an isoform annotated as nonfunctional (KTi1), which was determined to be synergistic with a previously identified mutation (KTi3–). We observed significant proteome rebalancing in all KTi mutant lines, resulting in dramatically increased BBI protein levels.