Molecular characterization of soybean meal trypsin inhibitors and lectins as a basis for developing approaches to mitigate their anti-nutritional effects

Abstract

Soybean is a crop that is widely eaten because of its nutritive properties. However, soybean cannot be consumed in its raw form because it contains anti-nutrients like trypsin inhibitors which bind to serine proteases (chymotrypsin, elastase and trypsin) and restrain their digestive functions leading to indigestion and stunted growth. It also contains lectin which binds to intestinal cells containing N-acetyl galactosamine (GalNAc) to impede nutrient uptake into the blood. Soybean needs to be processed to get rid of these anti-nutrients before human or animal consumption. The conventional method of soybean processing which involves the moist heat treatment of soybeans is time and energy demanding, leads to the loss of nutrients, and residual amount of anti-nutrients are retained in the processed soybean meal, making this process relatively inefficient. This research work aimed to provide alternative solutions to the conventional soybean processing methods. Using quick purification techniques, we isolated TIs [Bowman-Birk trypsin inhibitor (BBTI) and Kunitz trypsin inhibitor (KTI)] and lectin from soybean meal. Using biophysical techniques, we characterized the interactions between soybean anti-nutrients and their host ligands to offer alternative solutions to improve the nutritional quality of soybean meal. The known anti-nutritional pathway of soybean lectin involves its binding to GalNAc containing intestinal cells; however, we believe soybean lectin could be also targeting another ligand called sulfatide besides GalNAc in the small intestine due to some similarities between soybean lectin and galectin-4, a mammalian lectin that binds to sulfatide in the small intestine. Hence, we also explored soybean lectin to sulfatide interactions to find a new soybean lectin anti-nutritional pathway. Results from a soybean meal cultivar showed that KTI had a binding preference for chymotrypsin, while BBTI preferred binding to trypsin and elastase. This provides insights into the unique roles that both TIs play in soybean and why the plant retains both. After screening TIs from several soybean meal lines for their affinity to trypsin, we identified BBTI soybean lines with ~4 to 6-fold lower affinity for trypsin and a KTI soybean line with ~8-fold lower affinity for trypsin, compared to the standard BBTI and KTI, respectively. These promising soybean meal lines have been designed for soybean crossbreeding to produce hybrid soybean meal lines that would require little to no processing before animal consumption. We also identified the KTI amino acid sequence motif, SPLHALFI as a suitable motif for gene editing to produce soybean meal lines with improved nutritional qualities. After screening GalNAc and GalNAc analogs against lectin to find a higher lectin affinity ligand than GalNAc that could be introduced as an additive in soybean meal to bind to lectin and prevent lectin from binding to GalNAc containing intestinal cells, we found no higher affinity ligand than GalNAc for lectin. However, we found out that a suitable substitution on the anomeric carbon of GalNAc would lead to the design of higher lectin affinity ligands than GalNAc that could serve as additives in soybean meal to mitigate lectin adverse effects. We also found out that soybean lectin crosslinks and binds to sulfatides with a high affinity, in a process that could result in unwanted signaling events in the body, making this another soybean lectin antinutritional pathway. This discovery could also lead to the design of suitable additives to target the sulfatide binding site on soybean lectin and prevent soybean lectin from binding to sulfatide in the body. It is recommended that this newly discovered soybean lectin-sulfatide anti-nutritional pathway should also be considered when assessing the nutritional quality of soybean meal. Taken together, this research provides alternative solutions that would lead to the enhancement of the nutritional quality of soybean meal and the mitigation of the adverse effects of soybean's anti-nutrients.