Monoclonal antibodies against soybean Bowman-Birk protease inhibitor (BBI) have been generated and used to detect and quantify BBI in foods, soybean germplasm, and animal tissues and fluids. The purpose of this study was to determine the recognition sites of two monoclonal antibodies to BBI (mAb 238 and mAb 217) in relation to the protease-inhibitory sites of BBI. The results showed that (1) the binding of mAb 238 can be blocked by trypsin and that of mAb 217 by chymotrypsin; (2) the trypsin or chymotrypsin inhibitory activities of BBI are blocked by mAb 238 or mAb 217, respectively; and (3) mAb 238 failed to recognize a tryptic loop mutant BBI variant and mAb 217 was unable to bind a chymotryptic loop mutant BBI variant. These findings demonstrate that the epitopes recognized by mAb 238 and mAb 217 reside, at least in part, in the tryptic and chymotryptic loops of BBI, respectively.

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Ochratoxin A (OTA), a mycotoxin that is of utmost concern in food and feed safety, is produced by fungal species that mainly belong to the Aspergillus and Penicillium genera. The development of mitigation strategies to reduce OTA content along the supply chains is key to ensuring safer production of food and feed. Enzyme-based strategies are among the most promising methods due to their specificity, efficacy, and multi-situ applicability. In particular, some enzymes are already known for hydrolyzing OTA into ochratoxin alpha (OTα) and phenylalanine (Phe), eventually resulting in detoxification action. Therefore, the discovery of novel OTA hydrolyzing enzymes, along with the advancement of an innovative approach for their identification, could provide a broader basis to develop more effective mitigating strategies in the future. In the present study, a hybrid in silico/in vitro workflow coupling virtual screening with enzymatic assays was applied in order to identify novel OTA hydrolyzing enzymes. Among the various hits, porcine carboxypeptidase B was identified for the first time as an effective OTA hydrolyzing enzyme. The successful experimental endorsement of findings of the workflow confirms that the presented strategy is suitable for identifying novel OTA hydrolyzing enzymes, and it might be relevant for the discovery of other mycotoxin- mitigating enzymes.

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