Ochratoxin A (OTA) is an important mycotoxin that contaminates a variety of agricultural products. The cell-free supernatant of Bacillus subtilis CW14 liquid cultures were reported previously to be capable of removing OTA efficiently. In this work, we examined several substances that are probably involved in this removal of OTA using in vitro experiments. The strain CW14 culture supernatant that was separated by ultrafiltration showed that the fractions collected at >10 kDa and <3 kDa had a significant ability to reduce OTA (84.9 and 74.8%, respectively) when incubated with 6 μg/ml OTA at 37 °C for 24 h. A putative metalloenzyme was responsible for the activity of the >10-kDa fraction, which was confirmed by the detrimental effects of heat treatments or addition of SDS, proteinase K, or EDTA. Subsequently, a carboxypeptidase (CP) gene that was likely related to the enzymatic conversion of OTA by the >10-kDa fraction was cloned from the B. subtilis CW14 genome, and over-expressed in Escherichia coli. The recombinant CP degraded 71.3% of OTA at 37 °C for 24 h, and ochratoxin α (OTα) was confirmed as a degradation product. From the <3-kDa fraction, some small peptides (1.7 kDa >Mw >0.7 kDa) were purified and decreased OTA by 45.0% under the same conditions, but no product was detected. These peptides were presumed to be capable of binding OTA due to their affinity with the OTA molecule, and the OTA-peptide complexes escaped from the extraction procedures for OTA quantification. These results indicated there was a probable synergistic effect that was involved in removal of OTA by the strain CW14 culture supernatant, which included enzymatic degradation by a CP and physical adsorption by some small peptides.
Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy
