Patulin (PAT), a toxic secondary metabolite produced mainly by Penicillium species that frequently contaminates fruit and fruit-derived products, poses serious health risks to humans and animals. In the present study, three short-chain dehydrogenases/reductases (SDRs) with PAT-degrading ability, designated BsSDR1, BsSDR2, and BsSDR3, were identified from the genome of Bacillus subtilis 168. BsSDR1 and BsSDR2 showed powerful PAT elimination abilities, which can completely convert PAT to nontoxic E-ascladiol. Moreover, BsSDR1, BsSDR2, and BsSDR3 shared the highest sequence identity of 36.03% with the reported PAT-degrading enzymes, indicating that they are novel PAT-degrading enzymes. BsSDR1, BsSDR2, and BsSDR3 exhibited the highest activity against PAT at 40, 40, and 35 °C, respectively. Additionally, BsSDR1, BsSDR2, and BsSDR3 displayed remarkable thermostability, retaining 32.50, 24.63, and 46.74% residual activity, respectively, after incubation at 50 °C for 1 h. Three-dimensional (3D) simulation and site-directed mutagenesis indicated that the catalytic triad formed by the residues (Ser, Tyr, and Lys) was the key for SDR activity, and this conserved catalytic mechanism was followed in the catalytic process of novel PAT-degrading enzymes BsSDR1, BsSDR2, and BsSDR3. More importantly, BsSDR1, BsSDR2, and BsSDR3 can degrade PAT in apple juice at rates of 86.90, 90.17, and 61.57%, respectively. The identification of BsSDR1, BsSDR2, and BsSDR3 enriched the PAT-degrading enzyme libraries, providing promising candidates for PAT decontamination in the food industry.
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