Salt-Resistant and Ethanol-Resistant Monoamine Oxidases: New Sight for yobN Mining from Bacillus and Biogenic Amine Degradation Mechanism in Fermented Food

Amine oxidases have strong capabilities for the degradation of biogenic amines (BAs) in fermented foods. However, their application is limited by substrate specificity, and high concentrations of ethanol and salt can hinder their effectiveness. This study presents a novel approach utilizing comparative genomics, protein clustering analysis, and full homology modeling to identify three amine oxidases: KCYOBN from the salt-resistant Bacillus subtilis, and LYYOBN1 and LYYOBN2 from the ethanol-resistant Bacillus cereus. These enzymes are highly similar in structure and exhibit broad substrate specificities. KCYOBN maintains over 84% relative activity at 20% (w/v) NaCl, while LYYOBN1 and LYYOBN2 retain over 32 and 21% relative activity at 25% vol ethanol. Variations in key residues are one of the reasons for the differences in tolerance. In fermented foods, KCYOBN degraded 45.97% of the total BAs in cooking wine and 38.33% in fish sauce. LYYOBN1 achieved the highest degradation rate of 32.93% in huangjiu. LYYOBN2 exhibited degradation rates of 30.00% in soy sauce and 35.14% in wine with no significant impact on flavor compounds. The significance of this work lies in the identification of the novel salt-resistant KCYOBN and ethanol-resistant LYYOBN1 and LYYOBN2 through this new method, which can simultaneously degrade multiple BAs and have a broad application potential.