Mining and characterization of key amine oxidases from <em>Saccharopolyspora hirsuta</em> T14 based on full homology modeling, transcriptional analysis, and applications in <em>huangjiu</em>

Excessive biogenic amines (BAs) not only impact the quality of fermented foods but also harm health. Although enzymatic methods are effective in controlling the BAs content, research remains limited. <em>Saccharopolyspora hirsuta</em> T14 was identified for its superior ability to degrade BAs. A novel approach to predict the functional information of all proteins in the genome using homology modeling has screened several genes involved in BAs metabolism. Transcriptional analysis revealed that three critical amine oxidase genes, <em>PAO4</em>, <em>AO6</em>, and <em>MAO7</em>, play dominant roles in this degradation. The primary amine oxidase PAO4, along with amine oxidase AO6 and monoamine oxidase MAO7 with molecular weights of 71.0 kDa, 46.7 kDa, and 47.0 kDa, respectively, were cloned, expressed and purified. Enzymatic and kinetic studies demonstrated that these three enzymes cooperatively degrade major BAs with varying catalytic efficiencies and tolerances to substrate inhibition. Structural analysis suggests that increased hydrogen bonding and hydrophobic interactions contribute to substrate specificity and tolerance. When applied individually to <em>huangjiu </em>(18%vol), the enzymes achieved BAs degradation rates of 36.51% ± 3.68%, 18.93% ± 2.47%, and 22.09% ± 3.65%, respectively, as measured by high-performance liquid chromatography (HPLC). These findings provide practical methods and a theoretical foundation for specific and synergistic degradation of various BAs.