Enzymatic properties and inhibition tolerance analysis of key enzymes in β-phenylethanol anabolic pathway of HJ.

is known for its unique aroma, primarily attributed to its high concentration of β-phenylethanol (ranging from 40 to 130 mg/L). Phenylalanine aminotransferase Aro9p and phenylpyruvate decarboxylase Aro10p are key enzymes in the β-phenylethanol synthetic pathway of HJ. This study examined the enzymatic properties of these two enzymes derived from HJ and S288C. After substrate docking, Aro9p (-24.05 kJ/mol) and Aro10p (-14.33 kJ/mol) exhibited lower binding free energies compared to Aro9p (-21.93 kJ/mol) and Aro10p (-12.84 kJ/mol). and genes were heterologously expressed in BL21. Aro9p, which was purified via affinity chromatography, showed inhibition by l-phenylalanine (L-PHE), but the reaction rate (Aro9p: 23.89 μmol·(min∙g)) > Aro9p: 21.3 μmol·(min∙g)) and inhibition constant values (Aro9p: 0.28 mol L>Aro9p 0.26 mol L) indicated that Aro9p from HJ was more tolerant to substrate stress during fermentation. In the presence of the same substrate phenylpyruvate (PPY), Aro10p exhibited a stronger affinity than Aro10p. Furthermore, Aro9p and Aro10p were slightly more tolerant to the final metabolites β-phenylethanol and ethanol, respectively, compared to those from S288C. The study suggests that the mutations in Aro9p and Aro10p may contribute to the increased β-phenylethanol concentration in . This is the first study investigating enzyme tolerance mechanisms in terms of substrate and product, providing a theoretical basis for the regulation of the β-phenylethanol metabolic pathway.