Engineering an Unspecific Peroxygenase From <i>Thielavia terrestris</i> for Specific Terminal Oxidation of Xylene Derivatives

Abstract Xylene and its derivatives are bulk raw materials in the chemical industry, and their oxidation products, including p ‐toluic acid and terephthalic acid, are also crucial in the production of fine chemicals. Unspecific peroxygenases (UPOs) are heme‐thiolate enzymes that are capable of oxidizing diverse organic compounds. In this study, a UPO from Thielavia terrestris ( Tte UPO) showed the ability to oxidize p ‐xylene to p ‐toluic acid with &gt;99% chemoselectivity. To address the sensitivity of Tte UPO to H 2 O 2 during the reaction, a fusion protein of Tte UPO and formate oxidase from Aspergillus oryzae ( Ao FOx) was constructed for in situ H 2 O 2 regeneration. Additionally, site‐directed saturation mutagenesis of Tte UPO was performed, giving the mutant F63S/A155 V, which exhibited a 2.4‐fold increase in the specific activity toward p ‐xylene compared with wild‐type Tte UPO. The engineered fusion protein Tte UPO F63S/A155V ‐AoFOx achieved 3.8 mM p ‐toluic acid under 8 mM p ‐xylene loading, which is approximately 60‐fold higher than previously reported p ‐toluic acid concentrations through bio‐oxidation. It also showed the capability to convert other monocyclic and polycyclic aromatic hydrocarbons, indicating its potential for the high‐value conversion of xylene and its derivatives.