A Continuous Tandem Catalytic Platform Linking Direct H$_2$O$_2$ Synthesis to Yeast Surface-Displayed Peroxygenases

Continuous tandem catalysis enables the direct coupling of chemical hydrogen peroxide (H₂O₂) synthesis with
oxidative biocatalysis while avoiding external peroxide addition. In this study, a continuous-flow tandem system
combining palladium-catalyzed H₂O₂ direct synthesis with yeast surface–displayed unspecific peroxygenases
(YSD-UPOs) is presented. Peroxygenases were immobilized via a recently published surface-display on
Komagataella phaffii cells, which were solvent-inactivated, and freeze-dried to allow application in standard
chemical labs. A palladium-based catalyst was prepared on titanium dioxide as a support and evaluated for
H₂O₂ generation under conditions compatible with enzymatic reactions. Chemical and enzymatic steps were
first studied separately to identify suitable operating windows for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) oxidation and hydroxylation of 1,2-(methylenedioxy)-4-nitrobenzene (NBD). The combined system
was then operated in a continuous micro fixed-bed reactor proving the concept of continuous tandem catalysis
reaching turnover numbers of 3596 for NBD and 6350 for ABTS oxidation.