Continuous and sensitive monitoring of LPMO reactions using an optical H₂O₂sensor

We demonstrate the use of an optical sensor to follow the activity of H₂O₂-dependent enzymes in real-time, using quasi-continuous detection of H₂O₂. The sensor enables kinetic measurements of the H₂O₂-consuming enzyme lytic polysaccharide monooxygenase (LPMO) under homogeneous conditions at 1 mL scale conversion assays. By tracking H₂O₂concentration changes over time during 2-minute reaction intervals, we established quantitative assays and determined Michaelis–Menten kinetics for the LPMO from Lentinus similis to cellotetraose, with a K_Mvalue of 0.22 ± 0.08 mM and a maximum reaction rate (V_max) of 0.97 ± 0.64 μM s⁻¹. This method allows for continuous monitoring without reliance on time-consuming, discontinuous assays or post-reaction sample processing. We evaluate the capabilities of the sensor to monitor enzyme activity, benchmark it against established spectrophotometric methods, and discuss its limitations and advantages. This work provides a method for the real-time assessment of LPMO kinetics but also enables broader application to other redox enzymes that consume or release H₂O₂during the reaction.