Monitoring of reactions catalyzed by lytic polysaccharide monooxygenases using highly-sensitive fluorimetric assay of the oxygen consumption rate.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that catalyze the oxidative deconstruction of polysaccharides. However fast and reliable methods of determination of LPMO activity still need to be developed, especially those based on the initial reaction rates. A method based on the oxygen consumption rate (OCR) measurements, using a Seahorse XFp Analyzer with highly-sensitive fluorimetric sensors, was applied for monitoring the oxidation of amorphous cellulose by three fungal LPMOs: recombinant enzymes from Thielavia terrestris (GH61E), Trichoderma reesei (Cel61A), and a native LPMO9A from Myceliophthora thermophila. The turnover numbers for 4 μM enzymes acting on 4 mg mL cellulose at 37 °C were 0.88, 1.26 and 0.93 min, respectively. A possibility of feeding the dissolved reagents into the reaction system during measurements with obtaining a simultaneous response in the OCR allowed in situ monitoring the LPMO inhibition and activation by EDTA and Cu ions as well as studying other effects on the enzymatic reaction.