Kinetic and Substrate Specificity Determination of Bacterial LPMOs

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered in the past decade. Their importance in the degradation of recalcitrant substrates in sustainable processes is now well established; however, the catalytic peroxygenase mechanism has yet to be fully understood. This is also because the study of reaction kinetics has to deal with multiple variables, including the nature of the substrates, the presence of unwanted side reactions, and the low protein stability in the presence of H2O2 as the cosubstrate. In this work, three bacterial LPMOs from the AA10 family were investigated: Pseudomonas putida AA10 (PpAA10), Streptomyces coelicolor AA10B (ScAA10B), and AA10C (ScAA10C). Their activity against specific substrates was initially evaluated by ATR-FTIR for a qualitative characterization, and then, to determine electrochemically their kinetic constants, an amperometric assay based on the detection of the H2O2 consumption was used. This allowed the determination of turnover numbers (TNs) and total turnover numbers (TTNs) on different substrates. The best performance was obtained with ScAA10C and ScAAA10B on nanocrystalline cellulose with TNs of 3.81 and 2.88 s–1, respectively, and TTNs of 1208 and 735, respectively. PpAA10 is active on β-chitin with a TN of 1.02 s–1 and a TTN of 61, providing valuable insight into their substrate specificity and stability. Although the initial rates were found to be lower than those for fungal LPMOs, the enzyme stability over time increased on more crystalline substrates and in the presence of the carbohydrate-binding module (CBM), yielding activity values comparable to those for fungal LPMOs. Moreover, the presence of the CBM resulted in a more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and increased resistance to oxidative inactivation.