Recombinant Expression of Lytic Polysaccharide Monooxygenase and its Functional Characterization

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that can act on crystalline polysaccharides directly, which plays a critical role in cellulose degradation. In addition to reports on its structure and mechanism of action, it is important to study the auxiliary activity 9 (AA9) characteristics from different resources to support the mechanism research. The gene encoded ToLPMO9A was cloned from Trichoderma orientalis EU7-22 and first heterologously expressed in Pichia pastoris GS115. Both metal ions and reducing agent concentrations showed an important effect on ToLPMO9A. The ToLPMO9A exhibited maximal activity at 60 degrees C and a 6.0 pH. In addition, ToLPMO9A showed substrate specificity. The matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis showed that ToLPMO9A cleaved the glycosidic bonds at C-1 and C-4/C-6 position via oxidation. Concerning the synergistic effects on enzymic activity, ToLPMO9A exhibited promotion with endo-glucanase or exo-glucanase, but inhibition with beta-glucosidases. In conclusion, ToLPMO9A could be a good choice for enzyme cocktails and provide theoretical support for subsequent action mechanisms and broader applications.