Chapter Three - Production and spectroscopic characterization of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs, also known as PMOs) are a recently discovered family of enzymes that play a key role in the breakdown of polysaccharide substrates. The ability of LPMOs to introduce chain breaks, using an oxidative mechanism, has particularly attracted attention as the world seeks more cost-effective and environmentally friendly ways of producing second-generation biofuels for the future. LPMOs are copper-dependent enzymes and have an unusual active site which includes the N-terminal residue of the protein in the copper coordination sphere. This N-terminal histidine side chain is also methylated in fungal enzymes, the molecular reason for which is still a debated topic. The production of these enzymes poses several challenges if we are to understand their chemical mechanisms. Here, we describe the methods that have been used in the field to produce LPMOs and provide information on the workflows that we use for our electron paramagnetic resonance (EPR) spectroscopy experiments. EPR has been a particularly powerful tool in the study of these enzymes and our objective with this chapter is to provide some helpful information for researchers for whom this technique might be daunting or theoretically difficult to access