Expression, purification and properties of the enzymes involved in lanthanide-dependent alcohol oxidation: XoxF4, XoxF5, ExaF/PedH, and XoxG4.

In this chapter we describe logistics, protocols and conditions for expression, purification and characterization of Ln-dependent alcohol dehydrogenases representing three distinct phylogenetic clades of these enzymes, classified as XoxF4, XoxF5 and ExaF/PedH. We present data on the biochemical properties of a dozen enzymes, all generated by our group, in a comparative fashion. These enzymes display a range of properties in terms of substrate and metal specificities, pH and ammonium requirement, as well as catalytic constants. In addition, we describe a single novel cytochrome, XoxG4, that likely serves as a natural electron acceptor from XoxF5 in methanotrophs of the Gammaproteobacteria class.