Characterization and Engineering of bacterial Manganese-Oxidizing Peroxidases as Lignolytic Biocatalysts

Cost-effective lignolytic biocatalysts would facilitate the economic and sustainable development of biomass-derived products. Efforts to develop fungal-based biocatalysts, such as manganese peroxidase, have failed largely due to the difficulty of producing fungal enzymes. To overcome this problem, we are exploring a family of bacterial manganese-oxidizing peroxidases (MOPs), which represent an uncharacterized subfamily of the peroxidase-cyclooxygenase superfamily. We have heterologously produced two truncated versions of MopA from Leptothrix cholodnii SP-6, MopA₈₄₂ and MopA₁₀₂₄, both of which contain the peroxidase domain of the full length enzyme. Reconstituted MopA₈₄₂ has a Soret peak at 410 nm, and a covalently bound heme as is typical of peroxidase-cyclooxygenases. MopA₈₄₂ oxidized both 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and manganese in the presence of hydrogen peroxide. The ability of MopA₈₄₂ to transform lignin was investigated. Enzymatically transformed samples were analyzed for changes in molecular weight, using gel permeation chromatography, and for changes in chemical structure, using nuclear magnetic resonance spectroscopy. The production of lower molecular weight aromatic compounds will be investigated using high performance liquid chromatography. Stopped-flow spectroscopy and X-ray crystallography are also being used to gain insight into the activity and function of this poorly characterized family of peroxidases.