There is a continuous search for novel enzymes to complement the abilities of today’s commercially available enzyme and find tailor-fit alternatives to suit the diverse array of bio-based industries. One application could be to increase biogas yield by finding substrate degrading proteases that can be added to the anaerobic digestion process and survive degradation themselves. A novel enzyme identified as a hypothetical dipeptidyl peptidase III, a zinc dependent metallo-protease, was found by a metaproteogenomics approach to be produced by the microorganisms of a thermophilic biogas process. The aim of this study was to express and refold a recombinant variant of the novel DPP III to its active form after production in inclusion bodies in Escherichia Coli. Assaying of refolding conditions was performed by stepwise dialysis and drip dilution. Nine attempts were performed based on findings in literature, although no other variant of DPP III has earlier been successfully refolded from inclusion bodies. The study resulted in a limited set of conditions of temperature, volumes, metal ions, salts and other additives being tested in the refolding buffers. Enzyme refolding and activation was monitored by the hydrolysis of the DPP III fluorescent substrate Arg-Arg β-naphthylamide trihydrochloride, alongside with measurements of protein concentration and SDS-PAGE. The novel DPP III was successfully purified but no definite strategy of producing correctly folded protein was found.