In 2014 we described a new activity-based screen suited to seek active recombinant RubisCOs from the environment - independent of the native host's culturability. Within this study we aimed at seeking novel RubisCO active enzymes from the environment. For this purpose we screened six metagenomic fosmid libraries from hydrothermal vent environments, differing with respect to their (i) geographic origin (north or south Mid Atlantic Ridge) and their (ii) environmental characteristics (basalt vs. ultramafic hosted, temperature, and pH). Overall we screened roughly 12500 metagenomic fosmid clones and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.

Samples were collected within the DFG-SPP 1144 priority program “From Mantle to Ocean: Energy-, Material-, and Life-cycles at Spreading Axes".Specific enzyme activity measurements were performed within the scope of the DFG Project PE1549/5 1 "Linkage between the distribution and biochemical properties of RubisCO and CODH enzymes and abiotic properties in thermally and chemically distinct deep-sea hydrothermal vent systems".For anoxic activity measurements crude extractes were produced aerobically and allowed to become anoxic by placing them under N2/H2 atmosphere (95%/5%; v/v) at 4°C over night. The assay was then performed in an anaerobic chamber under the same atmosphere using anoxic buffer A (100 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 1 mM EDTA, 25 mM NaHCO3 and 1 mM DTT) and anoxic ribulose-1,5-bisphosphate substrate.