Tunnel-redesigned O2-tolerant CO dehydrogenase for removal of CO in real flue gas

Carbon monoxide dehydrogenase (CODH)-catalyzed oxidation of CO to CO 2 provides a promising means of removal of toxic and waste CO from industrial flue gas despite of the lack of active and stable enzymes in the atmosphere. Herein we present rationally and selectively redesigned Ch CODH-II ( Carboxydothermus hydrogenoformans ) variants by engineering gas tunnels in order for O 2 -tolerant CODHs to catalyze efficient CO oxidation under oxygen (O 2 ). Using the redesigned Ch CODH-II A559W and A559H variants showing 42- and 128-fold elevation of O 2 tolerance, respectively, complete CO removal was achieved under a near-atmospheric condition. Moreover, these variants efficiently removed CO from industrial flue gas (Linz–Donawiz converter Gas: LDG) discharged from a steel mill despite the high O 2 level (13.4%) during successful and repeated reuse after immobilized on Ni-NTA agarose beads. Our study will provide insights into redesigning the transformation of O 2 -sensitive CODHs into tolerant enzymes for use as workhorses for conversion of toxic or waste gases into safe or value-added chemicals.