Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli |
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Authors: | Balaji Sundara Sekar Subramanian Mohan Raj Eunhee Seol Satish Kumar Ainala Jungeun Lee Sunghoon Park |
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Affiliation: | 1. Department of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, Republic of Korea;2. Centre for Research and Development, PRIST University, Thanjavur, TN 613 403, India |
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Abstract: | Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H2). CO-dependent H2 production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H2ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein−1) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein−1). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein−1. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H2 from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H2ase for CO-dependent H2 production. |
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Keywords: | Carbon monoxide dehydrogenase Citrobacter amalonaticus Y19 Water&ndash gas shift reaction Recombinant Escherichia coli |
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