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Microb Cell Fact. 2010 Jul 7;9:54. doi: 10.1186/1475-2859-9-54.

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli.

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Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.



Hydrogenases catalyze reversible reaction between hydrogen (H2) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability.


Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h.L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (approximately 65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of approximately 12 nmol H2/( protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition.


This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H2 production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions.

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