Characterization and cloning of oxygen-tolerant hydrogenase from Klebsiella oxytoca HP1

Hydrogenase from a hot spring bacterium Klebsiella oxytoca HP1 was purified and found to have a specific activity of 199.8 U/mg of protein and a yield of 7.3%. The purified enzyme was determined to consist of six subunits (65, 33, 28, 23, 21 and 18 kDa), similar to hydrogenase-3 from Escherichia coli, and therefore it was named Hyd3. The enzyme displayed remarkable oxygen tolerance. For the purified enzyme, 50% maximal activity was maintained following incubation for 24 h in air at room temperature. The hydrogenase gene cluster (hyc) was cloned and found to consist of hycD, hycE, hycF, hycdG, hycH and hycI genes. hycE and hycG genes encode for the large and small subunit of the hydrogenase, respectively. A hycE gene deletion mutant, ΔhycE, was constructed for elucidating the function of the hyc-operon in hydrogen metabolism. Compared with the wild type strain HP1, the mutant strain showed a dramatic decrease in hydrogen production in the presence of formate, sodium pyruvate and glucose under O2-stressed conditions, while substantial activity was detected under anaerobic conditions. This strongly suggests that K. oxytoca HP1 carries a number of hydrogenases or hydrogen metabolic pathways independently of Hyd3. However, Hyd3 is the main factor responsible for hydrogen production under O2 stress conditions.