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

Hydrogen production by over-expression of hydrogenase subunit in oxygen-tolerant Klebsiella oxytoca HP1

“…i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 3 2 2 7 e1 3 2 3 3 oxytoca HP1 is 5.1 U/mg.protein; this value is close to that obtained in our previous work [18]. However, the hydrogenase activity of K. oxytoca HP1/hycE and K. oxytoca HP1/hycG increases to 10.7 U/mg.protein and 7.9 U/mg.protein, respectively.…”

“…Though E. coli and K. oxytoca HP1 are both members of Enterobacteriaceae, K. oxytoca HP1 lacks hydrogenase 1 (Hyd1), hydrogenase 2 (Hyd2) and hydrogenase 4 (Hyd4), all of which are present in E. coli. On the other hand, the hydrogenase 3 (Hyd3) gene cluster (hyc), reported in our previous work [18], was found in the K. oxytoca HP1 genome. The hyc operon in E. coli contains hycA, hycB, hycC, hycD, hycE, hycF, hycG, hycH and hycI genes [23].…”

“…Cells were broken by sonication (300 W, 20 kHz; JY92-II, Scientz) for 45 min in an ice bath. Cell debris was removed by centrifugation at 20,000 Â g for 30 min at 4 C, and the supernatants retained for hydrogenase activity analysis [18]. Hydrogenase activity was quantified by the amount of hydrogen evolved from methyl viologen, which had been reduced by sodium dithionite.…”

“…However, K. oxytoca HP1 possesses a certain degree of aerobic hydrogen production ability [17,18]. To analyze the effect of over-expression of hydrogenase subunits on K. oxytoca HP1 aerobic hydrogen production, and to further evaluate the role of the hydrogenase subunits HycE and HycG in aerobic hydrogen production by this microorganism, the hydrogen production of recombinant strains K. oxytoca HP1 and E. coli under 5% or 10% oxygen stress was investigated.…”

“…Klebsiella oxytoca HP1, an Enterobacteriaceae strain, was isolated from hot springs (Sanming, Fujian, China) as described in our previous work [17,18]. K. oxytoca HP1 is a strong candidate for dark hydrogen production due to its high hydrogen production rate (HPR) and aerobic hydrogen production character.…”