Purification And Characteristics Of Soluble Hydrogenase From Klebsiella Oxytoca HP1

Hydrogenases, which catalyze the reversible oxidation-reduction reaction between molecular hydrogen and proton (H2 ? 2H+ +2e-), are key enzymes for the hydrogen metabolism in many microbes. Klebsiella oxytoca HP1 is a high yield hydrogen producer with short grow duration and simple grow conditions. In this research, we reported the purification and properties of soluble hydrogenase from K.oxytoca HP1.20g of wet cells were suspended in Tris-HCl buffer (30mmol/L, pH8.0, 10mmol/L DTT) and disrupted by sixty times ( 35s exposure to 400W ultrasonic wave with 15s cooling intervals). Centrifugation at 8,000×g for15min gives a crude extra.The crude extract is centrifuged at 130,000×g for 30 min which gives a supernatant with a specific activity 0.088μmol H2/min·mg prot. The supernatant was treated by 20% and 60% of ammonium sulfate to precipitate the mixed proteins and hydrogenase, respectively. The crude enzyme was purified by three steps of column chromatography (DEAE-Sepharose F.F.,Sephacryl S-200 and TSK-DEAE). After three steps of purification,the soluble hydrogense of K. oxytoca HP1 was purified about 200 fodls with a yield of 15.3% . The specific activity of hydrogenase was achieved as 17.68μmol H2/min·mg prot.The catalytic property of soluble hydrogenase was studied in this research. The optimal temperature for hydrogen production was detected as 30oC. When the temperature increased to 65oC, the enzyme lost it's activity rapidly. The hydrogenase showed maximal hydrogen production activity at pH 7.5. Oxygen was an inhibitor for the hydrogen production of soluble hydrogenase. MV was the optimal electron carrier for the soluble hydrogenase's activity.