Characterization of hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina and its prolonged in vitro hydrogen evolution

Hydrogenase from a purple sulfur bacterium, Thiocapsa roseopersicina NCIB 8347, was purified, characterized and investigated for in vitro hydrogen ( H 2 ) production. The cell concentration and the specific hydrogenase activity were increased when T. roseopersicina NCIB 8347 was photo-autotropically cultivated with the addition of nutrients every 24 h. Crude extracts of the cells were prepared by sonication and then subjected sequentially to ultra-centrifugation, ammonium sulfate fractionation and heat-treatment to remove the impurities. Four-step separation of chromatographic procedures utilizing Q-Sepharose, Phenyl Superose, Mono-Q and Superdex resulted in a 9.56-fold purification with a 0.25% yield of crude cell extract. The optimum pH and temperature for the H 2 evolution of the purified enzyme was 7.0 and 70 °C, respectively, and those for optimum cell growth were 7.0 and 25 °C, respectively. The maximum H 2 -evolution activity was exhibited at 70 °C, and when the reaction temperature was decreased to 50 °C, the manifest activity fell to 44%. Hydrogenase activities were initially increased in the course of the 50–70 °C heat-treatment, whereas the remaining activities after 5-h heat-treatment at 50 and 70 °C were 110% and 60% of the initial ones, respectively. The purified hydrogenase, at 50 °C and pH 7.0, evolved H 2 gas for approximately 10 days under in vitro conditions, using sodium dithionite and methyl viologen as the electron donor and carrier, respectively.