Purification and characterization of hydrogenase from the marine green alga, Chlorococcum littorale

FEBS Letters

Kurano, Norihide¹  Miyachi, Shigetoh²  Ueno, Yoshiyuki¹ 

[1] Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi-shi, Iwate 026-0001, Japan;Marine Biotechnology Institute, 1-28-10 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

关键词: Hydrogenase;    Ferredoxin;    Hydrogen production;    Chlorococcum littorale;    DTT;    dithiothreitol;    PMSF;    phenylmethylsulfonyl fluoride;    PTH;    phenylthiohydantoin;    CAPS;    3-[cyclohexylamino]-1-propanesulfonic acid;   

DOI  :  10.1016/S0014-5793(98)01699-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 μmol/min/mg of protein at 25°C, using reduced methyl viologen as an electron donor. The K _m value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS-PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50°C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K _m value for ferredoxin was 0.68 μM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4°C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.

【 授权许可】

Unknown