ISSN:
1432-072X
Keywords:
NAD-Dependent Hydrogenase
;
Hydrogen Dehydrogenase
;
Nocardia opaca Strain 1 b
;
Hydrogen Bacteria
;
Chemolithoautotrophic Bacteria
;
Gram-Positive Hydrogen Bacteria
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract Nocardia opaca strain 1 b has a NAD-dependent hydrogenase (hydrogen dehydrogenase). The enzyme has been purified from autotrophically grown cells and tested for optimal assay conditions and stability. The purification procedure involved protamine sulfate treatment, ammonium sulfate precipitation, and separation by DEAE-cellulose and Sephadex G-200 chromatography and resulted in a 63-fold increase of specific activity at a 11.7% enzyme recovery. The final specific activity was 103 μmoles H2/min·mg protein. The purified enzyme was dependent on nickel and magnesium ions at 0.5 and 5.0 mM concentrations, respectively, as well as flavin mononucleotide at a 5–10 μM concentration. Straight enzyme kinetics were achieved by preincubating the enzyme in the presence of NADH2. A high stability of the enzyme was observed in 0.1 M potassium phosphate buffer, pH 6.5, in the presence of 0.5 mM nickel and 5 mM magnesium ions under hydrogen atmosphere. Even under air the enzyme was remarkably stable, although less than under hydrogen. From double reciprocal plots of substrate saturation curves the Michaelis-Menten constants were calculated: For saturating NAD-concentration the K m was 0.063 mM H2 and for saturating hydrogen concentration the K m was 0.123 mM NAD.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00446303
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