Salt tolerant and high pH resistant hydrogenase from the haloalkaliphilic, sulfate-reducing bacterium Desulfonatronum thiodismutans

摘要

Hydrogenase is the key enzyme of energetic metabolism in cells, catalyzing the converse reaction of hydrogen oxidation and responsible for the consumption and excretion of hydrogen in bacteria. Hydrogenases are proteins, most of which contain either nickel and iron or iron alone in their active center. Hydrogenases have been found in many microorganisms, such as methanogenic, acetogenic, nitrogen-fixing, sulfate-reducing, photosynthetic bacteria, and algae that use the hydrogen as an energy source or as an electron sink. Hydrogenases are the subject of wide physiological, biochemical, physico-chemical and genetic studies due to their abilities to produce molecular hydrogen as an alternative source of energy. Despite the large quantity of work dealing with the intracellular and extracellular enzymes of halophilic bacteria, the data about the response of hydrogenases to salts are practically absent. The study of hydrogenase in cell-free extracts of the extremely halophilic eubacterium Acetohalobium arabaticum showed a dramatic increase in the activity of the enzyme at high concentrations of NaC1 and KC1 (near saturated solutions). Here we present data about hydrogenase in a free-cell extract from the new haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans, which grows on a highly mineralized carbonate-bicarbonate medium in the salinity range from 1 to 7 % NaCl and at pH 8.0-10.0. The studied enzyme was active in concentration range from 0.0 to 4.3 M NaCl with the optimum at 1.0 M NaCl. At 1.0 M NaCl the enzyme expressed 20 % additional activity, with NaCl concentration changing from 2.1 M to 3.4 M, and then the activity decreased and reached a constant level. Although sodium bicarbonate decreases the hydrogenase activity, the enzyme still showed activity at 60 % of the maximum level at concentration in a near saturated solution (1.2 M NaHCO_3). The maximum enzyme activity was observed at pH 9.5 with limits of 7.5 and 11.5, which is practically equal to the pH optimum of bacterial growth. Therefore the hydrogenase of D. thiodismutans is extremely tolerant to high concentrations of sodium salts and resistant to high pH, which makes it a unique subject for biochemical research with the possibility of important biotechnological applications.