Purification and partial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus.
Cell-free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O6(2-) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm. Trithionate hydrolase was purified to near homogeneity from cell-free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5-4.5 and a temperature optimum of 70 degrees C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 microM at 30 degrees C and 270 microM at 70 degrees C. Enzyme activity was lost after 36 days at 0 degrees C, 27 days at 70 degrees C; but after 97 days at 30 degrees C, 40% of the initial activity was still present: The enzyme activity was inhibited by mercury chloride, N-ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O6(2-) was not hydrolysed by trithionate hydrolase.
Meulenberg
R
R
Department of Microbiology and Enzymology, Kluyver Laboratory of Biotechnology, Delft University of Technology, The Netherlands.
Pronk
J T
JT
Frank
J
J
Hazeu
W
W
Bos
P
P
Kuenen
J G
JG
D000645:Ammonium Sulfate / Q000494:pharmacology; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D013053:Spectrophotometry; D013696:Temperature; D013855:Thiobacillus / Q000201:enzymology*; D013885:Thiosulfates / Q000378:metabolism
D013885:Thiosulfates; D006867:Hydrolases; C077275:trithionate hydrolase; D000645:Ammonium Sulfate