| Project/Area Number |
03806017
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
応用微生物学・発酵学
|
|---|
| Research Institution | Tokyo University of Agriculture and Technology |
|---|
Principal Investigator |
KATAYAMA Yoko Tokyo University of Agriculture and Technology Faculty of Agriculture Assistant Professor., 農学部, 助手 (90165415)
|
|---|
| Project Period (FY) |
1991 – 1992
|
| Project Status |
Completed (Fiscal Year 1992)
|
| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
|
|---|
| Keywords | Sulfur Oxidizing Bacteria / Thiocyanate / Thiobacillus thioparus / Thiocyanate Hydrolase |
|---|
| Research Abstract |
Thiobacillus thioparus is an obligate chemolithotrophic bacterium which obtains energy by the oxidation of reduced inorganic sulfur compounds. Objective of this study is the biochemical characterization of thiocyanate hydrolase which was recently purified from T. thioparus. Thiocyanate hydrolase converted thiocyanate to carbonyl sulfide and ammonia. Purified enzyme has molecular mass of 142 kDa and was composed of three different subunits of alpha(19 kDa), beta (23 kDa) and gamma (32 kDa). The Km value for thiocyanate was -11 mM, and showed optimum pH around pH 7.5. Activity of this enzyme was inhibited by cyanide at low concentration even in the presence of its substrate. The inhibition of cyanide was non-competitive to thiocyanate with a Ki of 14.8 muM. This inhibition was reduced by the dilution of cyanide in a reaction mixture and eliminated by a gel filtration, indicating that cyanide does not bind to thiocyanate hydrolase by covalent bonds nor the inhibition does not results in permanent conformational changes on the enzyme. Mechanisms of cyanide inhibition on thiocyanate hydrolase is in progress.
T. thioparus library was constructed in Escherichia coli HB101. Positive clones were screened by using antibodies against subunit proteins of thiocyanate hydrolase.
One of the products of thiocyanate hydrolase is a volatile sulfur compound of carbonyl sulfide. To investigate the metabolism of carbonyl sulfide by T. thioparus, a new assay system was made which involves the enzymatic formation of carbonyl sulfide by thiocyanate hydrolase.
|
