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Decomposition of thiocyanate and related sulfur compounds by Thiobacillus and cloning of thiocyanate hydrolase gene.

硫杆菌对硫氰酸盐及相关硫化合物的分解及硫氰酸盐水解酶基因的克隆。

基本信息

批准号：
03806017
负责人：
KATAYAMA Yoko
金额：
$ 1.41万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03806017/
关键词：
Sulfur Oxidizing Bacteria Thiocyanate Thiobacillus thioparus Thiocyanate Hydrolase

项目摘要

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.

排硫硫杆菌是一种专性化能无机营养细菌，通过氧化还原的无机硫化合物获得能量。本研究的目的是对新近从T.硫中毒。硫氰酸盐水解酶将硫氰酸盐转化为硫化羰和氨。纯化的酶具有142 kDa的分子量，并且由α（19 kDa）、β（23 kDa）和γ（32 kDa）三种不同的亚基组成。硫氰酸盐的Km值为~ 11 mM，并显示出约pH 7.5的最佳pH。该酶的活性被抑制氰化物在低浓度下，即使在其底物的存在下。氰化物的抑制作用与硫氰酸盐是非竞争性的，Ki为14.8 μ M。这种抑制减少了氰化物在反应混合物中的稀释和消除的凝胶过滤，表明氰化物不结合硫氰酸盐水解酶的共价键，也没有抑制不会导致永久的构象变化的酶。氰化物对硫氰酸盐水解酶的抑制机理研究正在进行中。在大肠杆菌HB 101中构建了硫蛋白文库。用抗硫氰酸盐水解酶亚单位蛋白的抗体筛选阳性克隆，硫氰酸盐水解酶的产物之一是羰基硫的挥发性含硫化合物。研究T. thioparus，一个新的分析系统，它涉及通过硫氰酸盐水解酶的羰基硫的酶促形成。