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

硫代硫杆菌是一种专性化能营养细菌，通过还原无机硫化合物的氧化获得能量。本文研究了新纯化的硫氰酸酯水解酶的生化特性。硫氰酸酯水解酶将硫氰酸酯转化为羰基硫化物和氨。纯化后的酶分子质量为142 kDa，由α (19 kDa)、β (23 kDa)和γ (32 kDa)三个不同的亚基组成。硫氰酸盐的Km值为-11 mM， pH值在7.5左右为最佳。在低浓度氰化物的作用下，即使有底物存在，该酶的活性也受到抑制。氰化物对硫氰酸盐的抑制作用为非竞争性，Ki为14.8 muM。这种抑制作用通过在反应混合物中稀释氰化物而减弱，并通过凝胶过滤消除，这表明氰化物不会通过共价键与硫氰酸酯水解酶结合，也不会导致酶的永久构象变化。氰化物抑制硫氰酸酯水解酶的机制尚不清楚。用大肠杆菌HB101构建了硫代菌文库。利用抗硫氰酸酯水解酶亚基蛋白抗体筛选阳性克隆。硫氰酸酯水解酶的产物之一是羰基硫化物的挥发性硫化合物。为了研究硫氰酸酯水解酶对羰基硫化物的代谢，建立了硫氰酸酯水解酶对羰基硫化物的酶促反应体系。

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.