報告書番号

U98055

タイトル（和文）

石炭の生物加工-13- --黄鉄鉱吸着因子、ラスチシアニンの構造と機能--

タイトル（英文）

Bio-processing of coal -13-Structure and function of pyrite-binding protein, rusticyanin of iron-oxidizing bacterium

概要 （図表や脚注は「報告書全文」に掲載しております）

当所において開発中の微生物（鉄酸化細菌）による石炭の脱硫技術では、微生物が石炭中の無機硫黄である黄鉄鉱を特異的に認識し、吸着する性質を利用している。本報告では、鉄酸化細菌の黄鉄鉱吸着因子の機能部位を明らかにすることを目的に、まず黄鉄鉱吸着因子を生産する大腸菌の作成を行い黄鉄鉱吸着因子の性質を検討した。その結果、大腸菌由来の黄鉄鉱吸着因子を鉄酸化細菌に加えると、菌体の黄鉄鉱への吸着を促進する効果があることが分かった。さらに、黄鉄鉱吸着因子のアミノ酸を部分的に改変することによって、黄鉄鉱への結合に必要なアミノ酸を探索し、３つのアミノ酸（His85、Cys138、Met148）が結合に必須であることが分かった。黄鉄鉱吸着因子の立体構造より、これら３つのアミノ酸は黄鉄鉱表面の鉄原子に配位するための“鍵穴”のような構造を形成しているものと推定された。

概要 （英文）

We have been developing the coal desulfurization technique using iron-oxidizing bacterium, Thiobacillus ferrooxidans as a biological surfactant which can selectively adhere to pyrite existing in pulverized coal. In the former report, we isolated a pyrite-binding protein from T. ferrooxidans. The binding protein was identified as rusticyanin. Rusticyanin had been known as a blue copper protein but the binding protein was copper less in apo form. In this article, we report the characterization of several recombinant rusticyanins, which were replaced one of four ligands for a copper atom. The wild and mutated rusticyanins were clarified the structure and the function of binding sites on the protein to pyrite. Our results are summarized as follows: 1) In order to create expression plasmid, rusticyanin gene was isolated from T. ferrooxidans. The recombinant E. coli was permitted to express abundant amount of rusticyanin. 2) The recombinant rusticyanin in the wild type could bind to iron-bearing sulfide minerals including pyrite much, but not to other sulfide minerals. This selectivity of the recombinant in the binding to minerals was the same phenomenon that was observed in both the intact rusticyanin and the cells of T. ferrooxidans. In addition, the preincubation of the rusticyanin with native T. ferrooxidans cells increased the number of the adhered cells to pyrite. It was possible to increase the number of the adhesive cells to pyrite by the addition of exogenous rusticyanin. 3) An optical trapping system by laser beam enables to measure actually the physicochemical forces between a singe cell of T. ferrooxidans and pyrite in pico-Newton order. The binding forces of the cell to pyrite was estimated as 3.5 pN when the cell did not express the rusticyanin. In contrast, the forces was more than 5.2 pN at least when the cell expressed rusticyanin. It was proved directly from measurable physicochemical forces that the rusticyanin mediated the strong interactions between the cells and the pyrite. 4) Each of the four copper ligands (His85, Cys138, His143 and Met148) of the wild-type rusticyanin were individually replaced with alanine by site-directed mutagenesis. When each mutant were subjected to the pyrite to clarify which ligand was necessary in the binding to pyrite. The binding profile was analyzed with flow fluorescence spectrophotometer. As a result, only His143Ala mutant could bind to pyrite while the other mutants not. Those results suggested that His85, Cys138 and Met148 were account for the binding to pyrite. From the three dimensional model of the rusticyanin, it was expected that the three ligands formed the cavity for the binding to pyrite on the surface of the molecule. The binding of the rusticyanin was inhibited by exposure of either ferrous iron or some organic chelating agents. Therefore, the binding cavity was designed to make the coordination bonding between the ferrous iron in the pyrite and the corresponding ligand in the cavity.

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