Studies on the cofactors and reaction mechanisms of nitrile hydratase which is useful for the synthesis of amides

用于酰胺合成的腈水合酶的辅因子和反应机制的研究

• 批准号: 02660114
• 负责人: NAGASAWA Toru
• 金额: $ 1.15万
• 依托单位: Nagoya University (1991)Kyoto University (1990)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660114/
• 关键词: Nitrile hydratase; Acylamide; Iron enzyme; Cobalt enzyme; ESR; Iron-active centrer; hydrated PQQ; Pyrroloquinoline quinone; 省エネルギ-

We have established, the enzymatic production process of acrylamide using the microbial nitrile hydratase and the industrial production of acrylamide was started since several years ago. Compared to the high development of the applied works, the basic studies on nitrile hydratase do not progress fully. Thus, the present works focused on more basic works, the elucidation of the cofactors and reaction mechanisms of nitrile hydratase. The following results are summarized.(1) There are two kinds of nitrile hydratases, iron or cobalt is involved as a cofactor.(2) ESR studies showed the following : nitrile hydratase is the first known nonheme iron enzyme with a typical low-spin Fe (III) coordination environment, the axial position of the iron (III) site in the native enzyme may be occupied by thiolate and equo groups, and aliphatic nutrile-substrates directly bind to the iron (III)-active center through water substrate replacement. Cobalt also probably plays the same role, the binding of nitrile and activation of it.(3) Iron nitrile hydratase involves an active carbonyl cofactor as a second prosthetic group, probably pyrrologuinoline quinone. There may be an interaction, such as a hydrogen bond, between the ferric site and a form of hydrated PQQ.(4) The iron and cobalt nitrile hydratases were cloned. The sequence of these genes was determined and the extreme high homology of two genes was found in spite of the difference of confactors.

我们已经建立了利用微生物腈水合酶生产丙烯酰胺的酶促工艺，并于几年前开始了丙烯酰胺的工业化生产。与应用工作的高度发展相比，对腈水合酶的基础研究进展并不充分。因此，本文的工作主要集中在更基础的工作上，即阐明腈水合酶的辅因子和反应机理。总结了以下结果。(1)有两种腈水合酶，铁或钴作为辅因子参与。(2) ESR研究表明：腈水合酶是已知的第一个具有典型低自旋Fe （III）配位环境的非血红素铁酶，天然酶中铁（III）位点的轴向位置可能被硫代酸基和等分基占据，脂肪族营养底物通过水底物置换直接与铁（III）活性中心结合。钴可能也起着同样的作用，结合腈并活化它。(3)铁腈水合酶涉及一个活性羰基辅助因子作为第二假基，可能是吡咯喹啉醌。铁离子位点与水合PQQ之间可能存在相互作用，如氢键。(4)克隆了铁和钴腈水合酶。测定了这些基因的序列，发现尽管两个基因的因子不同，但具有极高的同源性。

项目成果

Toru Nagasawa: "eーCaprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase" Archieves of Microbiology. 155. 13-17 (1990)

Toru Nagasawa：“e-己内酰胺，一种用于形成红红球菌 J1 腈水解酶的新型强效诱导剂”，《微生物学档案》155. 13-17 (1990)。

Toru Nagasawa: "Optimum culture conditions for the production of cobaltーcontaining nitrile hydratase by Rhodococcus rhodochrous J1" Applied Microbiology Biotechnology. 34. 783-788 (1991)

Toru Nagasawa：“紫红红球菌 J1 生产含钴腈水合酶的最佳培养条件”应用微生物学生物技术 34. 783-788 (1991)。

Toru Nagasawa: "Microbial transformations of nitriles" Trends in Biotechnology. 7. 153-158 (1989)

Toru Nagasawa：“腈的微生物转化”生物技术趋势。

Toru Nagasawa: "Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodcoccus rhodochrous J1" Applied Microbiology Biotechnology. 34. 783-788 (1991)

Toru Nagasawa：“红球菌 J1 生产含钴腈水合酶的最佳培养条件”应用微生物学生物技术。

T. Nagasawa, J. Mauger, H. Yamada: "A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3" Eur. J. Biochem.194. 765-772 (1990)

T. Nagasawa、J. Mauger、H. Yamada：“粪产碱菌 JM3 的一种新型腈水解酶，芳基乙腈酶”Eur。