Novel assay method for dimethyl sulfoxide reductase using capillary electrophoresis

毛细管电泳测定二甲亚砜还原酶的新方法

• 批准号: 05660115
• 负责人: OKUBO Akira
• 金额: $ 1.34万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660115/
• 关键词: capillary electrophoresis; DMSO reductase; CZE; molybdenum containing enzyme; micellar electrokinetic chromatography; MEKC

Dimethyl sulfoxide reductase was obtained by anaerobic cultivation of a green mutant of Rhodobacter sphaeroides f.s.denitrificans under visible light in the presence of DMSO.The purified enzyme through several column techniques gave a single band in SDS PAGE.A novel assay method for DMSO reductase was developed using capillary electrophoresis. A micellar electrokinetic chromatography mode was quite effective for a quantitative determination below the nanogram level without any pretreatment of the reaction mixture of the enzyme. About 6nl of sample solution was introduced in the hydrostatic mode (20sec, 5cm height) from the anodic end of the capillary. A boratephosphate-SDS buffer, pH7, was used for MEKC.The sample solution was electrophoresed at 15kV with an electric current of around 25 uA,on-column detection being done at an absorbance of 200nm. The enzymatic activity calculated by MEKC was compared with that by conventional optical absorption method. Both methods was in close agreement, indicating that MEKC is useful for a quantitative assay of DMSO reductase. This novel assay method established enabled us to search the electron donor for DMSO reductase. In the water soluble fraction of the cell homogenate, DMSO dependent oxidation activity was found. The active compound was now being purified.

通过在DMSO存在的情况下，通过可见光的光线下的杜鹃花杆菌Sphaeroides f.s.s.s.s.s.s.s.s.s.s.s.s. dyphaeroides的绿色突变体获得了二甲基亚砜还原酶。多种列酶通过SDS Page中的单个谱带提供了一种纯化的酶。用于SDS页面中的单个频段。一种用于使用DMSO的小说分析方法，用于使用DMSO的小型分析方法，它使用了DMSO capill capillophorsis。胶束电动色谱模式对于低于纳米图水平的定量测定非常有效，而无需预处理酶的反应混合物。从毛细管的阳极末端的静静力模式（20秒，5厘米高）引入了约6NL样品溶液。 MEKC使用了磷酸-SD-SDS缓冲液PH7。样品溶液以15kV的电流为15kV，电流约为25 UA，在200nm的吸光度下进行柱上检测。通过MEKC计算的酶活性与常规光吸收方法进行了比较。两种方法都非常一致，表明MEKC对于DMSO还原酶的定量测定有用。这种新颖的测定方法建立了使我们能够搜索电子供体以寻找DMSO还原酶。在细胞匀浆的水溶性部分中，发现DMSO依赖性氧化活性。现在正在纯化活性化合物。

项目成果

M.Tachibana, H.onoe, A.Okubo, S.Yamazaki: "Application of micellar electrokinetic chromatography as a novel assay method for dimethyl sulfoxide reductase." Biosci.Biotech.Biochem.59. 282-284 (1995)

M.Tachibana、H.onoe、A.Okubo、S.Yamazaki：“胶束电动色谱法作为二甲亚砜还原酶的新型测定方法的应用。”

M.Abo, M.Tachibana, A.Okubo, S.Yamazaki: "Enantioselective reduction of the sulfoxide in methyl phenyl sulfoxide by DMSO reductase from R.sphaeroides." Biosci.Biotech.Biochem.58. 596-597 (1994)

M.Abo、M.Tachibana、A.Okubo、S.Yamazaki：“通过来自 R.sphaeroides 的 DMSO 还原酶对甲基苯基亚砜中的亚砜进行对映选择性还原。”

M.Abo, M.Tachibana, A.Okubo, S.Yamazaki: "Enantioselective deoxigenation of alkyl aryl by DMSO reductase from R.sphaeroides." Bioorg.Med.Chem.(in press). (1995)

M.Abo、M.Tachibana、A.Okubo、S.Yamazaki：“通过来自 R.sphaeroides 的 DMSO 还原酶对烷基芳基进行对映选择性脱氧。”

M.ABO,M.TACHIBANA,A.OKUBO,S.YAMAZAKI: "Enantioselective Reduction of the Sulfoxide to Sulfide in Methyl Phenyl Sulfoxide by DMSO Reductase from R.sphaeroides" Biosci.Biotech.Biochem.58. 596-597 (1994)

M.ABO、M.TACHIBANA、A.OKUBO、S.YAMAZAKI：“通过来自 R.sphaeroides 的 DMSO 还原酶对甲基苯亚砜中的亚砜对映选择性还原为硫化物”Biosci.Biotech.Biochem.58。

M. Abo et al: "Enantioselective Reduction of the Sulfoxide in Meltyl Pheuyl Suilfoxide by DMSO Reductase from R. sphaeroides." Biosci. Biotech. Biochem.58. 596-597 (1994)

M. Abo 等人：“通过来自 R. sphaeroides 的 DMSO 还原酶对 Meltyl Pheuyl Sulfides 中的亚砜进行对映选择性还原”。