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首页> 外文期刊>Applied Microbiology and Biotechnology >A rapid fluorescence-based assay for detecting soluble methane monooxygenase
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A rapid fluorescence-based assay for detecting soluble methane monooxygenase

机译：一种基于荧光的快速检测可溶性甲烷单加氧酶的方法

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摘要

A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be K m(app)=262 µM and V max(app)=821 nmol 7-hydroxycoumarin min–1 mg protein–1. The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.
机译：开发了一种基于荧光的测定法，以评估溶液中的可溶性甲烷单加氧酶（sMMO）活性。表达sMMO的甲基毛状孢子虫OB3b的全细胞用于氧化各种化合物以筛选荧光产物。在测试的12种化合物中，只有香豆素可产生荧光产物。该产物的UV吸收光谱与7-羟基香豆素相匹配，并且通过13 C-NMR光谱证实了该鉴定。通过与乙炔（一种已知的sMMO活性抑制剂）进行预孵育，研究了荧光反应对sMMO活性的依赖性。确定全细胞的表观动力学参数为K m（app） = 262 µM和V max（app） = 821 nmol 7-羟基香豆素min–1 mg蛋白–1 < / sup>。 sMMO的香豆素氧化速率与三氯乙烯降解和萘氧化速率密切相关。基于荧光的香豆素氧化测定法比萘氧化测定法的优势包括更稳定的产品，无需额外试剂即可直接检测产品，以及更高的速度和便利性。

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  • 来源
    《Applied Microbiology and Biotechnology》 |2002年第2期|183-188|共6页
  • 作者

    A. Miller; W. Keener; M. Watwood; F. Roberto;

  • 作者单位

    Idaho National Engineering and Environmental Laboratory P.O. Box 1625 MS-2203 Idaho Falls ID 83415–2203 USA;

    Idaho National Engineering and Environmental Laboratory P.O. Box 1625 MS-2203 Idaho Falls ID 83415–2203 USA;

    Idaho State University Pocatello ID 83209 USA;

    Idaho National Engineering and Environmental Laboratory P.O. Box 1625 MS-2203 Idaho Falls ID 83415–2203 USA;

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