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Corynebacterium glutamicum ATCC13032 유래 Flavin-containing Monooxygenase와 변이효소의 정제 및 특성연구 원문보기
Purification and Characterization of Flavin-containing Monooxygenase and mutant forms from Corynebacterium glutamicum ATCC13032

  • 저자

    정혜숙

  • 학위수여기관

    경성대학교 일반대학원

  • 학위구분

    국내석사

  • 학과

    식품생명공학과

  • 지도교수

    이진호

  • 발행년도

    2014

  • 총페이지

    vi, 55 p.

  • 키워드

    Flavin-containing monooxygenase purification characterization mutation;

  • 언어

    kor

  • 원문 URL

    http://www.riss.kr/link?id=T13535203&outLink=K  

  • 초록

    A flavin-containing monooxygenase from Corynebacterium glutamicum ATCC13032 (cFMO) was expressed in pMAL-C2X. cFMO was purified as a maltose binding protein(MBP)-fused form by amylose affinity chromatography. The recombinant enzyme showed a temperature optimum at 25℃ and had the highest activity at pH 8.0. According to the absorption spectra of MBP-cFMO in the oxidized and reduced forms, cFMO was classified in a typical flavoprotein and required more than 2 mM of exogenous FAD for maximal enzyme activity. When the effect of several metal ions was determined using trimethylamine as a substrate, 1 mM Cu2+ strongly inhibited FMO activity, and its activity was recoverd by addition of 1 mM EDTA. The enzyme can catalyze the oxidation of trimethylamine, thiourea, and cysteamine, but not glutathione and cysteine. The Km values for trimethylamine, thiourea, and cysteamine are 0.575, 0.38, and 5.95 mM, respectively; Kcat values are 156.1, 40.22 and 110.3 per min, respectively. Inaddition, cFMO had indoxyl-forming activity by oxidation of indole. Escherichia coli expressing cFMO produced 684 mg/l of indigo and 103 mg/l of indirubin in the LB medium containing 2.5 g/l of tryptophan after 36 hr cultivation. In order to increase enzyme activity, 6 types of mutants, A210G, A210S, A210C, S211K, F170Y, and T326S, were constructed based on based on the structure of the FMO protein from Methylophaga aminisulfidivorans and characterized their biochemical characteristics. Of them, T326S mutant showed a great decrease of NADPH oxidation activity (futile activity) in the absence of substrate compared to wild type FMO.


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