Journal of Microbiology and Biotechnology

  • 제19권4호
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  • Pages.362-367
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  • 2009
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Production, Purification, and Characterization of Soluble NADH-Flavin Oxidoreductase(StyB) from Pseudomonas putida SN1

  • Yeo, Yun-Ji (Department of Bio and Nanochemistry, Kookmin University) ;
  • Shin, Seung-Hee (Department of Chemical and Biochemical Engineering, and Institute for Environmental Technology and Industry, Pusan National University) ;
  • Lee, Sun-Gu (Department of Chemical and Biochemical Engineering, and Institute for Environmental Technology and Industry, Pusan National University) ;
  • Park, Sung-Hoon (Department of Chemical and Biochemical Engineering, and Institute for Environmental Technology and Industry, Pusan National University) ;
  • Jeong, Yong-Joo (Department of Bio and Nanochemistry, Kookmin University)
  • 발행 : 2009.04.30
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초록

In recombinant strains, many proteins and enzymes are expressed as inactive and insoluble inclusion bodies. For soluble expression of an active form of StyB, an NADH-flavin oxidoreductase, several recombinant Escherichia coli strains were developed and tested. Among them, strain BL21(DE3)pLysS effectively produced an active and soluble form of StyB as about 9% of the total protein content, when cultivated at $20^{\circ}C$ with 0.5 mM IPTG. The solubly expressed StyB has the highest oxidoreductase activity at pH 6.5-7.5 and $37^{\circ}C$. Substrate dependence profiles of the StyB-catalyzed reaction showed that the maximum specific activity($V_m$) and half saturation constant($K_m$) were $1,867{\pm}148\;U/mg$ protein and $51.6{\pm}11{\mu}M$ for NADH, and $1,274{\pm}34\;U/mg$ protein and $8.2{\pm}1.2{\mu}M$ for FAD, respectively. This indicates that solubly produced StyB has 6- to 9-fold higher oxidoreductase activities than the in vitro refolded StyB from inclusion bodies.

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참고문헌

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