Production, purification, and characterization of soluble NADH-flavin Oxidoreductase (StyB) from Pseudomonas putida SN1.
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 NADHflavin 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 degrees with 0.5 mM IPTG. The solubly expressed StyB has the highest oxidoreductase activity at pH 6.5-7.5 and 37 degrees . Substrate dependence profiles of the StyB-catalyzed reaction showed that the maximum specific activity (Vm) and half saturation constant (Km) were 1,867+/-148 U/mg protein and 51.6+/-11 microM for NADH, and 1,274+/-34 U/mg protein and 8.2+/-1.2 microM 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.
Yeo
Yun-Ji
YJ
Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Korea. jeongyj@kookmin.ac.kr
Shin
Seunghee
S
Lee
Sun-Gu
SG
Park
Sunghoon
S
Jeong
Yong-Joo
YJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000378:metabolism; D010105:Oxygenases; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D010105:Oxygenases; C026721:styrene monooxygenase; D038181:FMN Reductase