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.