JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.19, no.4, pp.362 - 367
Abstract
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 degrees C with 0.5 mM IPTC. The solubly expressed Styli has the highest oxidoreductase activity at pH 6.5-7.5 and 37 degrees 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 +/- 148 U/mg protein and 51.6 +/- 11 mu M for NADH, and 1,274 +/- 34 U/mg protein and 8.2 +/- 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.