| Flavin-containing monooxygenases (FMOs, E.C.1.14.13.8) are, after cytochromes P450 (CYP450, E.C.1.14.14.1), one of the most important monooxygenase system in humans. They are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO (bFMO) from Methylophaga sp. strain SKI has been solved at 2.6-A resolution.All mammalian FMOs have a very strong membrane combination, and poor water solubility, but so far not received any crystal structure of a mammalian FMO. Bacterial FMOs do not exist such problems, and thus more suitable for practical application. Because of their regioselective asymmetric catalysis and good stability, the source of biomass in metabolism of toxic exogenous compounds, pharmacokinetics and bio-catalytic synthesis and other fields has been widespread concern.The metagenomic library was established from crude oil contaminated soil samples, isolating a blue clone which could make the conversion of indole. This function fmo gene was designed bfmoⅡ. Bioinformatics analysis shows that, there are many differences between bFMOⅡand bFMO, reflected in the codon usage bias and so on. Alignment of the sequences showed that their structures were very similar. Phylogenetic tree was also constructed, confirmed the existence of the diversity of prokaryotic FMOs, the corresponding study for the future and lay the foundation for further development.E. coli DH5a harboring fmo gene and vector pMD18-T, could convert indole in the metabolism of tryptophan, into bio-indigo, and the production of bio-indigo made a preliminary determination. E. coli BL21 (DE3) harboring fmo gene and vector pET-28a, could covert indole and indole derivatives into different colors of pigment, as before had not been confirmed.This study established a complete set of bFMOⅡinduced protein expression and purification scheme. E. coli BL21 (DE3) harboring plasmid pET-28a-FMO expression system of enzymes was determined. Induced expression optimized conditions, was chosen for the low temperature induced (16℃), low concentration of inducer (final concentration of 0.3 mM IPTG), moderate expression of the environment (130 rpm). A a lot of soluble protein were obteined and prepared for subsequent foundation. Purification of the enzyme process, through experimental groping, found ammonium sulfate precipitation was the key process for the purification. By ammonium sulfate precipitation, more purified bFMOⅡwas captured and satisfactory results had been obtained.Basic properties of purified bFMOⅡwere studied. Learned through native-PAGE, active state of bFMOⅡwas a dimeric protein with the molecular weight of about 110 kDa, and each of the monomer molecular weight was about 55 kDa. Through the process of purification of pure color and the absorbance spectra of bFMOⅡ, it showed that bFMOⅡcontained non-covalent bound cofactor FAD which could be easily lost during purification. Quantitative analysis showed that the enzyme requires a combination of a FAD per subunit.Site-directed mutagenesis was performed containing bFMO as template. Four mutants were designed and theri activity were verified. By observing the direct effects of indole conversion, we established efficient detection of mutant activity. Results indicated that the four amino acid residues corresponding to experimental active sites played an important role in the activity of bFMOⅡ. By homology modeling of bFMOⅡtwo other bFMOⅡmutants, onstructed model was to assess the results and ideal structure. By comparsion of analog structure of part of the active site, the mechanism of enzyme inactivation was explained preliminary.
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