Clonine, Expression Sulfur Oxidases From N.Aromaticivorans And Research In Situ Cofactor Regeneration On Synthesis Of Chiral Sulfoxides

Flavin-containing monooxygenase (EC1.14.13.8) called sulfur oxidases in this paper has great potential in synthesis of chiral sulfoxide. Eight sulfur oxidases candiate genes ncml, ncm2, ncm3, nfm1, nfm2, nfm3, nfm4and nfm5from genomes N. aromaticivorans were identified based on analysis of all the bacterial genomes that reported. The ORF of the eight genes were1650bp,1638bp,1953bp,1623bp,1704bp,1524bp,1518bp and1722bp,encoding eight proteins61.6KD,61.38KD,72.47KD,60.16KD,63.02KD,57.31KD,57.48KD and62.13KD as predicted. Prokaryotic expression systems for these genes were constructed with pET-28a, and,soluble proteins NCM1,NCM2,NFM1,NFM2and NFM3were obtained after optimizing. NCM1,NCM2and NFM1were chosen for purication followed by Ni+-sepharose affinity chromatography.Using PMS as the substrate to test these three enzymes activity drawed a conclusion that only NCM1had a live activity. Further research indicated that it was NADPH-dependent with broad substrate specificity and high activity on aromatic sulfide, their derivates and1,3-dithiane but low activity on chain sulfide and cyclic ketone. The optimum temperature and pH for NCM1were55℃and8.87.Coexpressing CHMO and FDH in a two-plasmid system, and the whole-cell of the E. coli was used in the process of oxidizing sulfide to sulfoxide. After reaction optimization (temperature, pH, NADP+addition and cell concentration), many important result were obtained. The concentration of the substrate improved from10mM to20mM and the productivity was98.57%with a high enantioselectivity (ee99%). The final product concentration could access to5g/L when adding PMS in a fed-batch strategy.