Catalytic Characterizations Of Dibenzothiophene Monooxygenase And Its Application In Catalyzing Indole To Indigoids

Four enzymes are involved in the biocatalytic desulfurization via the "4S" pathway.Dibenzothiophene monooxygenase initiates the desulfurization by catalyzing dibenzothiophene(DBT) to dibenzothiophene sulfone(DBTO₂) through two consecutive oxidations.DszC requires an independent NADH:flavin oxidoreductase(DszD) to provide reduced flavins to sustain its activity.DszC and DszD consist of a flavin-dependent two component monooxygenase system.DszC is the monooxygenase unit,and DszD is the reductase one.Usually the monooxygenase unit is strictly dependent on FMNH₂ or FADH₂.Until now only two enzymes have been reported to have this unusual ability.One is the C₂ component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, and the other is the isobutylamine N-hydroxylase from Streptomyces viridifacienshave MG456-hF10.However,utilization of FADH₂ by DszC has never been elucidated clearly.Flavin dependent monooxygenase is one of the sources of reactive oxygen species(ROS).Overproduction of ROS would cause damage to the cell.Therefor, antioxidant proteins play an important role in keeping the ROS at proper levels and the cells work well.Any antioxidant protein related to "4S" pathway has never been reported.Biodegradtion and biocatalysis is always related.DszC has broader substrate than DBTs,and it is a potential biocatalyst in synthesis of valuble products.Biocatalytic desulfurization has been studied for a long time at our laboratory, and several valuable strains were isolated.In this study,dszC from a thermophilic biodesulfurizing strain Mycobacterium goodii X7B was cloned and expressed in Escherichia coli,and DszC was purified.Preference to reduced flavins by this DszC and factors that affected the DszC activity was discussed.Relationship between desulfurizing enzymes expression and antioxidant proteins expression was studied. Research was also conducted to estimate the potential application of DszC (microorganism containing of) in the biocatalytic synthesis of indigoids.Purified DszC showed a single band of about 45 kDa on the SDS-PAGE. Wavescan of the enzyme showed that DszC had strong absorbance at 280 nm,but no specific absorbtion in the range of 300-700 nm,indicating that no heine or flavin was bounded.As a monooxygenase unit of the flavin-dependent two-component monooxygenase,DszC activity is significantly dependent on the reduced flavins provided by DszD.Using a DszD that reduced either FMN or FAD,DszC was proved to be able to utilize FADH₂ as well as FMNH₂ to catalyze DBT to DBTO₂. DszC activity is dependent on the type and concentration of the flavin,and the ratio of DszC to DszD.DszC was much more active with FMNH₂ than that with FADH₂. Either flavin at low concentration stimulates the DszC activity but at high concentrations inhibits the activity of DszC due to the autocatalytic oxidation of reduced flavins.Autooxidation depressed the flow of reduced flavin to DszC,and hydrogen peroxide formed due to the autooxidation,which caused the inactivation of DszC and DszD and led to the decrease in DszC activity.The autooxidation was enhanced when flavins concentration increased,and released by the increase of DszC amount in the DszCD system.Addition of catalase destructed H₂O₂ as soon as it formed and thus increased the activity of DszC.Relationships between expressions of desulfurizing enzymes and the antioxidant proteins were investigated in both wild types of desulfurizing strains and engineering strains.Using undenaturing gel electrophoresis and active staining,a superoxide dismutase(SOD) induced by DBT or dimethyl sulfoxide was for the first time discovered in Rhodococcus erythropolis 1 awq.Expression of desulfurizing genes in heterogeneous engineering strains would cause a serious decrease in catalase activity; such phenomenon was not observed in the wild strains.Although more and more flavin dependent monooxygenase have been discovered in the past decade,seldom of them was applied in the biocatalytic synthesis of valuable compounds that were hard to obtain with traditionally chemical catalysts.Two obstacles exist on the way to explore scaled-up production by the flavin dependent monooxygenase.First,monooxygenase is usually difficult to express and purify,and second,expensive cofactors of NAD(P)H is required to drive the reaction,and stoichemsitroic addition of these cofactors are not economical. Glucose dehydrogenase(GDH) gene was cloned from Bacillus subtilis subsp,subtilis str.168 and transferred to E.coli for overexpression.Using GDH purified from the engineered E.coli,a system for regeneration of NADH was constructed.It could provide NADH continuously to the DszCD coupling reaction to catalyze DBT to DBTO₂.Effect of GDH on the DszC activity was investigated with indole as the substrate of DszC.Increase of DszC provided more NADH to DszCD,hence DszCD activity was enhanced.However,GDH should be kept at a proper ratio to DszD.DszD activity can be elevated by NADH supply,and thus more FMNH₂ is formed.As described previously,excessive FMNH₂ would inevitably lead to decrease of DszCD activity.Yield of indigo from indole was improved when whole cells of recombinant E. coli were used instead of isolated enzymes,in which case the autooxdiation of reduced flavins was depressed greatly.Co-expression of DszD and DszC significantly increased the activity of the biocatalysts,but further co-expression of GDH led to a decrease in the yield of indigo.It could be attributed to that the over expression of GDH made a sharp decrease in the expression of DszC and DszD. When the cells grew to the exponential phase,1 mM IPTG was added and the cells were further induced at 27℃for 8 h.The biotransformtion was carried out at pH 6-7;higher pH would reduce the indigo production.Cell density had an obvious effect on the production;an optimized cell density was OD₆₀₀ at about 20 when the reaction volume was one fifth of the total volume.In the range of 1-4 mM,increase of indole concentration led to indigo production increase,but the yield of product to substrate decreased.Higher indole concentration than 4 mM decreased the final concentration of indigo,which was possibly due to the cell toxicity of indole.A pink compound was primarily identified as inirubin,which was proved as an effective anti cancer drug,and might be applied in the therapeutics of several diseases. Whole cells expressing DszC also could catalyze various substituted indole at the C-5 site to form the blue and pink compounds,assumed to be derivatives of indigo and indirbin.It suggested that DszC(whole microorganism containing of) had great potential in the production of various valuable indigoids.