Study On The Transformationprocess Of Indole By Recombinant Srtain Bphab_LA-4

Recently, some studies have indicated that the introduction of hydroxyl groups into indole molecule by different mono-and dioxygenases leads to the production of indigo. This biotransformation has well researched using human cytochrome P450enzymes and naphthalene dioxygenase. As a well-known biocatalyst, biphenyl dioxygenase possessed the ability to transform indole to indigo. However, there has been little information about this enzymatic transformation process. In this research, these problems have been discussed, including the feasibility of this biotransformation, the optimum condition, the transformation mechanism and the transformation system.Indole can be bound with the mononuclear Fe(II) in the active area of BphA1_LA-4which is the same with biphenyl. The distance between Fe(II) and C2/C3of indole was4.7and4.6A, whereas the distance between Fe(II) and the C2/C3of biphenyl was3.6and4.1A. Besides, the nitrogen atom of indole in BphA1_LA-4was able to form hydrogen bonds with residues of Gln-231and Asp-235in BphAl_LA-4, respectively, which enhanced the interactions between indole and BphA1LA-4.The bphAB gene fragment from the genomic DNA of Dyella ginsengisoli LA-4, which expressed biphenyl dioxygenase (BphA) and bipheny1-2,3-dihydrodiol2,3-dehydrogenase (BphB) was successfully ligated into pBR322. This expression vector was transformed into Escherichia coli BL21(DE3) yielding the recombinant strain BphAB_LA-4.The BphAB_LA-4could transform indole into indigo with a poor efficiency. The transformation ratio reached the maximum (95%) when BphAB_LA-4was induced at lower temperature (15℃) and IPTG concentration (0.25mM) in M9after12h transformation. According to SDS-PAGE analysis, more expressed components were existed as soluble forms in the supernatant of BphAB_LA-4crude enzyme, especially BphA1_LA-4, which encoded the large submit of terminal dioxygenase.When the cell concentrations were0.28g/L, indole concentrations was200mg/L and the glucose concentration was0.2%(w/v), the indigo concentration increased to44mg/L within10h and the productivity of indigo was0.28mg/mg CDW (CDW:dry weight of cells). The products were identified as isatin. indigo and indirubin using HPLC-MS. Here, we made a hypothesis, the oxidation of indole by BphA leads to the production of indole-2,3-dihydrodiol which was then spontaneously dehydrated to indoxyl or dehydrogenized to isatin (through an unstable intimidate2,3-dihydroindole) by BphB. Condensation of two molecules of indoxyl followed by air oxidation leads to the formation of indigo, whereas, condensation of indoxyl and isatin yields indirubin.Three immobilization methods were used, and sodium alginate obtained the best results. The immobilized cell could be repeatedly used for two times in a biphasic system under the optimum immobilization condition:3.5%(w/v) sodium alginate,1%(w/v) CaCl2, and the indigo production was15.9mg/L after6h for the first transformation. Besides, the indigo decomposition could be avoided by the biphasic system with40%(v/v) dodecane as the optimal organic phase. The optimum indole tolerance concentration was400mg/L, and the productivity of indigo was0.28mg/mg CDW.