Preparation Of Indigo By Recombinant E. Coli BL21(pET28a(+)-P450 BM3) And E. Coli BL21(pET28a(+)-P450 BM3-gdh0310)

Indigo is the most important and oldest blue dyestuff. Recently, more and more biotechnological methods of indigo production have been performed to replace the chemical synthesis of indigo, since environment pollution could been controlled effectively. In this work, biotransformation of indole to indigo by the free and immobilized recombinant E.coli BL21(pET28a(+)-P450 BM3) was studied. In order to solve the problem of coenzyme NADPH regeneration during reaction, a recombinant E.coli BL21(pET28a(+)-P450 BM3-gdh0310) was constructed to coexpress P450 BM3 and glucose dehydrogenase, which is able to regenerate NADP⁺ to NADPH coupled with the oxidation of glucose.Factorial design and response surface technique were used to design and optimize the process to increase the P450 BM3 production by E. coli BL21(pET28a(+)-P450 BM3). Initially, Plackett-Burman design was used to evaluate the process variables which were relevant in relation to the production of P450 BM3. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM3 activity was improved up to 57.9 × 10^-3 U/ml by the best combinations of the physicochemical parameters at optimal levels of 0.12 mg/L FeCl₃, inoculum volume of 2.1 %, induction at OD₅₇₈=1.1, and with 6 h of induction.Indigo was synthesized from indole catalyzed by recombinant E. coli BL21(pET28a(+)-P450 BM3) strain. The effects on reaction of substrate, product, glucose concentration, pH and temperature were investigated. The results indicated that the optimal conditions of the reaction were pH 7.0-7.5 and 35 ℃, and indole concentration was 0.5 mmol/L. Indigo had toxicity to recombinant E. coli BL21 and would inhibit the biotransformation of indole into indigo. The yield of indigo was increased up to 44.08 % in the case of adding 5 g/L glucose into reaction buffer. The effect of coenzyme regeneration on the reaction catalyzed by recombinant E. coli BL21(pET28a(+)-P450 BM3) was investigated. The regeneration capacity of the cells was very low. While NADP⁺ and glucose dehydrogenase were added to the reactionsystem, NADPH was regenerated successfully and indigo yield was increased effectively.The ability of catalyzing indole into indigo of recombinant E.coli BL21(pET28a(+)-P450 BM3) immobilized by entrapment in calcium-alginate gel capsules was examined. And various characteristics of immobilized cells were investigated. The optimum conditions for cells activity were not affected after immobilization, and optimum pH and temperature for immobilized cells were found to be pH7.5 and 35 ℃, respectively. The immobilized cells exhibited a markedly improved thermal stability than free cells. After five repeated experiments, the yield of indigo with the immobilized cells retained over 94 % of their original activity, which indicated the operational stability for recycling in batch processes was improved.A recombinant E. coli BL21(pET28a(+)-P450 BM3-gdh0310) was constructed to regenerate NADPH, which coexpressed P450 BM3 and glucose dehydrogenase. After optimization of cultivation and induction conditions, the specific activities of P450 BM3 and glucose dehydrogenase expressed by E.coli BL21(pET28a(+)-P450 BM3-gdh0310) were increased up to 8173.13 U/mg protein and 0.045 U/mg protein, respectively. The whole cells of E. coli BL21(pET28a(+)-P450 BM3-gdh0310) were used not only to transform indole to indigo, but also as the system for NADPH regeneration. In the reaction system catalyzed by E.coli BL21(pET28a(+)-P450 BM3-gdh0310), indole could be converted to indigo efficiently with no addition of NADP⁺ and glucose dehydrogenase.The transformation from indole to indigo was completed effectively without any addition of glucose dehydrogenase or NADPH/NADP⁺ when initial concentration of indole was not higher than 5 mmol/L. The optimized biotransformation conditions were: 35 ℃, 100 r/min of rotate speed, with addition of 100 mmol/L glucose. And indigo formed amounted to 4.7 mmol/L (1235.2 mg/L) with batch indole and glucose feeding, with a yield of 81 %.The kinetics of enzyme catalyzing reaction was studied. The ordered Bi Bi mechanism was adopted to the systems with two-substrate paticipating in the reaction. The kinetic equations of P450 BM3 and glucose dehydrogenase were obtained.Comparing with the experiment data, these equations could excellently simulate to the Experimented data. Applying the equations to the two-enzyme coupled system :d[NADPH]/dt = V_GDH-V_{P450 BM3}d[NADP+]/dt = -V_GDH+V_{P450 BM3}The model could predict the process successfully.