재조합 대장균을 이용한 인디고의 최적 생산

Abstract

Flavin-containing monooxygense has been identified in organisms from bacteria to humans. A fragment of FMO from Methylophage aminisulfidivorans MPT has been cloned and experssed in Escherichia coli DH5a. Growth of the recombinant Escherichia coli DH5a pBlue 1.7 in tryptophan medium(2 g tryptophan, 10 g NaCl, 5 g yeast extract, and 50 mg ampicillin per liter) resulted in the formation of indigo. In the batch reaction, the recombinant E. coli DH5a pBlue 1.7 produced 920 mg/L of bio indigo for 20 h cultivation in 5 L reactor. Cultured in the 3000 L reactor containing tryptophan medium in the same culture conditions, the same concentration of indigo could be obtained. To enhance indigo production, a novel medium system was constructed. The cultivated medium containing indigo was transferred to the bottle and mixed with diphenylmethane. Next, the mixture was applied to the cylindrical column (3 X 30 cm) filled with diphenylmethane, and indigo was moved into the diphenylmethane layer. The isolated medium was circulated into the bioreactor. In the water-organic system, 1.4 g/L of indigo was produced from the medium containing 3ｇ/L of tryptophan. In addition, continues system was constructed. It investigated the optimum continues culture conditions such as medium pH, temperature, tryptophan concentration, and feeding rate. The optimum conditions were as follows: pH 7.0, temperature 35℃, and tryptophan concentration 3 g/l. After indigo concentration reached to 0.8 g/l in the 2 g/l tryptophan medium, a fresh medium containing 3 g/l tryptophan was continuously fed at a rate of 2.8 ml/min during 140 h. Under this condition, the recombinant E. coli cells produced 5.6 g/L/day of indigo. Bio indigo was applied to dye several fabrics such as cotton, silk, hemp, and ramie. Colour fastness value to light, washing, and perspiration were 3, 4-5, and 4-5, respectively. We concluded that bio indigo appeared to have a lot of benefits that enable the dyed materials to exhibit their natural colors.