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Fig. 1.
Changes in 4-HIL-producting activity of the Dabrafenib Mesylate of B. thuringiensis strain 2e2 during cultivation. Washed cells of B. thuringiensis strain 2e2 collected at several time points during cultivation were applied as catalysts for the l-Ile hydroxylating reaction, and amount of 4-HIL production was measured. Cell densities at O.D.660 (open triangles) and 4-HIL production (open squares) are shown.
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Purification and preliminary characterization of IDO
Through the steps presented in Table 1, IDO was purified from the lysate of the cells obtained at logarithmic growth phase to homogeneity as shown in Fig. 2. The molecular weight of the subunit was 28 kDa and that of native enzyme observed on gel-filtration analysis with Superdex 75 was about 31 kDa; indicating that the enzyme is a monomeric protein. Using the purified IDO, l-Ile hydroxylation reactions were examined under several conditions (Table 2), and it was revealed that the enzyme strictly required α-KG for the hydroxylation of l-Ile. A ferrous ion was also required for the reaction, and addition of EDTA completely inhibited the activity. The activity was also inhibited by ZnCl2 and CuCl2. Ascorbic acid was not necessarily required for the reaction; however, in the presence of ascorbic acid, the reaction rate was greatly stimulated.