Nuclear magnetic resonance (NMR) analysis. 4-HIL prepared using the purified enzyme was purified from the reaction mixtures by the methods reported previously [5] and dissolved in D2O, and their 1H-NMR spectra were recorded using an Avance 400 (Bruker, Billerica, MA, USA).
Results
l-Ile hydroxylating activity in the cell lysate of B. thuringiensis strain 2e2
The metabolites of various microorganisms cultivated in medium containing l-Ile (IDO broth) were investigated, and it ITF 2357 was found that B. thuringiensis strain 2e2 accumulated 4-HIL and AMKP as l-Ile metabolites (data not shown). As shown in Fig. 1, the activity to produce 4-HIL from l-Ile was found only in the cells of B. thuringiensis strain 2e2 obtained at logarithmic growth phase. The 4-HIL-producing activity was confirmed with the lysate of the cells obtained at logarithmic growth phase in the l-Ile-transforming reaction with α-KG, ferric ions, and ascorbic acid. These results indicated that B. thuringiensis strain 2e2 produces α-KG-dependent l-isoleucine dioxygenase, IDO.
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