N-Acetyltryptophan → D-Tryptophan

참여 물질

반응 단계

Cells of the E. coli transformant obtained in Example 9 were inoculated on an LB agar plate. Each colony grown on the plate was inoculated with a toothpick to a well of a microtiter plate which contained 150 μl of LB culture medium containing 50 μg/ml ampicillin and 0.02 mM IPTG, and then the microtiter plate was covered with Breathable Sealing Membrane (Nalgene). The microtiter plate was incubated at 37° C. for 18 hours while being shaken. 10 μl of the culture medium solution was added to the reaction solution containing 300 mM Tris-hydrochloride buffer (pH 8.0), 0.1% cetylpyridinium bromide, 2% N-acetyl-DL-tryptophan, 3.5% N-acetyl-L-tryptophan, and distilled water in the wells of a microtiter plate. The plate was incubated at 25° C. for 30 minutes while being shaken. Then, D-tryptophan produced from N-acetyl-D-tryptophan was quantified by colorimetry according to the TNBS method using D-tryptophan as standard. The same reaction as described above was conducted simultaneously using wild-type E. coli strain. Then, mutants in which the activity for producing D-tryptophan was increased compared with wild-type strain were isolated as mutants with reduced inhibition. Screening of about 200 colonies yielded a number of mutants whose competitive inhibition had been reduced.

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