反应详情

Three types of the E. coli transformant libraries obtained in Example 11 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, 5% N-acetyl-DL-tryptophan, 1% N-acetyl-L-tryptophan, and distilled water in the wells of a microtiter plate. The plate was incubated at 25° C. for 1 hour while being shaken. Then, D-tryptophan produced from N-acetyl-D-tryptophan was quantified by colorimetry according to the TNBS method. The same reaction as described above was conducted simultaneously using wild-type E.coli strain. Then, mutants with increased activity for producing D-tryptophan 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.

1. customcoli transformant libraries obtained in Example 11
2. addition10 μl of the culture medium solution was added to the reaction solution
3. distillationdistilled water in the wells of a microtiter plate
4. waitThe plate was incubated at 25° C. for 1 hour
5. stirringwhile being shaken