反应详情

The recombinant E. coli obtained in Example 14 was examined for resistance to (S)—N-benzyl-3-aminopyrrolidinone. A cell-free extract of each multiply-mutated strain was obtained using the same process as in Example 13. Subsequently, 900 μL of a 0.1 M potassium phosphate buffer (pH 6.3) containing 0.83% of (S)—N-benzyl-3-aminopyrrolidinone was added to 100 μL of the cell-free extract and incubated at 35° C. The mixture was sampled in an amount of 100 μL at reaction times 0 and 2.5 hours, respectively, and each sample was diluted 200 times with an aqueous 0.1 M potassium phosphate solution (pH 7.5). Subsequently, 200 μL of each dilution was added to 800 μL of a substrate solution having the composition shown below. After the mixture was allowed to react at 30° C. for 1 hour, 50 μL of 6 N hydrochloric acid was added to stop the reaction. The concentration of the produced acetophenone was measured using HPLC under the same conditions as in Example 4. Table 15 shows the remaining activity at reaction time 2.5 hours, which was determined relative to the activity at reaction time 0 normalized as 100%. As a result, the mutated enzymes shown in Table 15 had (S)-7-methoxy-2-aminotetralin resistance higher than that of the wild-type enzyme.

1. customcoli obtained in Example 14
2. extractionA cell-free extract of each multiply-mutated strain
3. customwas obtained
4. additionwas added to 100 μL of the
5. extractioncell-free extract
6. customincubated at 35° C
7. aliquotThe mixture was sampled in an amount of 100 μL at reaction times 0 and 2.5 hours
8. additionrespectively, and each sample was diluted 200 times with an aqueous 0.1 M potassium phosphate solution (pH 7.5)
9. additionSubsequently, 200 μL of each dilution was added to 800 μL of a substrate solution
10. customto react at 30° C. for 1 hour
11. customthe reaction
12. customTable 15 shows the remaining activity at reaction time 2.5 hours, which
13. customwas determined relative to the activity at reaction time 0 normalized as 100%