反応 #1122664

反応方程式

α-ketoglutarate

sodium phosphate

sodium pyruvate

tryptophan

pyridoxal phosphate

MgCl2

→

monatin

反応条件

後処理

1. 1
   その他the following reaction conditions
2. 2
   その他Each 1 mL reaction
3. 3
   workup.ADDITIONadded as a cell
4. 4
   抽出free extract) and
5. 5
   濃縮concentrations (approximately 50 and 500 μg/mL) of aminotransferase (
6. 6
   workup.ADDITIONadded as a cell
7. 7
   抽出free extract)
8. 8
   その他to prepare the stock solutions
9. 9
   workup.ADDITIONto the addition of the enzymes
10. 10
    その他The reaction tubes
11. 11
    その他were incubated at 30° C.
12. 12
    その他Samples (0.01 mL) were withdrawn at 1, 2, and 4 h
13. 13
    workup.ADDITIONafter the addition of the enzymes
14. 14
    ろ過filtered

実験手順

The activity of the HEXaspC mutant proteins for the production of S,S-monatin was measured using the following reaction conditions: Each 1 mL reaction contained 50 mM TAPS, pH 8.2, 4 mM MgCl2, 3 mM sodium phosphate, pH 8.0, 200 mM sodium pyruvate (pH adjusted to 8), 5 mM α-ketoglutarate (pH adjusted to 8), 50 mM tryptophan, 0.05 mM pyridoxal 3-phosphate, 50 μg/mL ProA aldolase (added as a cell free extract) and varying concentrations (approximately 50 and 500 μg/mL) of aminotransferase (added as a cell free extract). Deaerated water was used to prepare the stock solutions and to adjust the volume of the reaction mixtures to 1.0 mL. The pyridoxal phosphate was added just prior to the addition of the enzymes. The reaction tubes were incubated at 30° C. with gentle shaking for 4 h. Samples (0.01 mL) were withdrawn at 1, 2, and 4 h after the addition of the enzymes, filtered, and analyzed by LC/MS/MS, as described in Example 1. Monatin production was normalized based on the amount of aminotransferase present in the reactions. Under the conditions of these assays, the HEXaspC and the HEXaspCT156A produced the most total monatin per mg of aminotransferase while the P9T/R122G protein produced the least, followed by HEXaspCW130F. The HEXaspCW130F and P9T/R122G enzymes showed the greatest stereoselectivity for S-MP (greater than 98% S,S-monatin), even when high enzyme concentrations were used (greater than 300 μg/mL). The percentage of S,S-monatin product decreased to less than 90% in the enzymatic reactions containing the P9T/T156A enzyme at high concentration. The other mutants showed a product stereoselectivity very similar to the original HEXaspC mutant (approximately 95% S,S-monatin). Analysis of the product of the reaction containing the HEXaspC enzyme using the FDAA derivitazation reagent described in Example 1 showed that the second stereoisomer formed is R,S-monatin.

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