10.1002/cctc.201902194
ChemCatChem
amounts of CPA. The error bars represent the standard deviation of triplicate
experiments.
We suspected substrate inhibition to account for this and
therefore determined the CiVCPO activity in the presence of
different concentrations of glutamate (Figure 5). Very much to our
surprise, increasing glutamate concentrations showed limited
influence on the activity of CiVCPO; even in the presence of 500
mM glutamate, its activity in the MCD assay was reduced by only
23%.
Lastly, the oxidative decarboxylation of Glu by CiVCPO was
performed at semi-preparative scale. From a 200 mL reaction
scale (100 mM Glu), 0.827 g CPA (42% isolated yield, 96% pure)
was obtained after 5 h reaction with 100 nM CiVCPO. CPA was
isolated by extraction in organic solvents, however, the extraction
efficiency was low (see Experimental section). Based on the
isolated yield, 420000 turnovers were performed which is less
than in the small scale (Figure 4), however, CPA remained in the
aqueous phase even after the second extraction. The isolated
yield is in agreement with previously reported chemical reaction
with NaOCl/NaBr (43%),[11] but higher selectivity towards the
nitrile was obtained by using CiVCPO. Derivatisation to the
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corresponding ester or amide would certainly increase the
[12]
efficiency of the extraction as demonstrated previously.
Also
continuous liquid-liquid extraction appears to be a promising
method to increase the isolated yield. It is worth to mention that
the semi-preparative reaction was performed without additional
buffer (therefore less waste) and instead the substrate, sodium
glutamate, was used as a buffer (where the pH was adjusted to
pH 5.6 with H2SO4).
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100
200
300
400
500
[NaGlu] / mM
Figure 5. Influence of sodium glutamate (NaGlu) on the activity of CiVCPO.
Assay conditions: [MCD]= 50 µM, [H2O2]= 1 mM, [NaBr]= 0.5 mM, [Na3VO4]=
100 µM, 50 mM sodium citrate buffer (pH 5), T= 25 oC, 290 nm. The reaction
was started with addition of 0.8 nM CiVCPO that was pre-incubated for 5 min
with different amounts of NaGlu. The error bars represent the standard deviation
of triplicate experiments.
In conclusion, we demonstrate that the chemoenzymatic
oxidative decarboxylation of glutamate is indeed a possible
alternative to the established chemical and the new catalytic
methods. The product inhibition and the isolation of the product
are currently the main bottlenecks of this reaction which could be
solved by selective in situ solid phase extraction or by using a
packed bed reactor with immobilised VCPO. Product isolation
could be circumvented by direct conversion of CPA to a more
hydrophobic product. Furthermore, this preparative scale opens
the route towards the oxidative decarboxylation of other amino
acids with different side chain functionalities and their
corresponding nitriles.
Next, the possibility of CiVCPO inhibition by the product, 3-
cyanopropanoic acid, was investigated (Figure 6). With increasing
CPA concentration, the observed activity of CiVCPO decreased.
In the presence of 75 mM CPA the enzyme activity was reduced
by 50%, whereas in the presence of 200 mM the enzyme lost
almost completely its activity in the MCD assay. It can be
concluded that CPA, the product of oxidative decarboxylation,
significantly inhibits CiVCPO. Possibly, CPA coordinates to the
prosthetic vanadate thereby preventing the coordination of H2O2
to initiate the catalytic cycle but further studies will be necessary
to fully elucidate the inhibitory mechanism.
Experimental Section
Enzyme preparation. Vanadium chloroperoxidase from Curvularia
inaequalis (CiVCPO) was obtained from heterologous expression in
recombinant Escherichia coli and purified by heat treatment (see
Supplementary information).
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Enzymatic reaction conditions. In a 4 mL gals vials a solution (2 mL
starting volume) containing 0.5 mM NaBr, 55 nM CiVCPO, different
concentration of glutamic acid or sodium glutamate monohydrate in 20 mM
sodium citrate buffer (pH 5.6) was prepared. The reaction was started by
addition of H2O2, which was added with a continuous flow rate (see
captions of figures) at room temperature (about 22°C). The reaction was
quenched by adding Na2S2O3. For each time point a separate reaction vial
was prepared. The conversion of Glu and formation of CPA was analysed
by two different HPLC methods (see Supplementary information).
Enzyme activity assay. To assess CiVCPO activity, a standardised assay
reported previously was used.[13] In short: in a disposable UV plastic
cuvette a solution (1 mL) containing 50 µM monochlorodimedone (MCD),
1 mM H2O2, 0.5 mM NaBr, 100 µM Na3VO4 in 50 mM sodium citrate (pH
5.6) was prepared. The absorbance of MCD solution was followed at 290
nm, 25°C. The reaction was started with the addition of CiVCPO. The
enzyme activity was calculated using a molar extinction coefficient for
MCD of 20 (mM·cm)-1. For the inhibition tests, the enzyme was incubated
0
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50
75
100
200
[CPA] / mM
Figure 6. Inhibition of CiVCPO by 3-cyanopropanoic acid (CPA). Assay
conditions: [MCD]= 50 µM, [H2O2]= 1 mM, [NaBr]= 0.5 mM, [Na3VO4]= 100 µM,
50 mM sodium citrate buffer (pH 5), T= 25 oC, 290 nm. The reaction was started
with addition of 0.8 nM CiVCPO that was pre-incubated for 5 min with different
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