Biomedical Engineering Reference
In-Depth Information
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Time of incubation (h)
Figure 5. Effect of incubation time in 50 % (v/v) ethyl acetate on the residual activity of de
Acacia
caven
CE, at 37° C.
In order to maximize the concentration of the unionized amine form of the phenyl
nucleophile, the peptide synthesis was performed at pH 8.5. In this respect, pH values above 9
might have been optimum. However, such conditions reduced the overall activity of the
enzyme (Figure 1). The chosen temperature of reaction (37° C) was based on the optimum
temperature of
Acacia caven
CE and the remarkable thermostability of the enzyme extract at
that temperature (Figure 2).
The peptide synthesis catalyzed by
Acacia caven
CE was carried out in the biphasic
medium of 0.1M Tris-HCl buffer pH 8.5 and ethyl acetate (50:50 ratio). In these conditions, a
clear decreasing of 60 % on the activity enzyme was observed after 24h at 37° C and 160 rpm
(Figure 5). Nevertheless, enzyme inactivation by the organic solvent did not influence on the
synthesis yield and high dipeptide yields were obtained before 1 h.
N-protected (N-benzyloxycarbonyl-L-amino acids (Phe, Ala, Gln)) acyl donors were
selected in base of the highest preferences showed by
Acacia caven
CE by those amino acids
derivatives.
Under such reaction conditions,
Acacia caven
CE was able to catalyze the synthesis of
different bioactive peptides: Z-Gln-Phe.OMe, Z-Phe-Phe-OMe (substrate for cathepsin A [36]
and Z-Ala-Phe.OMe (bitter dipeptide precursor with commercial interest for the food industry
[37]).
When Z-Ala and Phe.OMe were used as carboxylic and nucleophilic components in the
assayed biphasic medium,
Acacia caven
CE catalyzed the synthesis of the bitter dipeptide
precursor Z-Ala-Phe.OMe, and a maximum product conversion (Xp) of 50% was obtained
after 10 min of reaction. Xp parameter represents the amount of product obtained as a
function of the limitant reactive and it was defined as the relation between the dipeptide
concentration (mM) and the initial concentration (mM) of the limitant reactive (Phe.OMe).
