Biomedical Engineering Reference
In-Depth Information
Enzymatic Synthesis Reaction Conditions
The enzymatic synthesis was carried out in a mixture of Tris-HCl buffer (0.1M, pH 8.5)
and ethyl acetate in 50:50 ratios. The condensation reaction was initiated by mixing the
aqueous phase containing the enzyme (0.11 IU/ml), 4mM Phe.OMe, 20mM 2-
mercaptoethanol as activator and 4mM TEA (since the amino component is commercially
available as hydrochloride derivative), with the organic phase containing 4mM carboxylic
component (Z-Ala, Z-Phe or Z-Gln). The reaction was conducted at 37º C in stopper flask
under magnetic stirring at 160 rpm during 24 h at pH 8.5. At time intervals, aliquots were
taken and analyzed by HPLC-MS. Simultaneously, blanks with identical composition but
without the enzyme and with only the enzyme in the organic system, were carried out.
Analytical Control of Enzymatic Synthesis
RP-HPLC
The enzymatic synthesis was followed by RP-HPLC. Analyses were performed on a
Gilson System (Model 712) equipped with a C-18 Luna (5 ￿), 250mm x 4.60mm column
(Varian). For all the experiences, the volume of injection was of 20 μL, the speed of flow was
0.8 ml/min and the eluted material was monitored spectrophotometrically at 254 nm and 25º
C. The mobile phase consisted of 50% acetonitrile and pH was adjusted to 3 by the aggregate
of 0.1% trifluoroacetic acid (TFA). The quantification of products and substrates was carried
out using the corresponding patterns.
HPLC-MS
Reaction products were identified by HPLC-MS. Analyses were performed on VG-
Quattro (Micromass Instruments S.A.), with a C-18 Nucleosil (120-5) (5 ￿m), 250mm ×
40mm column (Scharlan). The technique of electrospray was used with positive ion reading
(100-1000 uma). Nebulizer gas: N 2 (flow: 10 l/h). Drying gas: N 2 (flow: 450 l/h).
Temperature of the source: 80º C. Voltage of the capillary: 3.5 kV. Voltage focus: 55V. Flow:
15 (ml/min). Elution: solvent A, H 2 O containing 1% formic acid and solvent B, CH 3 CN; A:B
= 50:50. Volume of injection: 10-20 ￿l.
Results and Discussion
In the present study, the performance of a proteolytic crude extract (CE) of Acacia caven
(Mol.) Molina was investigated as both, -additive of laundry detergents formulations and -
catalyst for the peptide synthesis in aqueous-organic media.
Initially, a partial characterization of Acacia caven CE was carried out and it showed
maxima proteolytic activities (3 IU/mg of protein) at pH 8 and 40° C (Figure 1 and 2a).
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