Abstract: Two commercial proteases from Bacillus
licheniformis (Alcalase 2.4 L FG and Alcalase 2.5 L, Type DX) were
screened for the production of Z-Ala-Phe-NH2 in batch reaction.
Alcalase 2.4 L FG was the most efficient enzyme for the C-terminal
amidation of Z-Ala-Phe-OMe using ammonium carbamate as
ammonium source. Immobilization of protease has been achieved by
the sol-gel method, using dimethyldimethoxysilane (DMDMOS) and
tetramethoxysilane (TMOS) as precursors (unpublished results). In
batch production, about 95% of Z-Ala-Phe-NH2 was obtained at
30°C after 24 hours of incubation. Reproducibility of different
batches of commercial Alcalase 2.4 L FG preparations was also
investigated by evaluating the amidation activity and the entrapment
yields in the case of immobilization. A packed-bed reactor (0.68 cm
ID, 15.0 cm long) was operated successfully for the continuous
synthesis of peptide amides. The immobilized enzyme retained the
initial activity over 10 cycles of repeated use in continuous reactor at
ambient temperature. At 0.75 mL/min flow rate of the substrate
mixture, the total conversion of Z-Ala-Phe-OMe was achieved after 5
hours of substrate recycling. The product contained about 90%
peptide amide and 10% hydrolysis byproduct.