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Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.

The separation of drugs cephalexin, ampicillin and their biosynthetic precursors L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val was investigated by using capillary zone electrophoresis in acidic Tris-H3PO4 buffer at pH 2.3. With 50 mmol/L Tris-H3PO4 (pH 2.3) buffer, L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val were only partially separated whereas near baseline separation was achieved between cephalexin and ampicillin. The experiments showed that the addition of beta-type cyclodextrins (beta-CD) to the running buffer can influence the separation selectivity. When beta-CD was used as buffer additives at 12 mmol/L, the separation between L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val was much improved, however, the separation between cephalexin and ampicillin was impaired. When using 50 mmol/L Tris-H3PO4 containing 12 mmol/L DM-beta-CD (pH 2.3) buffer system, all compounds investigated were completely separated. This separation method is simple, fast and easy to operate. It involves only the conventional reagents with a much lower run cost than using HPLC. It is expected to be used in the synthetic process monitoring.