USE OF AQUEOUS 2-PHASE SYSTEMS FOR IN-SITU EXTRACTION OF WATER-SOLUBLE ANTIBIOTICS DURING THEIR SYNTHESIS BY ENZYMES IMMOBILIZED ON POROUS SUPPORTS

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coil (PGA) have been greatly in creased by continuous extraction of water soluble products (cephalexin ) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reac tion is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilib rated biocatalyst is mixed with the second phase (where the reaction p roduct will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Part ition coefficients (K) of cephalexin in very different aqueous two-pha se systems were firstly evaluated. High K values were obtained under d rastic conditions. The best K value for cephalexin (23) was found in 1 00% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl e ster and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In th is reaction system, the immobilized enzyme remains in the ammonium sul fate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synth etic yields of a similar process carried out in monophasic systems wer e much lower (55%) because of a rapid enzymatic hydrolysis of cephalex in. (C) 1998 John Wiley % Sons, Inc.