ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Effect of pH in the synthesis of ampicillin by penicillin acylase Sonia Ospina,* Eduardo Barzaua,’ Octavia T. Ramirez,* and Agustin Lbpez-Munguia* zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA *Departamento de Farmacia, Instituto de Biotecnologia, Universidad National de Colombia, Santa@ de Bogotd, Colombia tFacultad de Quimica, Universidad National Autcinoma de Mkxico, Cd. ~n~versitaria, M&co ~Ins~ituto de Biote~nolog~a, Univers~dad National Aut~noma de Mexico, ~ue~ava~a Morelos, M&ico Recombinant Escherichia coli cells with high penicillin acylase (PA) activity were immobilized by gel entrapment with agar. This biocatalyst was used to study the effect of pH on the synthesis of ampicillin from phenylglycine methylester (PGME) and 6-aminopenicillanic acid (GAPA). The parallel hydrolysis reactions of PGME and ampicillin were also studied. A selective inhibition of the hydrolysis of the ester was possible by controlling the pH at 6.0. At such conditions, and using 6-APA solutions ranging from W-200 mM, a 75% conversion to ampicillin was obtained. This yield was higher than obtained with other strategies. The reaction kinetics was described by a second-order model for ampicillin synthesis with experimentally determined Michaelis-Menten constants of 27 and 2.5 rnMfor 6-APA and PGME, respectively. In addition, ampicillin and PGME were hydrolyzed by the enz_yme following Michaelis-Menten kinetics with K, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML values of 40.5 and 30 mM, respectively. A good correlation was found between experimental results of synthesis reactions and rhe kinetic model derived from initial rate experiments with on& slight deviations at high substrate concentrations. This is the~rst report where the specific effect of pH on the synthesis of ampicillin was studied in detail. It is shown that, by controlling the pH, it is possible to inhibit the lateral undesirable reactions increasing the yield of the main reaction. Keywords: Penicillin amidase; pH; ampicillin synthesis Introduction Ampicillin is one of the most widely used p-iactam anti- biotics with an annual production of 5,600 ton year-‘.’ It has the advantage of a high water solubility, a high absorp- tion rate when orally administered and high stability at acidic pH.* It is also a broad spectrum antibiotic particularly when combined with clavuianic acid, an inhibitor of the @lactamases produced by ~croorganisms as a resistance m~hanism.3 Ampiciliin is produced by chemical synthesis in a complex process requiring protection of the a-amino group of phenylglycine (PG), the use of highly reactive derivatives of PG (phenylglycine chloride hydrochloride which is highly unstable), very low temperature (-30”(Z), Address reprint requests to Dr. Agustin Lbpez-Mungoia, Instituto de Bio- tecnologia, UNAM, Apartado Postal 510-3, Cuemavaca, Morelos, 62271, MCxico Received 9 November 1995; revised 6 February 1996; accepted 8 February 1996 anhydrous conditions, and the use of highly toxic com- pounds (pyridine, dimethylaniline, and dichloromethane).4*s Accordingly, an enzymatic process can be an attractive al- ternative for replacing the chemical synthesis process. Although the enzymatic synthesis of ampicillin has been reported since 1969,6 among other factors, the low yields and concentrations of ampicillin obtained to date have pre- vented the enzymatic processes from reaching a commercial stage. Enzymatic synthesis of ~picillin has been attained with penicillin acylase [PA; EC 3.5.1.11; also known as penicillin amidase (PA)], penicillin amidohydrolase, and penicillin acyltransferase.“-8 From the various existing penicillin acylases (penicillin V, penicillin G, and ampicillin amidase), penicillin G amidase is industrially used for the hydrolysis of penicillin G.9 This acylase is produced from various microorganisms but mainly ~sc~eric~~a coli’“*’ ’ and Bacillus megaterium. 12,13 Although such an enzyme is used as a hydrolase for the production of &-aminopenicil- lanic acid (BAPA) and phenylacetic acid (PAA), it can also catalyze the reverse reaction if low pH values are main- Enzyme and Microbial Technology 19:462-469, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0141-0229/96/$15.00 PII SO141-0229(96)00032-4