Penicillinacylase.

Vol. 30, No. 4 Printed in U.S.A. BACTERIOLOGICAL REVIEWS, Dec., 1966 Copyright © 1966 American Society for Microbiology Penicillinacylase J. M. T. HAMILTON-MILLER Departmenit of Bacteriology, Guy's Hospital Medical School, London, England INTRODUCTION ................................................................ HISTORICAL BACKGROUND. .................................................... PROPERTIES OF ACYLASES. ..................................................... Type I Acylases ("Fungal" Type).............................................. Type II Acylases ("Bacterial" Type)........................................... Synthetic Ability of Acylases................................................ ROLE OF ACYLASES IN MICROBIAL ECONOMY ................................. QUANTITATIVE ASPECTs. ....................................................... COEXISTENCE OF ACYLASE AND IS-LACTAMASE (PENICILLINASE) .................. ASSAY METHODS.............................................................. Biological Methods........................................................ Butyl Acetate Extraction Technique.......................................... Use of Radioactive Substrate................................................ Detection of X After Treating Reaction Mixture with Penicillinase.................. Liberation of Ammonia or Amino Groups from Substrates Other than Benzylpenicillin Direct Estimation of 6APA.................................................. PENICILLINAMIDASE............................................................ RETROSPECT AND PROSPECT.................................................... LITERATURE CITED............................................................ INTRODUCTION The penicillin molecule is susceptible to hydrolytic cleavage not only at the,-lactam bond (the process which is carried out by the enzyme penicillinase, or by treatment with dilute alkali), but also at the peptide linkage (see Fig. 1) by which the side chain (R group) of the penicillin (I) is joined to the "nucleus," 6-aminopenicillanic acid (6APA, II). The latter reaction (equation 1, Fig. 2) can be brought about enzymatically; the enzyme responsible has been given several different names (13), including "penicillin amidase," "penamidase," "benzylpenicillin acylase," "penicillin splitting and synthesizing enzyme," "acyltransferase," and, perhaps the least satisfactory name of all, the official (Enzyme Commission) nomenclature "benzylpenicillin amidohydrolase" (EC 3.5.1.11). This last name is unsatisfactory for two main reasons: (i) it is too similar to the name for penicillin ,B-lactamase (EC 3.5.2.6), which is "penicillin amidohydrolase," and (ii) it suggests that benzylpenicillin is the best, or even the only, substrate, a fact which, as will be shown below, is totally erroneous. The enzyme will be referred to here simply as "acylase." Acylase is an enzyme of great commercial importance, as it is because of its existence that 6APA is available in amounts large enough to enable the large-scale production of semisynthetic penicillins to be an economic proposition (19). 761 761 761 762 762 763 764 765 766 766 766 767 767 767 767 767 768 768 769 770 HISTORICAL BACKGROUND Acylase activity was first described by workers in Japan (48), in Penicillium chrysogenum Q 176; they reported that the mycelia of this strain were capable of converting benzylpenicillin into phenylacetic acid and a compound that they called "penicin." Further details of this reaction were subsequently given by Murao (42), who showed that penicin was degraded by penicillinase to "penicic acid," which was strongly ninhydrinpositive; the same author reported that "penicin" had a melting point of 157 to 160 C. Meanwhile, it had been suggested (34) that penicin was in fact the penicillin nucleus 6APA. The matter rested here for some years until the report by Batchelor and his colleagues (4) at the Beecham Research Laboratories to the effect that 6APA had been detected in a fermentation brew of P. chrysogenum W51.20 to which no side chain precursors had been added; the identity of this 6APA was proved by these workers by virtue of the fact that, on phenylacetylation, benzylpenicillin was produced. Purification and isolation of 6APA from the brew were described in great detail in a subsequent paper (6). The melting point of 6APA was given by Batchelor et al. (4) as 208 to 209 C (decomposition), which does not agree with the figure given by Murao (42). There was further doubt in many minds as to whether the "penicin" described by Murao and his colleagues was indeed 6APA because, 762 HAMILTON-MILLER (1). ~~~ ~ CH R * CO 4 NH. CH_C/ \C-,CH3 V\CH3 I, I CO-N -- CH* COOH (2) Side-chain 6-amino penicillanic acid residue FIG. 1. Linkage of penicillin side chain to 6-aminopenicillanic acid residue. (I) Site of action ofpenicillin acylase (splitting the amide linkage). (2) Site of action of penicillinase (giving rise to penicilloic acid). I CONH CH -'CH C (CH3)2 NH2 CH -CH 2 =RCOOH C (CH3)2 + CO- N- CH COOSi CO- N- CH COOH FIG. 2. Equation 1. Side chain (R group) of the penicillin (I) is irreversibly removed from the "nutcleus" 6-aminopenicillanic acid (II). although acylase activity had been demonstrated independently in 1960 by several groups of workers (11, 30, 36, 46) in various different microbial species (see subsequent sections), no one had succeeded in repeating the experiments performed by the Japanese workers; i.e., despite detailed searches, no acylase activity could be found in P. chrysogenum Q 176. However, the Japanese workers were vindicated when, in 1961, both Erickson and Bennett (22) and Murao and Kishida (43) succeeded in demonstrating that this strain is capable of hydrolyzing benzylpenicillin to 6APA. Erickson and Bennett (22) also suggested reasons for the failure of previous workers to repeat the original work. PROPERTIES OF ACYLAsES Acylases differ, broadly speaking, in their properties according to whether they come from bacteria, or from yeasts, molds, or fungi. The distinction is not clear-cut; thus, to avoid confusion and contradictions, the properties of the various acylases will be subsumed under type I and type II, according to the nomenclature of Claridge, Luttinger, and Lein (12). Type I Acylases ("Fungal" Type) The chief characteristic of type I acylases is that they hydrolyze phenoxymethylpenicillin much more rapidly than benzylpenicillin. The enzyme has been described in many species of actinomycetes, filamentous fungi, and yeasts, the following genera having been reported to exhibit acylase activity: Alternatia, Aspergillus, Botrytis, Cephalosporium, Cryptococcus, Emericellopsis, Epicoccum, Epidermophyton, Fusarium, B BACTERIOL. REV. Mucor, Penicillium, Phoma, Trichoderma, Trichophyton, and Trichosporon (7, 10, 12, 14, 15, 46, 48, 55, 58). The enzyme of Streptomyces lavendulae BRL 198 has been studied in detail by the Beecham team (7,46; U.S. Patent 3,014,845 (...truncated)