Penicillin-acylase

In two recent studies, Braiuca et al. applied GRID/PCA to the investigation of substrate selectivity of different forms of penicillin acylase (PA), an important enzyme in the 9-lactam antibiotics industry [12, 17]. Several microbiological sources of PA exist, the enzymes differing in selectivity, activity, or stability. The authors used GRID-MIFs to explain the differences in PA from different sources, E. coli (PA-EC), P. rettgeri (PA-PR), and A. faecalis (PA-AF). GRID/PCA was employed to focus on the important parts in the active site and to reduce the noise in the untreated MIFs. 

An important aspect of this analysis was that the authors decided to build up to four different sub-models for different probe types (e.g. donor, acceptor, hydro-phobic, and halogen probes) to circumvent the known problem of underestimating hydrophobic interactions in the GRID/PCA approach. 

The first study compared PA-EC with PA-AF [12]. The mutation of S67A in one part of the active site leads to weaker interactions with H-bonding probes in PA-AF. Several other amino acid differences between the enzymes translate into different interaction strengths or even structural differences of the protein backbone, which are reflected in the shape of the MIFs and the interaction energy maxima. Together with docking calculations of model substrates, the authors were able to explain the experimental selectivity profile and the enantioselectivity of the enzymes. 

The second publication analyzes the differences between PA-EC and PA-PR [17]. In the score plots of all sub-models the second component discriminates the enzymes, while the first component differentiates the probes, indicative of very similar enzymes. 

Indeed, the major difference is due to a mutation of Metl42 (PA-EC) into a Leu (PA-PR), leading to a smaller binding site in PA-PR, consistent with the observed substrate selectivity. 

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This amide, readily formed from an amine and the anhydride, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This depro-tection procedure works on peptides as well as on nonpeptide substrates. 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

Figure 6.11 Hydrolysis of penicillin G by penicillin acylase and p-lactamase.

Table 6.3 Some sources of penicillin acylases used for the large scale production of 6-APA.

The latter two points usually tip the balance in favour of using purified enzymes. Ideally the enzyme should be easy to isolate. The penicillin acylase from Bacillus megaterium is, for example, an extracelluar enzyme and can be readily absorbed into bentonite. 

In section 6.6.1, we described how enzymatic methods have come to dominate the production of the important intermediates used in the manufacture of semi-synthetic -lactams. In principle, the hydrolytic penicillin acylases may be used in the reverse direction to add acyl groups to 6-APA. For example, a two-step enzymatic process has been described for the preparation of ampiciilin (D-(-)-a-aminobenzylpenidllin structure shown in Figure 6.17). 

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

Stereoselective hydrolysis of racemic l-(//-phenylacetylamino) alkanephos-phonic acids performed in the presence of penicillin acylase under the kinetic resolution conditions gave both the unreacted substrates and the products - the corresponding 1-aminophosphonic acids in high yields and with full enantioselec-tivity. The unreacted A -acyl derivatives were hydrolysed chemically and in this way each enantiomer of the free acid was obtained (Scheme 5). ... 

Rasor and Tischer (1998) have brought out the advantages of enzyme immobilization. Examples of penicillin-G to 6-APA, hydrolysis of cephalospwrin C into 7-ACA, hydrolysis of isosorbide diacetate and hydrolysis of 5-(4-hydroxy phenyl) hydantom are cited. De Vroom (1998) has reported covalent attachment of penicillin acylase (EC 3.51.11) from E.Coli in a gelatine-based carrier to give a water insoluble catalyst assemblase which can be recycled many times, and is suitable for the production of semi-synthetic antibiotics in an aqueous environment. The enzyme can be applied both in a hydrolytic fashion and a synthetic fashion. 6-APA was produced from penicillin-G similarly, 7-ADCA was produced from desa acetoxycephalosporin G, a ring expansion product of penicillin G. 

Y. L. Lee, H. N. Chang (1990) High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor. Biotechnol. Bioeng., 36 330-337. 

Wu et al. [183] studied the reversible hydrolysis of penicillin G into 6-aminopeni-cillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor. E. coli cells containing penicillin acylase (the catalyst) were immobilized by entrapment into gelatine and further cross-linking with glutaraldehyde. The ad-... 

M. Cole, Hydrolysis of Penicillins and Related Compounds by the Cell-Bound Penicillin Acylase of Escherichia coli , Biochem. J. 1969, 115, 733-739. 

Penicillin acylases or amidohydrolases, which cleave the amide side chain of penicillin G, have been known for almost 50 years. " As one of the first enzymes to be developed for use at scale in the pharmaceutical industry, penicillin G acylase (PGA) has often been used as a model system for academic studies from molecular biology to biochemical engineering. Despite extensive screening, however, for decades there was no equivalent enzyme to generate 7-ACA by cleaving the polar D-a-aminoadipoyl side chain from cephalosporin C. 

Penicillin acylase catalyzes the hydrolysis of phenylacetamides and has been used in peptide synthesis for the cleavage of protecting groups [46—47]. In linker (40) developed by Flitsch et al. [41—42] (Scheme 10.8) the group -XR represents the alcohol or amine group of the target molecule. Hydrolysis of the phenylaceta-... 

Scheme 10.8 Loading and cleavage of a penicillin acylase scissile linker.

A two-step approach, involving repeated use of the same enzyme, has been reported for the resolution of rac-l-phenylethylamine 56a (Scheme 2.34). Penicillin acylase, from Alcaligenes faecalis, was initially used in aqueous medium with (R)-phenylglycine amide 67 as the acyl donor. Under these conditions, the enzyme catalyzed the enantioselective acylation of 56a at pH 10-11. The product amide 68 was insoluble, and was collected and re-exposed to the enzyme at pH below 7.5. This resulted in the cleavage of the phenylglycinyl substituent. Excellent conversions, E values and enantiomeric excesses were achieved [36]. 

Scheme 2.34 Dual use of penicillin acylase to generate (R)-l -phenylethylamine 56a.

This process has many benefits in the context of green chemistry it involves two enzymatic steps, in a one-pot procedure, in water as solvent at ambient temperature. It has one shortcoming, however-penicillin acylase generally works well only with amines containing an aromatic moiety and poor enantioselectivities are often observed with simple aliphatic amines. Hence, for the easy-on/easy-off resolution of aliphatic amines a hybrid form was developed in which a hpase [Candida antarctica hpase B (CALB)] was used for the acylation step and peniciUin acylase for the deacylahon step [22]. The structure of the acyl donor was also optimized to combine a high enanhoselectivity in the first step with facile deacylation in the second step. It was found that pyridyl-3-acetic acid esters gave optimum results (see Scheme 6.8). 

Penicillin acylase is an intracellular enzyme so that its isolation is relatively difficnlt... 

The market for the penicillin acylase is still quite moderate at ca. 8-10x10 in 1988. The armual use for the immobilized enzyme was estimated in 1993 at 1000 kg/a. 

Processes operate using immobilised penicillin V or G acylases derived from fungi such as Bovista plumbea, and such as E. coli respectively. Following the discovery ot penicillin acylases commercial processes were developed very rapidly. Productivities ot up to 2,000 kg of 6-APA/kg immobilized enzyme are obtained with operating lifetimes in excess of 10 h. 

These processes have operated successfully for 25 years and now result in over 10x10 sales of penicillin derivatives world-wide. In many cases penicillin acylases have been developed in-house by the user companies such as SmithKline Beecham. 

Martin, J., Slade, A., Aitken, A., Anche, R. and Virden, R. (1991) Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila. Biochem. J., 280, 659-662. 

Braggink, A., Roos, E.C. and De Vroom, E. (1998) Penicillin acylase in the industrial production of -lactam antibiotics. Organic Process Research Development, 2, 128-133. 

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