Penicillin-G-amidase

In a similar way, several cephalosporins have been hydrolyzed to 7-aminodeacetoxycephalosporanic acid (72), and nocardicin C to 6-aminonocardicinic acid (73). Penicillin G amidase from Pscherichia coli has been used in an efficient resolution of a racemic cis intermediate required for a preparation of the synthon required for synthesis of the antibiotic Loracarbef (74). The racemic intermediate (21) underwent selective acylation to yield the cis derivative (22) in 44% yield the product displayed a 97% enantiomeric excess (ee). 

Penicillin G acylase (PGA, EC 3.5.1.11, penicillin G amidase) catalyzes the hydrolysis of the phenylacetyl side chain of penicillin to give 6-aminopenicillanic acid. PGA accepts only phenylacetyl and structurally similar groups (phenoxyacetyl, 4-pyridylacetyl) in the acyl moiety of the substrates, whereas a wide range of structures are tolerated in the amine part [100]. A representative selection of amide substrates, which have been hydrolyzed in a highly selective fashion, is depicted in Figure 6.36. 

Patent literature reports on the analogous resolutions of phosphinotricin using, among others, penicillin G-acylase, penicillin G-amidase, subtilisin or microorganisms such as Enterobacter aerogenes, Klebsiella oxytoca, Corynebac-terium sp., Rhodococcus rubropertinctus and others7°... 

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

We also wanted to evaluate the disassembly of our dendritic system under physiological conditions. Thus, we synthesized a self-immolative AB6 dendron 32 with water-soluble tryptophan tail units and a phenylacetamide head as a trigger (Fig. 5.26) to evaluate disassembly in aqueous conditions. The phenylacetamide is selectively cleaved by the bacterial enzyme penicillin G amidase (PGA). The trigger was designed to disassemble through azaquinone methide rearrangement and cyclic dimethylurea elimination to release a phenol intermediate that will undergo six quinone methide elimination reactions to release the tryptophan tail units. 

FIGURE 5.37 Chemical structure of a molecular probe with UV-Vis and fluorescence outputs for penicillin G amidase activity. The phenylacetamide group (red) is a substrate for PGA. The reporter units, 4-nitrophenol and 6-aminoquinoline, provide a visible signal and a fluorescence signal, respectively, upon release. (See the color version of this figure in Color Plates section.)... 

Scheme 6.7 Easy-on/easy-off resolution of amines with penicillin G amidase.

Scheme 6.7). Penicillin G amidase from Mcaligenes faecalis, which is used in the manufacture of semisynthetic penicillins and cephalosporins, was used in both steps to afford a one-pot cascade process [21]. The acylation was performed in an aqueous medium at pH 10-11 and, after separation of the remaining amine enantiomer, the acylated amine was hydrolyzed with the same enzyme by lowering the pH to 7. 

Lactams, i.e., penicillins and cephalosporins, represent the most important class of antibiotics. Penicillins consist of a common core, 6-aminopenicillanic acid (6-APA) and different side chains. Penicillin G (pen G), with a phenyl acetate side chain, and penicillin V (pen V), with a phenoxyacetate side chain, are fermented from the fungus Penicillium chrysogenum all the others are produced from 6-APA, which nowadays is produced mostly from pen G via penicillin G amidase (pen G amidase, pen G acylase, PGA, E.C. 3.5.1.11 Figure 7.33). Cephalosporins feature 7-aminocephalosporanic acid (7-ACA) or its deacetyl-form, 7-aminodesacetyl-cephalosporanic add (7-ADCA) as their common core cephalosporin C (Ceph C) is obtained through fermentation, and all the others are derived from 7-A(D)CA. 

Recombinant penicillin G amidase 4-Hydroxy-D-phenylglycine Immobilized recombinant penicillin G amidase... 

The enzyme mixture of 20 ml containing immobilized recombinant penicillin G amidase as the enzyme, 10% hydroxyethyl ester of 4-hydroxy-D-phenylglycine, 4% cefprozil (amine source), and 8% enzyme (immobilized recombinant penicillin G amidase, equivalent to 32 IU/ml of enzyme) was made up without buffer. The above prepared ester solution (6.9 ml) was mixed with water (2 ml) and adjusted to pH 7.5 with 10 N NH4OH. Then the amine source (0.8 g) was added to it and the pH adjusted to 7.5 with 1 N NH4OH and the volume to 18.4 ml. Then the mixture was cooled to 5-15°C and solid enzyme (1.6 g 640 IU) was added to it. The pH was not maintained at 7.5 and fell about 0.6 units during the reaction. The reaction mixture was analyzed by HPLC on a C18 Reverse Phase column. The mobile phase was 10% acetonitrile/0.3% H3P04. The isomers of cefprozil appeared at 2.9 minutes (cis) and at 5.1 minutes (trans). The final product was obtained with a maximum yield of 92-96%. The whole experiment was completed in 25-50 min. 

Synthesis of cefprozil may be carried out at 15°C using Boehringer penicillin G amidase as the enzyme and hydroxyethyl ester of 4-hydroxy-D-phenylglycine. A maximum yield of about 95%. The experiment was completed in 35 minutes. 

Solvent extraction of penicillin from fermentation broths has been well documented in the literature. Penicillin G and penicillin V can be efficiently extracted with amyl acetate or butyl acetate at pH 2.5-3.0 and at 0° to 3°C.33 Schiigerl1 systematically reviewed solvent extraction of different forms of penicillin from fermentation broths. Figure 1 shows an integrated process for the extraction of penicillin G from clarified broth of Penicillium chryso-genurn fermentation.1 Penicillin G is converted to 6-amino penicillanic acid and phenylacetic acid at pH 8 in a 10 L Kiihni extractor by penicillin G-amidase immobilized in an emulsion liquid membrane. The 6-amino penicillanic acid is subsequently converted to ampicillin at pH 6 and the enzyme is recycled. 

In this way, penicillin G amidase was first coupled to dextran (see below) with the modified enzyme showing significantly increased thermostability, and this preparation was then immobilized on amino-activated silica gels [49]. 

In a different approach penicillin G amidase was coupled to Nylon 6, which was partially hydrolyzed and activated by conversion of the liberated amino groups with N-hydroxysuccinimide and dicyclohexylcarbodiimide [66]. Immobilization yields were fairly high, and preliminary data showed a good operational stability in a column reactor. 

Compared to glutardialdehyde mediated coupling, reactions with oxirane groups are slower. However, here again coupling times can have a considerable influence on stability. This is illustrated in Fig. 5 which shows the storage stability of penicillin G amidase immobilized on a polymethacrylate carrier in relation to the coupling time. 

Fig. 4. Operational stability of penicillin G amidase immobilized on epoxy carrier. Batch cycles were run in a 1.5-1 laboratory reactor at 10% penicillin G, 6 kU L 36 °C. pH was maintained at 8.0 by the addition of 2 N NH3. Initial splitting time was 65 min. The initial activity (relative base consumption per min at the beginning of each batch conversion) is plotted against the cycle number. PGA was immobilized on polymethacrylate with an epoxy density >2000 pmol g" (squares) or of 600 pmol g" (triangles)...

Fig. 5. Storage stability of penicillin G amidase immobilized on epoxy-activated polymethacrylate in relation to the immobilization time. The immobilization reaction was allowed to proceed for 17,41, and 65 h, respectively, and was terminated by the addition of ethanolamine, the catalysts were washed and the activity (5 mM potassium phosphate pH 8, 28 °C, 10% penicillin G, pH-stat 8.0) was determined immediately (t = 0), and after storage in a sealed vessel at 25 °C for 21 and 120 d...

Table 3. Effectiveness factors zj for immobilized penicillin G amidases" ...

Penicillin G amidase was immobilized on pre-fabricated carriers or insolubUized as crosslinked crystals. Eupergit-related value for R (mean particle radius of swelled carrier) was 80 pm [87]. V , (assuming maximum intrinsic activity per accessible catalyst volume, based on active enzyme molecules 1 unit=l pmol min at 28°C) was 90 and 170 U cm for Eupergit C and 250L, respectively [87]. D ff (effective diffusion coefficient) was taken from literature [87] or calculated as shown in the text. Km (intrinsic Michaelis constant) was uniformly taken as 13 mM [87] and S = 268 mM corresponds to the substrate concentration at catalyst surface of a 10 % solution of penicillin G salt, q was calculated according to Atkinson et al. for spherical particles [85]. For simplification, surface and pore related indices have been omitted. 

To demonstrate limitations by porous diffusion, available data for penicillin G amidase on various carrier materials were collected to calculate and compare efficiency coefficients by insertion into Eqs. (3-5) (Table 3). 

Assuming that the above values are representative for carrier-fixed enzymes, the effectiveness factors for penicillin G amidase from E. coli are presented in Table 3. 

Carrier-fixed penicillin G amidase in the multi-ton hydrolysis of penicillin G is a useful example to illustrate enzyme consumption. The enzyme is applied in stirred tanks with sieve plates at the bottom to retain the enzyme particles when the product solution is drained off. The pH-value is kept constant by controlled feed of ammonia solution. Fresh substrate solution is refilled about a thousand times or more. The consumption of enzyme in such a process is below 10 mg kg (0.2 ku kg ) of isolated 6-APA when the enzyme activity is determined with penicillin G solutions at 28° C and pH 8.0. Under identical conditions the consumption of soluble enzyme for each tank filling would be beyond all reasonable cost. 

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