Amidases penicillin amidase

APA may be either obtained directly from special Penicillium strains or by hydrolysis of penicillin Q with the aid of amidase enzymes. A major problem in the synthesis of different amides from 6-APA is the acid- and base-sensitivity of its -lactam ring which is usually very unstable outside of the pH range from 3 to 6. One synthesis of ampidllin applies the condensation of 6-APA with a mixed anhydride of N-protected phenylglydne. Catalytic hydrogenation removes the N-protecting group. Yields are low (2 30%) (without scheme). 

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). 

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. ... 

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. 

A seeond method of producing 6-APA came with the diseovery that certain mieroorganisms produee enzymes, penicillin amidases (acylases), which catalyse the removal of the side ehain fiom benzylpenicillin (Fig. 5. IB). 

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°... 

If one of the species is anionic and we need to transport it to the organic phase, then a phase-transfer catalyst may be employed. Consider the example of benzyl penicillin where the reaction between phenyl acetic acid and the penicillin carboxylate ion, with penicillin amidase as a catalyst, is relevant, and which at pH 4.5 - 5.0 is shifted in the desired direction. Here a catalyst like tetrabutylammonium halide works, and with chloroform as a solvent 60% yield can be realized in contrast to a yield of only 5 - 10 % in water. 

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.)... 

Like with primary amides (see Sect. 4.2.1), bacterial amidases can be useful for the transformation of secondary amides in drug synthesis. Bacterial amidases have been extensively studied in the presence of penicillins and other [i-lactam antibiotics, for which two hydrolysis reactions are possible. One of these is carried out by enzymes known as penicillinases or /3-lactamases that open the /3-lactam ring this aspect will be discussed in Chapt. 5. The second type of hydrolysis involves cleavage of the side-chain amide bond (4.47 to 4.48) and is carried out by an enzyme called penicillinacylase (penicillin amidohydrolase, EC 3.5.1.11). Both types of hydrolysis inactivate the antibiotic [29-31],... 

K. Balasingham, D. Warburton, P. Dunhill, M. D. Lilly, The Isolation and Kinetics of Penicillin Amidase from Escherichia coli , Biochem. Biophys. Acta 1972, 276, 250-256. 

Matsumoto, K., Production of 6-APA, 7-ACA and 7-ADCA hy immobilised penicillin and cephalosporin amidases. Bioproc. Technol. 1993,16, 67-88. 

An exo-linker according to Fig. 10.1 must contain an enzyme labile group R, which is recognized and attacked by the biocatalyst Possible combinations could be phenylacetamide/penicillin amidase, ester/esterase, monosaccharide/glycosid-ase, phosphate/phosphatase, sulfate/sulfatase and peptides/peptidases [41]. The following systems have been worked out (Tab. 10.2). 

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. 

The microbial sources of penicillin amidases/acylases required for side-chain removal were found and were quickly commercialised as whole-cell biocatalysts. 

Genetic engineering techniques to improve penicillin amidase yields during fermentation are now employed thereby reducing biocatalyst process costs. 

The penicillin amidase reaction generates an acid product and so the pH control of... 

Two other immobilized enzymes have reached large scale industrial application penicillin amidase and lipase. 

Penicillin amidase is used industrially to produce 6-aminopenicillanic acid (6-APA) from penicillin G or V (see section 4.5). Acid is produced during the process and this will inactivate the enzyme. One way of overcoming this problem is by using a fixed bed reactor with immobilized enzyme. The substrate is pumped very rapidly... 

Forney, L.J. and Wong, B.C. (1989) Alteration of the catalytic efficiency of penicillin amidase from. Escherichia coli. Appl Environ. Microb., 55, 2556-2560. 

This may be illustrated by the following process, catalyzed by penicillin amidases (EC 3.5.1.1 1) from various sources... 

The optimum yield of a condensation product is obtained at the pH where Ka has a maximum. For peptide synthesis with serine proteases this coincides with the pH where the enzyme kinetic properties have their maxima. For the synthesis of penicillins with penicillin amidase, or esters with serine proteases or esterases, the pH of maximum product yield is much lower than the pH optimum of the enzymes. For penicillin amidase the pH stability is also markedly reduced at pH 4-5. Thus, in these cases, thermodynamically controlled processes for the synthesis of the condensation products are not favorable. When these enzymes are used as catalysts in thermodynamically controlled hydrolysis reactions an increase in pH increases the product yield. Penicilhn hydrolysis is generally carried out at pH about 8.0, where the enzyme has its optimum. At this pH the equiUbrium yield of hydrolysis product is about 97%. It could be further increased by increasing the pH. Due to the limited stability of the enzyme and the product 6-aminopenicillanic acid at pH>8, a higher pH is not used in the biotechnological process. 

Core structures of four B-lactam antibiotic families. The ring marked in each structure is the -lactam ring. The penicillins are susceptible to bacterial metabolism and inactivation by amidases and lactamases at the points shown. Note that the carbapenems have a different stereochemical configuration in the lactam ring that apparently imparts resistance to lactamases. Substituents for the penicillin and cephalosporin families are shown in Figures 43-2 and 43-6, respectively. 

---