Penicillin amidases

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. ... 

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). 

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. 

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. 

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). 

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. 

Similar results were also found for penicillin amidase-catalyzed transesterification of phenoxyacetate methyl ester with 1-propanol in hexane (Scheme 3.5) [46]. The initial rates of various penicillin amidase preparations containing acetate or chloride salts and their 1 1 binary mixtures were measured, which resulted in the identification of formulations with various degrees of activation. For example, the... 

Scheme 3.5 Transesterification of phenoxyacetate methyl ester with 1-PrOH in hexane by penicillin amidase.

Figure 3.10 Correlation between the observed reactivity of penicillin amidase formulations and the difference in the Jones-Dole coefficients of the kosmotropic anion and the chaotropic cation for single salt and binary salt mixtures [46].

Table 12.6 summarizes results obtained in the synthesis of antibiotics in the presence of undissolved substrates catalyzed by penicillin amidase, and... 

Recently, the kinetically controlled synthesis of cephalexin with penicillin amidase was studied at very high concentrations of substrates to the limit of... 

AL Margolin, VK Svedas, IV Berezin. Substrate specificity of penicillin amidase from coli. Biochim Biophys Acta 616 283-289, 1980. 

Semi-Synthetic Antibiotics. In 1959, Batchelor and coworkers in the Beecham Research Laboratories in England discovered that the penicillin nucleus, 6-aminopenicil-lanic acid (6-APA), accumulated during fermentation when side chain precursors were omitted. This 6-APA could be used for the chemical synthesis of entirely new types of penicillin by coupling with new side chains. Shortly thereafter, several sources of penicillin amidase were found that would cleave the phenylacetyl side chain from penicillin G, thus producing a more economical source of 6-APA. A vast number of synthetic penicillins have been generated, and a few have achieved clinical importance. Several objectives were sought ... 

Immobilized forms of penicillin amidases and acylases have replaced whole-cell biocatalysts for the production of 6-APA and 7-ACA as they can be reused many times, in some cases for over 1000 cycles. Another major advantage is the purity of the enzyme, lacking the /3-lactamase contaminants often present in whole cells. The productivity of these biocatalysts exceeds 2000 kg prod-uct/kg catalyst. A typical process for the production of 6-APA employs immobilized penicillin G acylase covalently attached to a macroporous resin. The process can be run in either batch or continuous modes. The pH of the reaction must be maintained at a value between 7.5 and 8 and requires continuous adjustment to compensate for the drop caused by the phenylacetic acid generated during the course of the reaction. Recycle reactors have been used, as they allow both pH control and the use of packed bed reactors containing the immobilized catalyst. The enzymatic process is cheaper, although not... 

Pressure activation and inactivation is investigated for several enzymes like penicillin amidase (E.coli), glutamate dehydrogenase (P. woesei) and lipase (Rhizopus arrhizus) in the pressure range between 1 bar and 4000 bar. In dependance of pressure and temperature the enzymes are acivated or inactivated and hence their enantioselectiviy can be directed. The activation of the enzymes corresponds to a decrease in the KM value which results in a higher substrate affinity. ... 

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