Enzymes penicillin

Class D enzymes (Mr of about 27,000) exhibit a high activity versus isoxazolyl penicillins, such as oxacillin and are referred to as the OXA-family. Surprisingly, the amino group of the SXXK lysine residues is carboxylated in the most active forms of the enzymes. Penicillins are generally better substrates than cephalosporins but mutations have been found which confer extended activity spectra to these enzymes. 

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). 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

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. 

Source of enzyme penicillin cillin cillin cillin cillin 6-APA sporin C loridine Ref. 

The penicillin antibiotics inhibit transpeptidase enzymes (penicillin-binding proteins (PBPs)) by acylation of the serinyl residue at their active site, which leads to cell wall lysis, since blocking PBPs circumvents proper murein membrane formation [3]. Several peptides and peptidomimetics containing the (3-lactam ring have been recently described as effective protease inhibitors and, consequently, as potential drugs for a wide range of diseases implicating proteases [5-8]. 

In a multiphase membrane reactor, the conversion of benzylpenicillin to 6-aminopenidllinic acid is performed. The type of microstructured reactor used is a fermentation reactor which contains the enzyme penicillin acylase immobilized on the wall of a hollow-fiber tube. The hollow-fiber tube extracts 6-aminopenicillinic acid at the same time selectively. Benzylpenicillin is converted at the outer wall of the hollow fiber into the desired product, which passes into the sweep stream inside the fiber where it can be purified, e.g. by ion exchange. The non-converted benzylpenicillin is recycled back through the reactor [84],... 

For the N-terminal deprotection of peptides, the enzyme penicillin G acylase from E. coli has been applied. This attacks phenylacetic acid (PhAc) amides and esters but does not hydrolyze peptide bonds [12-14,25]. The danger of a competitive cleavage of the peptide backbone at an undesired site, which always exists when proteases like trypsin and chymotrypsin are used, is overcome by using the acylase. The penicillin G acylase accepts a broad range of protected dipeptides (27) as substrates, and selectively liberates the N-terminal amino group under almost neutral conditions (pH 7-8, room temperature), leaving the peptide bonds as well as the C-terminal methyl-, allyl-, benzyl-, and tert-butyl ester unaffected (Fig. 8) [25a,bj. On the other hand, the phenylacetamide... 

Mode of action. Penicillins act by inhibiting the enzymes (Penicillin Binding Proteins, PBPs) involved in the crosslinking of the peptidoglycan layer of the cell wall which protects the bacterium from its environment incapable of withstanding... 

Two distinct types of enzymes (penicillin amidases) capable of removing the side chain attached to the amino position of 6-aminopeni-cillanic acid have been encountered in microorganisms 3 3, 55. 

APA is now produced by hydrolysing penicillin G or penicillin V with an enzyme (penicillin acylase) (Fig. 10.22) or by chemical methods (see later). These are more efficient procedures than fermentation. 

Development of a biocatalytic process for the resolution of (R)- and (S)-ethyl 3-amino-4-pentynoate isomers using enzyme penicillin G amidohydro-lase... 

Development of a Biocatalytic Process for the Resolution of (/ )- and (S)-Ethyl 3-amino-4-pentynoate Isomers Using Enzyme Penicillin C Amidohydrolase... 

The beta-amino acid, (S)-ethyl 3-amino-4-pentynoate, a chiral synthon, was an intermediate in the synthesis of an anti-platelet agent, xemilofiban. A biocatalytic process using the enzyme Penicillin G amidohydrolase was developed to resolve (R)-and (S)-enantiomers of ethyl 3-amino-4-pentynoate in enantiomerically pure form. The design of experimental approach used was one to optimize the critical reaction parameters to control the stereoselectivity of the enzyme Penicillin G amidohydrolase. 

As a part of ongoing efforts to synthesize a potent, orally active anti-platelet agent, xemilofiban 1 [1], development of an efficient chemoenzymatic process for 2, the chiral yS-amino acid ester synthon (Fig. 1) was proposed. The scheme emphasized the creation of the stereogenic center as the key step. In parallel with the enzymatic approach, chemical synthesis of the / -amino acid ester synthon emphasized formation of a chiral imine, nucleophilic addition of the Reformatsky reagent, and oxidative removal of the chiral auxiliary. This chapter describes a selective amida-tion/amide hydrolysis using the enzyme Penicillin G amidohydrolase from E. coli to synthesize (R)- and (S)-enantiomers of ethyl 3-amino-5-(trimethylsilyl)-4-pen-tynoate in an optically pure form. The design of the experimental approach was applied in order to optimize the critical reaction parameters to control the stereoselectivity of the enzyme Penicillin G amidohydrolase. 

The commercial supply is primarily derived from expressing the enzyme Penicillin G amidohydrolase (E.C. 3.5.1.11) ATCC 9637 in E. coli. Soluble enzyme was obtained from Calbiochem and Boehringer-Mannheim, and immobilized enzyme was obtained from Sigma, Rohm-Pharma and Boehringer-Mannheim. The recovery of the immobilized enzyme made it cost effective compared with the soluble enzyme. 

Figure 12.2-4. Enzymatic production of 6-APA and 7-ADCA. 6-APA is produced from penicillin C (or V) using the enzyme penicillin amidase. For the production of 7-ADCA, penicillin C is transformed chemically into phenylacetyl 7-ADCA. This transformation involves oxidation of penicillin C followed by a ring expansion...

Scheme 19.18 Production of the semisynthetic penicillins. This process takes advantage of large-scale fermentation to obtain penicillin G (61) from microbes in order to establish the requisite stereochemistry at positions 3, 5, and 6 as indicated on 6-APA. An isolated enzyme (Penicillin Acylase) is then used to cleave the phenylacetic acid group off of the 6-position amide in 61. The resulting 6-amino-penicillanic acid (6-APA) can then be readily acylated with any acid chloride under buffered conditions so as to prevent a build-up in acidity during the reaction. Table 19.8 lists some representative semi-synthetic penicillins.

In the first step, an enzymatic C-H bond cleavage occurs in the cysteine residue with formation of a C-N bond. From the intermediate 3, which is bound to the enzyme, penicillin is formed by a C-H bond cleavage in the valine residue and subsequently a C-S linkage (3 4) is formed. 

Basso A, De Martin L, Ebert C et al. (2003) Organically modified xerogels as novel tailor-made supports for covalent immobilisation of enzymes (penicillin G acylase). Tetrahed Lett 44 5889-... 

This reaction effectively acylates an OH group at the active site of the enzyme, and the acylated enzyme is inactive. By inactivating this enzyme, penicillin drugs are able to prevent bacteria from producing functional cell walls. Under these conditions, the bacteria are not able to reproduce, which allows the body s natural immune system to take control. 

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