Acylase cephalosporin

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. 

Cephalosporins are a class of antibiotic produced via the intermediate 7-aminocephalos-poranic acid (7-ACA), or 7-aminodesacetoxycephalosporanic acid (7-ADCA). Directed evolution has been used to improve the activity of cephalosporin acylases to produce these intermediates from adipyl-7-ACA or cephalosporin C [68]. Using site-directed saturation mutagenesis and a selection system whereby the E. coli host is dependent on leucine liberated from derivatives of the cephalosporin side-chains, a mutant was found that increased the catalytic efficiency toward adipyl-7-ADCA by 36-fold. 

Otten, L.G., Sio, C.F., Reis, C.R. et al. (2007) A highly active adipyl-cephalosporin acylase obtained via rational randomization. FEBS Journal, 274, 5600-5610. 

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. 

Matsuda, A., Matsuyama, K., Yamamoto, K., Ichikawa, S. and Komatsu, K.-I., Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. J. BacterioL, 1987,169, 5815-5820. 

ACA cephalosporin C D-amino acid oxidase + glutaryl acylase... 

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. 

ECB deacylase is an 81-83-kDa heterodimer consisting of 63- and 18-20-kDa subunits. Penicillin G acylase from Escherichia coli is an 87-kDa heterodimer with 65- and 22-kDa subunits [32], For comparison, cephalosporin acylase from a Pseudomonas strain is an 83-kDa heterodimer consisting of 57- and 26-kDa subunits [33], The essential absence of any external catalytic requirement, cofactor stimulation, or product inhibition of ECB deacylase is also an intrinsic property of penicillin acylase [34], Based on the amino-terminal sequences of the two subunits of ECB deacylase, a 48% sequence similarity has been observed between the small subunit of ECB deacylase and a penicillin acylase [25]. This statistically significant albeit moderate sequence similarity from two short segments of the enzymes suggests an evolutionary relationship between ECB deacylase and peni-... 

Previous efforts have failed to identify an enzyme with robust Ceph C amidase activity. Some glutaryl-7-ACA acylases can directly convert Ceph C to 7-ACA, but they do so with very poor efficiency and have not been considered for a single-enzyme manufacturing process.30-33 Nonetheless, glutary 1-7-AC A acylases with measurable activity on Ceph C are classified as cephalosporin C acylases. Mutagenesis approaches such as ePCR have been used in an attempt to improve the activity of these enzymes on Ceph C, but only marginal improvements in the desired activity have... 

Terreni, M., Pagani, G., Ubiali, D., et al. 2001. Modulation of penicillin acylase properties via immobilization techniques One-pot chemoenzymatic synthesis of Cephamandole from Cephalosporin C. Bioorganic and Medicinal Chemistry Letters, 11 2429-32. 

Aminocephalosporanic acid (15, Scheme 9) is an important intermediate in the production of many semisynthetic cephalosporin antibiotics (66, 67). However, direct deacylation of cephalosporin C (13) to 15 by cephalosporin C acy-lase is unfavorable, so an enzymatic process is used involving D-amino acid oxidase (DAAO) oxidation of 13 to A-glutaryl-7-aminocephalosporanic acid (14, GL-7-ACA) followed by deacylation to 15 and glutaric acid, catalyzed by GL-7-ACA acylase from Pseudomonas sp. 130 (Scheme 9) (68, 69). GL-7-ACA acylase underwent pseudo first-order time-dependent inactivation by 7 3-bromoacetyl aminocephalos-poranic acid (16) (70). Dialysis did not regenerate enzyme activity, indicating irreversible inhibition. The rate of inactivation was lowered by the presence of either glutaric acid or 15,... 

Kim Y, Hoi WGJ. Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate insight into the basis of its substrate specificity. Chem. Biol. 2001 8(12) 1253-1264. 

The /3-lactam antibiotics may be susceptible to attack by three bacterial enzymes acylases, esterases and /3-lactamases. However, only the /8-lactamases play a significant role in the resistance of bacteria to penicillins and cephalosporins. 

ACA (36) is produced in a three-step process using two enzymes, starting from cephalosporin C (Scheme 20). After deamination by a D-amino acid oxidase, the a-ketoadipyl-7-ACA spontaneously decarboxylates and the remaining glutaryl side-chain is hydrolyzed by glutary 1-7-ACA-acylase. 

Operation of hydrolases in the reverse direction to effect acylation is also viable, as shown by the preparation of 26 from 25.46 Hog kidney acylase has been used to resolve (2RS,3RS)-valine-[ H]3 for cephalosporin biosynthesis.47... 

Penicillins and cephalosporins are charaeterized by (3-lactam structures and are the antibiotics that have traditionally been those most eommonly used in the treatment of infeetions. Pharmaceutical companies have synthesized a variety of semisynthetic (3-lactam compounds for use as oral antibiotics, for example, ampicillin and amoxieillin. These penicillin derivatives are prepared by aeylation of 6-amino-penicillanie acid (6-APA) derived from penicillin G (benzyl penicillin) or penicillin V (phenoxymethyl penicillin). An immobilized penicillin amidase (penicillin acylase) from Escherichia coli or Bacillus megaterium is used to prepare the 6-APA in nearly quantitative yield (Fig. 4). This substance is used as the starting material for the produetion of a number of other penieillins. The immobilized enzyme can be reused more than... 

A similar reaction scheme can be used to produce derivatives of cephalosporin via acylation of 7-amino-cephalosporamic acid or 7-amino-desacetoxycephalos-poramic acid. These compounds could be produced from a natural cephalosporin using an immobilized cephalosporin acylase, but alternative routes to these compounds are more cost-effective. 

This consideration is significant for hydrolytic reactions with hydrolases such as lipases, esterases, and amidases. These include penicillin amidases (synonymous with penicillin acylases) and cephalosporin acylases which are used for hydrolytic cleavage of penicillins and cephalosporins in thousands of tons per year [98]. These hydrolyses have to be performed at a pH-value of 8 which is close to the optimum pH of the enzyme. Lower pH-values lead to lower reaction rates and reversibility of the reaction, and hence to a significant loss in product formation. Higher pH-values are not advisable owing to the instability of the reaction partners. Moreover, addition of buffers is not accepted because of the costly removal of the buffer components. 

ADCA (cephalosporins) Fermentation and biocatalysis metabolic engineering, acylase... 

Like the penicillins, the side-chain of the cephalosporins has been cleaved from the cepham core by cephalosporin acylases and a series of semi-synthetic cephalosporins have been prepared. 

The direct cleavage of Ceph C to 7-ACA and D-a-aminoadipic acid by a cephalosporin acylase would be the preferred route. A large number of enzymes were isolated from different microorganisms, e.g., Pseudomonas diminuta, BaciUus megateri-um, but substrate specificity for Ceph C is low. The conversion obtained was on a low level, eg., 8% in 4 hours [8-10], Therefore, today, the direct one-step cleavage is not feasible on an industrial scale and a two-step process had to be developed. 

Research is in progress, by means of recombinant DNA technology, to gain access to an industrial relevant cephalosporin C acylase [16]. This one-step synthesis would further improve the performance of the process with respect to economy, quality and ecology. 

The use of an expandase to convert penicillins to cephalosporins (9.36) also replaced a more expensive multistep chemical synthesis.238 Penicillin acylase has also been used to remove phenylacetyl protecting groups from amines in the synthesis of oligonucleotides.239... 

The enzymatic removal of acyl groups plays an important role in the industrial production of semisynthetic penicillins and cephalosporins. To this end, penicillin G 12 (R = CH2-Ph) and penicillin V12 (R = CHj-O-Ph), or the respective cephalosporins are first deacylated by means of penicillin acylases (Fig. 18-6)[67, 681. The 6-aminopenicillanic acid and the 7-aminocephalosporanic acid thus obtained are subsequently acylated by non-enzymatic or enzymatic methods to give the semisynthetic antibiotics 13. 

---