Progress in One-pot Bioconversion of Cephalosporin C to 7-Aminocephalosporanic Acid.

BACKGROUND: Cephalosporins are the most widely used semisynthetic antibiotics, which acted on bacterial cell wall (peptidoglycan) synthesis. The key intermediate for fabricating about twothirds of cephalosporins in clinical use is 7-aminocephalosporanic acid (7-ACA), which is derived from chemical or enzymatic deacylation of the natural antibiotic cephalosporin C (CPC). The chemical deacylation process has been replaced by the enzymatic deacylation process because the chemical process required harsh conditions and released toxic waste. METHODS: A two-step enzymatic process that utilized D-amino acid oxidase (DAAO) and 7-ß-(4carboxybutanamido)-cephalosporanic acid acylase (GLA) for two successive reactions has been applied for the conversion of CPC to 7-ACA in an industrial scale. RESULTS: To simplify the process and lower costs, the one-pot enzymatic processes were developed by the application of the mono-enzymatic process (application of cephalosporin C acylase or the variants of GLA), di-enzymatic process (simultaneous action of DAAO and GLA) or the tri-enzymatic process (simultaneous action of DAAO, GLA and catalase) for direct conversion of CPC to 7-ACA. CONCLUSION: Here, we mainly focused on the description of these one-pot enzymatic processes and emphasized on the preparation of the involved biocatalysts.

New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase.

BACKGROUND: Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic beta-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem. RESULTS: Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell. CONCLUSION: The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues on a predetermined region of the enzyme. The newly designed biocatalysts display improved synthetic performances and are able to maintain a similar activity to the free enzymes. Finally, we found that the activity of the immobilized enzyme proportionally improves with the number of introduced Lys.

[Penicillin G acylase--synthesis, regulation, production]. / Penicilin G acylasa--syntéza, regulace, produkce.

Penicillin G acylase (PGA) is one of very important industrial enzymes used in the production of polysynthetic beta-lactam antibiotics. This enzyme catalyzes the hydrolysis of the amidic bond of penicillin G with the development of 6-aminopenicillanic acid which serves as the initial substance for the production of semisynthetic penicillins. In the strain Escherichia coli W ATCC 11105 and ATCC 9637, PGA is coded by the pga gene on the chromozome and synthesized as the pre-pro-PGA (pp PGA) precursor, which is transported, with probable participation of the chaperon system, to the periplasmatic space of the cell. Here after a series of proteolytic reactions the active enzyme PGA develops, consisting of two subunits alpha and beta. Expression of the pga gene is subject to several regulatory mechanisms: temperature repression, catabolic repression by glucose, repression by oxygen, and induction by phenylacetic acid (FOK). The formation of active PGA is also influenced at the post-translation level, where an important role is played by intracellular proteolytic reactions and the transport system of pre-pro-PGA across the cytoplasmatic membrane. The chromozomal area of the pga gene of the E. coli W strain was employed for the construction of many recombinant plasmids. These plasmids served to transform suitable host strains, some of which are now used in industry as highly productive microorganisms.

Cephalosporin acylases: enzyme production, structure and application in the production of 7-ACA.

Cephalosporin acylases have application in the production of 7-aminocephalosporanic acid which forms a key raw material for the preparation of semisynthetic injectable cephalosporins. The enzymes are of industrial importance and hyperproducing genetically engineered strains have been constructed. Different aspects of these enzymes such as subunit structure, post translational modification, primary structure, substrate specificity and their importance in pharmaceutical industry are discussed.