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Penicillin amidase

About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.


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Journal ArticleDOI
TL;DR: 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.
Abstract: 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 β-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. 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. 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.

30 citations

Journal ArticleDOI
TL;DR: By marker exchange mutagenesis, Bacillus megaterium strain UN‐1 was used to prepare a mutant strain B. megateria UN‐cat (Bm‐UNcat) lacking the penicillin G acylase gene (pac), and the resultant plasmid, pBA402, was introduced into Bm‐ UNcat and Bacillus subtilis.
Abstract: By marker exchange mutagenesis, Bacillus megaterium strain UN-1 (Bm-UN1) was used to prepare a mutant strain B. megaterium UN-cat (Bm-UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm-UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm-UNcat and Bacillus subtilis. Bm-UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13·7, 19·5 and 20·4 U ml−1 at 24, 36 and 48 h of culture, respectively. This was two- to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20-fold higher than PAC produced by the parent strain, Bm-UN1.

30 citations

Journal ArticleDOI
TL;DR: Thermodialysis is an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved and the diffusion limitations in the immobilised enzyme on the membrane are reduced.
Abstract: The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase ® ) and for the free enzyme were used. For Assemblase ® , it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed. The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase ® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane. The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.

29 citations

Journal ArticleDOI
TL;DR: In this paper, a new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer.
Abstract: A new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer. Penicillin G acylase (PGA) and cephalexin were employed as catalyst and substrate, respectively. Cephalexin hydrolysis was studied in bioreactors operated under isothermal and non-isothermal conditions. A hydrolysis increase was found when the temperature of the warm membrane surface was kept constant and the temperature of the other membrane surface was kept at a lower value. The hydrolysis increase was linearly proportional to the applied temperature difference. Cephalexin hydrolysis increased to about 10% when a temperature difference of 1°C was applied across the catalytic membrane. These results have been attributed to the non-isothermal cephalexin transport across the membrane, i.e., to the process of thermodialysis. In this way, the enzyme immobilized on and into the membrane reacts with a substrate concentration higher than that produced by simple diffusion under isothermal conditions.

29 citations

Journal ArticleDOI
TL;DR: In this paper, the modulation of hydrolytic activity of penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae by organic solvents is reported.

29 citations

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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20234
20222
20183
20175
20165
20153