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

<p>Abstract</p> <p>Background</p> <p>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 <it>E. coli </it>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.</p> <p>Results</p> <p>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.</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Proceedings of the Fifth Italian Conference on Computational Linguistics CLiC-it 2018

On behalf of the Program Committee, a very warm welcome to the Fifth Italian Conference on Computational Linguistics (CLiC-­‐it 2018). This edition of the conference is held in Torino. The conference is locally organised by the University of Torino and hosted into its prestigious main lecture hall “Cavallerizza Reale”. The CLiC-­‐it conference series is an initiative of the Italian Association for Computational Linguistics (AILC) which, after five years of activity, has clearly established itself as the premier national forum for research and development in the fields of Computational Linguistics and Natural Language Processing, where leading researchers and practitioners from academia and industry meet to share their research results, experiences, and challenges

Relatório de estágio em farmácia comunitária

Relatório de estágio realizado no âmbito do Mestrado Integrado em Ciências Farmacêuticas, apresentado à Faculdade de Farmácia da Universidade de Coimbr

A Comparison between Immobilized Pyrimidine Nucleoside Phosphorylase from Bacillus subtilis and Thymidine Phosphorylase from Escherichia coli in the Synthesis of 5-Substituted Pyrimidine 2’-Deoxyribonucleosides

Pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli (EcTP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2'-deoxyribonucleosides (14–18) by transglycosylation in fully aqueous medium. From the comparative study ofthe two biocatalysts, no remarkable differences emerged about their substrate specificity, bioconversion yield, stability in organic cosolvents(DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2',3'- dideoxynucleosides as substrates, whereas they catalyze bioconversions involving 5-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14–18 proceeded at a similar conversion (33–68% for BsPyNP and 25–62% for EcTP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2'-deoxyuridine (14) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% (EcTP) or 76% (BsPyNP) conversion (about 9g/L

Coupling of Site-Directed Mutagenesis and Immobilization for the Rational Design of More Efficient Biocatalysts: The Case of Immobilized 3G3K PGA from E. coli

We have investigated the performances of the immobilized 3G3K mutant of the Penicillin G acylase (PGA) from E. coli obtained by site-directed mutagenesis. The 3G3K mutant, characterized by a tag consisting of three lysines alternating with three glycines at the end of the beta-chain, was previously reported to have a higher ratio between the rate of the antibiotic synthesis and the rate of the acylating agent hydrolysis than the wild type enzyme (vs/vh1 value). New immobilization studies were carried out with the 3G3K mutant by using different glyoxyl supports (activated with aldehyde groups). The catalytic properties of the new immobilized preparations were tested in the synthesis of Cefamandole and Cefonicid by kinetically controlled N-acylation (kcNa). Compared to the commercial wild type PGA, the immobilized 3G3K acylase on glyoxyl agarose showed higher synthetic performances, in all the tested reactions, in terms of reaction rates and yields