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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 β-lactam antibiotics. One of the most used supports for immobilization is glyox...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2007
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045090/ https://www.ncbi.nlm.nih.gov/pubmed/17845725 http://dx.doi.org/10.1186/1472-6750-7-54 |
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author | Cecchini, Davide A Serra, Immacolata Ubiali, Daniela Terreni, Marco Albertini, Alessandra M |
author_facet | Cecchini, Davide A Serra, Immacolata Ubiali, Daniela Terreni, Marco Albertini, Alessandra M |
author_sort | Cecchini, Davide A |
collection | PubMed |
description | 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 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. 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. |
format | Text |
id | pubmed-2045090 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-20450902007-10-30 New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase Cecchini, Davide A Serra, Immacolata Ubiali, Daniela Terreni, Marco Albertini, Alessandra M BMC Biotechnol Research Article 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 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. 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. BioMed Central 2007-09-10 /pmc/articles/PMC2045090/ /pubmed/17845725 http://dx.doi.org/10.1186/1472-6750-7-54 Text en Copyright © 2007 Cecchini et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Cecchini, Davide A Serra, Immacolata Ubiali, Daniela Terreni, Marco Albertini, Alessandra M New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase |
title | New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase |
title_full | New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase |
title_fullStr | New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase |
title_full_unstemmed | New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase |
title_short | New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase |
title_sort | new active site oriented glyoxyl-agarose derivatives of escherichia coli penicillin g acylase |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045090/ https://www.ncbi.nlm.nih.gov/pubmed/17845725 http://dx.doi.org/10.1186/1472-6750-7-54 |
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