Stabilization of Penicillin G Acylase from
            <i>Escherichia coli</i>
            : Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

Abián, Olga; Grazú, Valeria; Hermoso, J.A.; González, R. N.; Garcı́a, José Luis; Fernández‐Lafuente, Roberto; Guisán, José M.

doi:10.1128/aem.70.2.1249-1251.2004

Cited by 107 publications

(62 citation statements)

References 12 publications

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“…18,[86][87][88][89][90] In this case, we can focus on the area 535 where we intend to immobilize the enzyme, leaving the other areas of the protein unaltered. 536…”

Section: -78 533mentioning

confidence: 99%

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

et al. 2013

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27A heterofunctional support for enzyme immobilization may be defined as that which 28 possesses several distinct functionalities on its surface able to interact with a protein. We will 29 focus on those supports in which a final covalent attachment between the enzyme and the 30 support is achieved. Heterofunctionality sometimes has been featured in very old 31 immobilization techniques, even though in many instances it has been overlooked, giving rise to 32 some misunderstandings. In this respect, glutaraldehyde activated supports are the oldest 33 multifunctional supports. Their matrix has primary amino groups, the hydrophobic 34 glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. 35Thus, immobilization may start (first event of the immobilization) via different causes and may 36 involve different positions of the enzyme surface depending on the activation degree and 37 immobilization conditions. Other "classical" heterofunctional supports are epoxy commercial 38 supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization 39 is performed at high ionic strength to permit protein adsorption, so that covalent attachment may 40 take place at a later stage. Starting from these old immobilization techniques, tailor-made 41 heterofunctional supports have been designed to permit a stricter control of the enzyme 42 immobilization process. The requirement is to find conditions where the main covalent reactive 43 moieties may have very low reactivity towards the enzyme. In this review we will discuss the 44 suitable properties of the groups able to give the covalent attachment (intending a multipoint 45 covalent attachment), and the groups able to produce the first enzyme adsorption on the support. 46Prospects, limitations and likely pathways for the evolution (e.g., coupling of site-directed 47 mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional 48 supports) will be discussed in this review. 49 50

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“…18,[86][87][88][89][90] In this case, we can focus on the area 535 where we intend to immobilize the enzyme, leaving the other areas of the protein unaltered. 536…”

Section: -78 533mentioning

confidence: 99%

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

et al. 2013

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“…Multipoint covalent attachment on proper supports may permit a higher stabilization rigidification of the enzyme, with higher stabilization in thermal inactivations (1,15,32). However, the generation of hydrophilic nanoenvironments by this new strategy yields stabilization factors in the presence of dioxane that are very similar to those obtained by multipoint covalent attachment, permitting full use of the advantages of the reversible immobilization.…”

Section: Discussionmentioning

confidence: 97%

“…Plasmid pET101/D-TOPO (Invitrogen, Paisley, United Kingdom) was used for cloning purposes according to the supplier's procedures (Invitrogen, Paisley, United Kingdom). Plasmid pOAF, a pET101/D-TOPO plasmid derivative containing the wild-type pac gene from E. coli ATCC 11105, was previously described (1).…”

Section: Methodsmentioning

confidence: 99%

“…This approach does not require a knowledge of protein structure as deep as that required to directly improve the stability of soluble enzymes, since in this case we only have to be able to predict the groups that will be exposed to the medium. Moreover, it is assumed that site-directed mutagenesis on these surface groups should not cause dramatic negative changes on the enzyme properties (1).…”

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confidence: 99%

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Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers

Montes

Grazú

López‐Gallego

et al. 2007

Appl Environ Microbiol

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A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE-or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.

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“…Due to the industrial interest in PGA, mutational studies have been performed for over 20 years with the aim of altering its thermostability (23,24) and substrate specificity (25)(26)(27)(28)(29)(30)(31). To date, crystal structures of E. coli (32), P. rettgeri (28), and A. faecalis (33) PGAs are available, which have contributed either to rational design or to the explanation of results obtained by random mutagenesis techniques.…”

Section: Discussionmentioning

confidence: 99%

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

Torres

Cantero

Valle

et al. 2013

Appl Environ Microbiol

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A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position ␣188, ␣189, or ␤24 improved the K m for penicillin G between 9-and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.

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Stabilization of Penicillin G Acylase from Escherichia coli : Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

Cited by 107 publications

References 12 publications

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

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