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

Serra, Immacolata; Cecchini, Davide A.; Ubiali, Daniela; Manazza, Elena M.; Albertini, Alessandra M.; Terreni, Marco

doi:10.1002/ejoc.200801204

Cited by 23 publications

(17 citation statements)

References 36 publications

(42 reference statements)

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“…3.5.1.11) is used as a bulk enzyme in the production of 6 aminopenicillanic acid (6-APA) by hydrolysis of penicillin G or V (Shewale and Sudhakaran, 1997;Parmar et al 2000) and more recently in the synthesis of 6-APA and 7 amino-desacetoxycephalosporanic acid (7-ADCA) derived semi-synthetic penicillins and cephalosporins (Illanes and Wilson, 2006;Du et al 2009;Pchelintsev et al 2009). Biocatalyst engineering has been a central issue in penicillin acylase biocatalysis (Kallenberg et al 2005;Chandel et al 2008); substantial improvements in penicillin acylase stabilization have been obtained by directed immobilization to solid supports (Basso et al 2003;Montes et al 2007;Sun et al 2009), aggregation (Rajendhran and Gunasekaran, 2007), site-directed mutagenesis and directed evolution (Rajendhran and Gunasekaran, 2004;Serra et al 2009), and functional screening (Gabor et al 2005). Immobilization is the most powerful approach for increasing operational stability and multi-point covalent attachment to activated agarose gels is one of the most effective systems, being extensively used for immobilizing penicillin G acylase (Mateo et al 2005).…”

mentioning

confidence: 99%

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading

Illanes¹,

González²,

Gómez³

et al. 2010

Electro Journal of Biotech

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factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

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mentioning

confidence: 99%

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading

Illanes¹,

González²,

Gómez³

et al. 2010

Electro Journal of Biotech

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“…For example, it has been proposed a mutant enzyme designed to have a determined orientation after immobilization on glyoxyl-agarose, which allowed having a better behavior as biocatalysts of this process [153,154]. The authors recently were able to show the different orientations of the enzyme on the support by using tryptic hydrolysis and identifying the released peptides [155].…”

Section: Effect Of the Immobilization Protocol On The Enzyme Propertiesmentioning

confidence: 98%

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

Volpato¹,

Rodrigues

Fernández‐Lafuente

2010

CMC

110

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Semi-synthetic β-lactamic antibiotics are the most used anti-bacteria agents, produced in hundreds tons/year scale. It may be assumed that this situation will even increase during the next years, with new β-lactamic antibiotics under development. They are usually produced by the hydrolysis of natural antibiotics (penicillin G or cephalosporin C) and the further amidation of natural or modified antibiotic nuclei with different carboxylic acyl donor chains. Due to the contaminant reagents used in conventional chemical route, as well as the high energetic consumption, biocatalytic approaches have been studied for both steps in the production of these very interesting medicaments during the last decades. Recent successes in some of these methodologies may produce some significant advances in the antibiotics industry. In fact, the hydrolysis of penicillin G to produce 6-APA catalyzed by penicillin G acylase is one of the most successful historical examples of the enzymatic biocatalysis, and much effort has been devoted to find enzymatic routes to hydrolyze cephalosporin C. Initially this could be accomplished in a quite complex system, using a two enzyme system (D-amino acid oxidase plus glutaryl acylase), but very recently an efficient cephalosporin acylase has been designed by genetic tools. Other strategies, including metabolic engineering to produce other antibiotic nuclei, have been also reported. Regarding the amidation step, much effort has been devoted to the improvement of penicillin acylases for these reactions since 1960. New reaction strategies, continuous product extraction or new penicillin acylases with better properties have proven to be the key to have competitive biocatalytic processes. In this review, a critical discussion of these very interesting advances in the application of enzymes for the industrial synthesis of semi-synthetic antibiotics will be presented.

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“…For example, using glyoxyl supports, this may be performed by adding poly-Lys tags or by adding new Lys on rich-Lys areas of proteins, this can be a simple way to get the one pot immobilization, orientation, purification and, at least in the second case, the hyper-stabilization of enzymes (Abian et al, 2004, Cecchini et al, 2007, Cecchini et al, 2012, Scaramozzino et al, 2005, Serra et al, 2009, Serra et al, 2013, Temporini et al, 2010. (Mateo et al, 2005).…”

Section: Immobilization-purification Based On Different Immobilizatiomentioning

confidence: 99%

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

Barbosa

Ortíz

Berenguer-Murcia

et al. 2015

Biotechnology Advances

582

318

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Enzyme immobilization and purification are two steps that are usually required in the development of any industrial biocatalyst. In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies (versus the target enzyme or versus some domains), the development of specific domains with affinity for some specific supports or just to increase the affinity for standard ones (ionic exchangers, silicates) will be revised. We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailormade heterofunctional supports as a tool to immobilize-stabilize-purify some proteins will be discussed in deep, using low concentration of adsorbents groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be a the last part of the review.

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

Cited by 23 publications

References 36 publications

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

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