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Open AccessArticle

Modulation of the Microenvironment Surrounding the Active Site of Penicillin G Acylase Immobilized on Acrylic Carriers Improves the Enzymatic Synthesis of Cephalosporins

Dipartimento di Scienze del Farmaco and Italian Biocatalysis Center, Università degli Studi di Pavia, Via Taramelli 12, Pavia 27100, Italy
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Present address: UTC—Université de Technologie de Compiègne, rue du Dr Schweitzer, Compiègne 60200, France
Molecules 2013, 18(11), 14349-14365; https://doi.org/10.3390/molecules181114349
Received: 9 September 2013 / Revised: 20 October 2013 / Accepted: 28 October 2013 / Published: 20 November 2013
(This article belongs to the Special Issue Enzyme Chemistry)
The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion. View Full-Text
Keywords: penicillin G acylase; immobilization; microenvironment; epoxy acrylic carriers; glutaraldehyde; hydrophilization penicillin G acylase; immobilization; microenvironment; epoxy acrylic carriers; glutaraldehyde; hydrophilization
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Bonomi, P.; Bavaro, T.; Serra, I.; Tagliani, A.; Terreni, M.; Ubiali, D. Modulation of the Microenvironment Surrounding the Active Site of Penicillin G Acylase Immobilized on Acrylic Carriers Improves the Enzymatic Synthesis of Cephalosporins. Molecules 2013, 18, 14349-14365.

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