Abstract
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.
Highlights
Penicillin G acylase (PGA, E.C. 3.5.1.11) from E. coli is a N-terminal serine hydrolase mainly used in the industrial production of 6-aminopenicillanic acid (6-APA) and 7-aminodesacetoxycephaloporanic acid (7-ADCA) for the synthesis of β-lactam antibiotics [1]
Acrylic carriers bearing epoxy groups such as Eupergit® C and Sepabeads® EC-EP rely on a straightforward procedure for enzyme immobilization
Immobilization through glutaraldehyde is a complex multi-step procedure which is hardly feasible for applicative purposes
Summary
Penicillin G acylase (PGA, E.C. 3.5.1.11) from E. coli is a N-terminal serine hydrolase mainly used in the industrial production of 6-aminopenicillanic acid (6-APA) and 7-aminodesacetoxycephaloporanic acid (7-ADCA) for the synthesis of β-lactam antibiotics [1].Different strategies have been developed to enhance the properties of biocatalysts (e.g., stability), including multimeric enzymes, to tailor them for an industrial application [2,3]. Immobilized PGA on solid supports results in a more stable biocatalyst, which can be recovered and reused [4,5,6,7,8,9,10]. Immobilization generally exerts a detrimental effect on the synthetic performance of PGA in the kinetically controlled acylation of β-lactam nuclei, when epoxy acrylic carriers such as Eupergit® C or Sepabeads® EC-EP are used. More hydrophilic carriers such as glyoxyl agarose have been found superior because they generate a microenvironment more favorable to the diffusion of the β-lactam nucleus into the active site [11,12].
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