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

Abstract

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/vh 1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit ® C and Sepabeads ® EC-EP, respectively) with respect to the unmodified support (vs/vh 1 = 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/vh 1 compared to the untreated biocatalyst, although the OPEN ACCESSMolecules 2013, 18 14350 immobilization yield decreased from 70% (untreated Eupergit ® C) to 20%. Glutaraldehydeand 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.

Tuning the catalytic properties of lipases immobilized on divinylsulfone activated agarose by altering its nanoenvironment

“…Thus, we should have lipase molecules with identical orientation and degree of enzyme support multipoint covalent attachment. In a previous paper, the properties of the enzyme penicillin G acylase immobilized on epoxy activated supports were tuned in a kinetically controlled synthesis of antibiotics by changing the blocking reagent [53]. Results were explained mainly as partition of the substrates and products, the enzyme immobilization involved the areas near to the active center [54].…”

Design and synthesis study of the thermo‐sensitive copolymer carrier of penicillin G acylase

“…Immobilized penicillin G acylase (PGA) has been widely used in the process of the catalyzing hydrolysis of penicillin G potassium to produce 6‐aminopenicillanic acid (6‐APA) . Driven by the intensive and extensive application evolution, researchers have found that the properties of carriers play a key role in immobilized PGA, not only influence the activity of PGA at a large state, but also decide the loading amount, response rate, and recovery of PGA directly …”

Immobilization of γ‐Glutamyl Transpeptidase from Equine Kidney for the Synthesis of kokumi Compounds

“…On the same binding chemistry, however, GA‐EDA‐agarose is characterized by a spacer that may impart a higher flexibility to the immobilized enzyme. In principle, this scenario could assist in minimizing the distortion of the protein upon immobilization, thus preserving the enzyme activity . The immobilization yield was 100 % but, surprisingly, the immobilized enzyme lost almost completely its activity (3 % activity recovery).…”