期刊
MOLECULES
卷 18, 期 11, 页码 14349-14365出版社
MDPI AG
DOI: 10.3390/molecules181114349
关键词
penicillin G acylase; immobilization; microenvironment; epoxy acrylic carriers; glutaraldehyde; hydrophilization
The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of beta-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 (R) C and Sepabeads (R) 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 (R) 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 immobilization yield decreased from 70% (untreated Eupergit (R) C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit (R) C catalyzed the synthesis of cefazolin in 88% (+/- 0.9) and 87% (+/- 1.6) conversion, respectively, whereas untreated Eupergit (R) C afforded this antibiotic in 79% (+/- 1.2) conversion.
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