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Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: A step toward a thermosensitive industrial biocatalyst

Biotechnology Progress, ISSN: 8756-7938, Vol: 19, Issue: 4, Page: 1167-1175
2003
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Article Description

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10 and, presumably, cross-linked protein-polymer aggregates of MW > 10. Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 μM) was found to be slightly higher than that of the free enzyme (12 μM), and evaluation of V testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from D-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of β-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).

Bibliographic Details

Ivanov, Alexander E; Edink, Ewald; Kumar, Ashok; Galaev, Igor Yu; Arendsen, Alexander F; Bruggink, Alle; Mattiasson, Bo

Wiley

Biochemistry, Genetics and Molecular Biology

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