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Biotechnology and Applied Biochemistry (2001) 33, (201–207) (Printed in Great Britain)

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Immobilization of native and poly(ethylene glycol)-treated ('PEGylated') bovine serum amine oxidase into a biocompatible hydrogel

Nicole Demers*, Enzo Agostinelli†, Diana A. Averill-Bates* and Guy Fortier*¹

*Département de Chimie-Biochimie, Université du Québec à Montréal, C.P. 8888, Succ. Centre-Ville, Montréal (Québec), Canada H3C 3P8, and †Department of Biochemical Sciences 'A. Rossi Fanelli', University of Rome 'La Sapienza' and CNR Centre of Molecular Biology, 00185 Rome, Italy

Abbreviations used: BSAO, bovine serum amine oxidase; PEG, poly(ethylene glycol); mPEG, monomethoxy-PEG; PEGylated, PEG-treated.

¹ To whom correspondence should be addressed (e-mail fortier.guy@uqam.ca).

Native bovine serum amine oxidase (BSAO) and poly(ethylene glycol) (PEG)-treated ('PEGylated') BSAO were immobilized into a hydrogel during its synthesis. The hydrogel was obtained by cross-linking of BSA with PEG di-nitrophenyl carbonates with a molecular mass of 10 kDa. Approx. 60% of the amino groups at the surface of BSAO were modified by monomethoxy-PEG with a molecular mass of 5 kDa when the reaction was carried out for 5 h in borate buffer, pH 9. The number of anchorage points of BSAO in the matrix, which was determined as minimal when PEGylated BSAO was used or maximal when native BSAO was used, did not influence the apparent K_m and V_max values of the different preparations. The apparent K_m values of both forms of the enzyme were decreased due to preconcentration of benzylamine substrate by the negatively charged hydrogel. V_max values were generally lower upon immobilization. We can therefore conclude that the hydrogel swelling has no significant effect on the enzyme's structure. The operational stability, evaluated in the presence of substrate, was generally increased upon enzyme immobilization into the hydrogel. Enzymic hydrogels were very stable during storage in solution at 4 °C, maintaining a high activity even after several weeks. The immobilization of both forms of BSAO did not improve their thermostability at 65 °C. The BSA-PEG hydrogel is a good matrix for immobilization of enzymes with therapeutic potential such as BSAO.

Received 26 January 2001; accepted 20 March 2001

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