Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biotechnology and Applied Biochemistry (2002) 36, (163–170) (Printed in Great Britain)

PDF

Legacy HTML

Modified high amylose starch for immobilization of uricase for therapeutic application

Jérome Mulhbacher*, Kevin McGeeney†, Pompilia Ispas-Szabo*, Vincent Lenaerts‡ and Mircea-Alexandru Mateescu*¹

*Department of Chemistry and Biochemistry, Université du Québec à Montréal, C.P. 8888, Succ. A, Montréal, Québec, Canada H3C 3P8, †Department of Medicine and Therapeutics, University College Dublin, Dublin, Ireland, and ‡Labopharm Inc., Laval, Québec, Canada

Key words: enzyme therapy, gout, joints inflammation, uricase, uric acid.

Abbreviations used: AE-HASCL-35, aminoethyl high amylose starch cross-linked 35; CM-HASCL-35, carboxymethyl high amylose starch cross-linked 35; DNS, dinitrosalicylic acid; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide.

¹To whom correspondence should be addressed (e-mail mateescu.m-alexandru@uqam.ca).

Urate oxidase (uricase) was immobilized on carboxymethyl high amylose starch cross-linked 35 (CM-HASCL-35), on aminoethyl high amylose starch cross-linked 35, as well as on commercial supports, CNBr-activated Sepharose and diaminodipropylamine agarose. The N-ethyl-5-phenylisoxazolium-3´-sulphonate (Woodward reagent K) gave a high binding but totally inhibited the enzyme activity. Best results were obtained with CM-HASCL-35 using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide as a coupling agent. The immobilized enzyme retained 88% of its initial limit rate [V_max(app) = 16 EU/mg for immobilized uricase versus V_max = 18 EU/mg for the free enzyme], with an apparent decrease of affinity for urate substrate [K_m(app) = 0.17 mM versus K_m = 0.03 mM for the free enzyme]. The coupling yield was 60% and the modified uricase was found more resistant to proteolysis than the free enzyme. The immobilized uricase retained 25% of its initial activity after 60 min in pancreatic proteolysis medium (pancreatin), whereas the free enzyme retained only 5% of its initial activity. The best immobilization yield was obtained with the polymeric support based on CM-HASCL-35 (53%), which gave better results than commercial supports based on agarose.

Received 9 April 2002/23 July 2002; accepted 25 July 2002

Portland Press Ltd © 2002