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Biotechnology and Applied Biochemistry (2003) 38, (53–59) (Printed in Great Britain)

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Thermal stabilization of trypsin by enzymic modification with b-cyclodextrin derivatives

Reynaldo Villalonga*¹, Michael Fernández*, Alex Fragoso†, Roberto Cao†, Loredana Mariniello‡ and Raffaele Porta‡

*Enzyme Technology Group, Center for Biotechnological Studies, University of Matanzas, Autopista a Varadero km 3 1/2, Matanzas, C.P. 44740, Cuba, †Laboratory of Bioinorganic Chemistry, Faculty of Chemistry, Havana University, Havana 10400, Cuba, and ‡Dipartimento di Scienza degli Alimenti, Università di Napoli “Federico II”, Via Università 100, Portici, Naples, Italy

Key words: enzyme stability, oligosaccharide, serine protease, transglutaminase.

Abbreviations used: TGase, transglutaminase; CD, b-cyclodextrin; CDEN, mono-6-ethylenediamino-6-deoxy-b-cyclodextrin; CDPN, mono-6-propylenediamino-6-deoxy-b-cyclodextrin; CDBN, mono-6-butylenediamino-6-deoxy-b-cyclodextrin; CDHN, mono-6-hexylenediamino-6-deoxy-b-cyclodextrin; BAEE, N-a-benzoyl-L-arginine ethyl ester hydrochloride.

¹To whom correspondence should be addressed (e-mail reynaldo.villalonga@umcc.cu).

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several b-cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono-6-ethylenediamino-6-deoxy-b-cyclodextrin, mono-6-propylenediamino-6-deoxy-b-cyclodextrin, mono-6-butylenediamino-6-deoxy-b-cyclodextrin and mono-6-hexylenediamino-6-deoxy-b-cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4–21% after conjugation. The K_m values for cyclodextrin–trypsin complexes represented about 58–87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5–10 °C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 °C for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 °C was markedly increased for the oligosaccharide–trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.

Received 14 October 2002/4 February 2003; accepted 20 February 2003

Published as Immediate Publication 20 February 2003, DOI 10.1042/BA20020096

© 2003 Portland Press Ltd