Biotechnol. Appl. Biochem. (2003) 38, 53–59 (Printed in Great Britain) 53 Thermal stabilization of trypsin by enzymic modification with β -cyclodextrin derivatives Reynaldo Villalonga* 1 , Michael Fern´ andez*, 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, Universit` a di Napoli “Federico II”, Via Universit` a 100, Portici, Naples, Italy Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several β -cyclodextrin derivatives to the glutamine residues in bovine pancre- atic trypsin. The modifying agents used were mono-6- ethylenediamino-6-deoxy-β -cyclodextrin, mono-6-pro- pylenediamino-6-deoxy-β -cyclodextrin, mono-6-butyl- enediamino-6-deoxy-β -cyclodextrin and mono-6-hexy- lenediamino-6-deoxy-β -cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligo- saccharides/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 in- creased by about 5–10 ◦ C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 ◦ C for the modified trypsin. Ther- mal inactivation at different temperatures ranging from 45 to 60 ◦ C was markedly increased for the oligo- saccharide–trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH. Introduction Proteases are an important class of enzymes with widespread use in food manufacturing and processing industry [1]. Their uses in the food industry include cheese manufacture, meat tenderization, beer chill-proofing, flavour development in Oriental fermentation and viscoelasticity modification of bread dough [2]. They are also valuable for the production of protein hydrolysates for nutritional and therapeutic diets [3]. For many of these applications, highly stable proteolytic enzymes able to work in homogeneous systems are specially required. Much attention has been devoted to increasing the thermal resistance of these hydrolases [4,5]. In fact, there are important advantages that arise by carrying out enzymic reactions at high temperatures, such as (i) the prevention of microbial contamination, (ii) the higher conversion rates, (iii) the increase in substrates and products solubility and (iv) the decrease in the viscosity of the reaction medium [6,7]. Among the strategies reported for preparing water- soluble enzyme adducts having high thermal stability, the procedures based on the manipulation of their protein surface by chemical modification constitute one of the most promising [8–12]. In general, the effectiveness of these methods depends on choosing the appropriate conditions based on (i) the type, size and structure of the enzyme, (ii) the structure and size of the modifier compound and (iii) the type and conditions for the chemical reactions involved in the modification procedure [4]. In recent decades, a great number of studies have been devoted to improving the functional stability of proteolytic enzymes by the chemical attachment of low-molecular- mass compounds [8,12], as well as water-soluble polymers [4,8,13,14]. However, chemical modification of enzymes has often been reported to provoke significant losses of catalytic activity [8,10,12]. For this reason, the development of new modification methods for stabilizing enzymes, preserving their catalytic properties, is receiving considerable attention in enzyme technology. In the present paper we describe the use of Streptiverticillum sp. transglutaminase (TGase; EC 2.3.2.13) as a catalyst for preparing several trypsin–oligosaccharide conjugates, as well as the effect of this transformation on the catalytic and thermal stability properties of the conjugated enzymes. As modifying agents here we report Key words: enzyme stability, oligosaccharide, serine protease, transglutaminase. Abbreviations used: TGase, transglutaminase; CD, β-cyclodextrin; CDEN, mono-6-ethylenediamino-6-deoxy-β-cyclodextrin; CDPN, mono-6-propylenediamino-6-deoxy-β-cyclodextrin; CDBN, mono-6-butylenediamino-6-deoxy-β-cyclodextrin; CDHN, mono-6-hexylenediamino-6-deoxy-β-cyclodextrin; BAEE, N-α-benzoyl-L-arginine ethyl ester hydrochloride. 1 To whom correspondence should be addressed (e-mail reynaldo.villalonga@umcc.cu). C  2003 Portland Press Ltd