Cyclodextrin glucantransferase (CGTase; EC 2.4.1.19) catalyzes the convertion of starch and related a (1�¨4) glucans to cyclodextrins (CDs) through an intramolecular transglycosylation reaction. Three different CDs are known a-, b- and g-CD, which consist of 6, 7 and 8 a (1�¨4) linked D-glucose units, respectively. They have doughnut-shaped with an hydrophobic cavity. Due to this structure they are able to form inclusion complexes with different substances, improving their physical and chemical properties such as solubility and stability against oxidation, making them valuable for food, cosmetic and pharmaceutical industries. Apart for producing CDs, CGTases also catalyze other three reactions: a) coupling, where the CD molecule is opened and combined with a linear carbohydrate; b) disproportionation, which is the transfer of part of a linear oligosaccharide chain to an acceptor; and c) hydrolysis of starch. In spite of these activities, the use of CGTases has been limited by the cost and stability of the enzymes. Enzyme immobilization is one of the most useful approaches to overcome such difficulties. Due to our experience on reversible covalent immobilization, we tried the immobilization of native CGTase from Thermoanaerobacter sp onto a thiolreactive support (thiolsulfinate-agarose), but no immobilization was achieved, confirming that native SH groups were buried and couldn't react with the support. In this work, we compare two ways of increasing the SH content of native CGTase from Thermoanaerobacter sp, in order to allow its reversible covalent immobilization. As the enzyme has one disulfide bond we assayed its reduction with dithiotreitol (DTT) under different conditions of pH (7.5 to 8.5), ionic strength (10 to 100 mM) and DTT concentration (25 to 100 mM). The other alternative involved a controlled thiolation, where the enzyme was provided with "de novo" thiol groups by reaction with the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The reduction process did not affect b-CD cyclization activity but no increase in the SH content was achieved. In the other hand, when the SPDP/ CGTase molar ratio was changed between 3 to 100, and time reaction between 30 to 60 minutes , an eight- fold increase in the SH content of the native enzyme was achieved keeping nearly 80% of the native b-CD cyclization activity. In conclusion, the thiolation process would be of great importance for allowing the reversible covalent immobilization of this enzyme.