Screening of several immobilization carriers for trehalose synthase (TSase) from Thermus caldophilus GK24 has been performed for the efficient production of trehalose from maltose. This is the first report on immobilization of recombinant TSase for the production of trehalose. Taking account of yields of trehalose produced, Eupergit C250L was selected as a carrier in this work. The immobilization capacity reached 11 units/g-supports (92% of immobilization yield) when the conditions were as follows: coupling time of 14 h at 25 degrees C in 40 mM potassium phosphate buffer (pH 7.0) and enzyme loading of 12 units/g-supports. The optimum pH was not affected by immobilization, but optimum temperature was shifted from 45 to 65 degrees C. Immobilized enzyme was stable at high temperature (70 degrees C) for 16 days, whereas free enzyme retained 13% of its original activity after 6 days of incubation. The immobilized TSase could be used in the repetitive manner more than 10 times in batch reaction. Using the continuous system (bed volume: 25 mL), a maximum yield of 42% trehalose has been reached from 50 g/L maltose when the flow rate was 0.25 ml/min. (c) 2005 Elsevier Inc. All rights reserved.