Trehalose is a natural functional disaccharide, and can be synthesized by the trehalose synthase from Pseudomonas putida. In this paper, the properties of the trehalose synthase in the permeabilized cells of P. putida were studied. The enzyme exhibited an optimum temperature of 35 degrees C, and an optimum pH of 7.4, respectively. The enzyme was not thermostable, and its activity was increased by Mg2+ and K+, while inhibited by Zn2+, Mn2+ and Cu2+. The reaction condition for the production of trehalose by trehalose synthase was studied in details. The maltose concentration had little influence on the yield of trehalose. The optimum conditions of reaction were gained with orthogonal experimental design as follows: temperature 30 degrees C, pH 7.0, reaction time 24 h, and amount of enzyme 375 U/g maltose. (c) 2005 Elsevier Ltd. All rights reserved.