Purified trehalose synthase from Thermus caldophilus GK24 produced 18-86% trehalose from 10 mM-1 M maltose. The enzyme also catalyzed the conversion of alpha,alpha-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40 degrees C than at 80 degrees C and was independent of the substrate concentration. The maximum yield of alpha,alpha-trehalose from 10 mM maltose reached 86% at 40 degrees C. In addition, alpha,beta-trehalose was also formed from maltose or alpha,alpha-trehalose at 3.5% yield at 80 degrees C.