D-Psicose was produced from D-fructose using an immobilized D-tagatose S-epimerase (D-TE), and used as a substrate for the production of D-allose by immobilized L-rhamnose isomerase (L-RI). D-Allose was produced from D-psicose by L-RI, which was constitutively produced by a mutant strain of Pseudomonas sp. LL172 and immobilized on chitopearl beads of BCW 2603. The isomerization reaction progressed steadily while the concentration of D-psicose was increased from 2.5 to 20% in Tris-I-ICI buffer (0.05 M, pH 7.5), and about 40% of D-psicose was converted to D-allose in all the cases. The immobilized enzyme was stable even after repeated use over 20 d at 40 degrees C in the preparation of D-allose without detectable decrease in the enzyme activity, indicating the high stability of the enzyme in the batch reaction. The crystallized product was confirmed as D-allose by analysis of the IR spectra, NMR spectra, melting point and optical rotation.