L-Rhamnose isomerase (L-RhI) from Pseudomonas stutzeri LL172 can convert D-psicose to D-allose. Partially purified recombinant L-RhI from Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce D-allose. Total 20,000 units of immobilized enzyme converted D-psicose to D-allose without remarkable decrease in the enzyme activity over 17 days. When 50% D-psicose (w/w) was applied to a column with a flow rate of 0.8 ml/min at 42 degrees C, approximately 30% D-psicose was isomerized to D-allose for 17 days. However, by reducing the flow rate to 0.4 ml/min after 17 days, D-allose was transformed at the same rate for 13 days. The total of 27 1 reaction mixture was separated by simulated-Moving-Bed Chromatograph system. Approximately 2.2 l/d of 50% (w/w) reaction mixture was separated continuously. After separation, D-allose and D-psicose fractions were 31 of approximately 10% (w/w) with 95% purity and 101 of approximately 8% (w/w) with 95% purity per day, respectively. The separated D-allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature. Crystals Of D-allose were separated from the syrup by filtration and 1.65 kg crystals of 100% purity were obtained. The D-allose crystal yield from the D-psicose substrate was approximately 10%. (c) 2005 Elsevier Inc. All rights reserved.