The bacterial strain Gluconobacter oxydans GO112 exhibiting enhanced production of 2-keto-L-gulonic acid (2-KLG) from L-sorbose was bred by this laboratory. L-Sorbose dehydrogenase is one of the key enzymes responsible for the production of 2-KLG. In this study, we purified L-sorbose dehydrogenase from GO 112 to homogeneity through sequential chromatographical steps: DEAE-Sepharose Fast Flow, Mono Q anion exchange and Superose 12 gel filtration. The purified L-Sorbose dehydrogenase was single subunit protein with apparent molecular weight of about 60 kDa by SDS-PAGE and about 116 kDa by gel filtration chromatography, indicating the L-sorbose dehydrogenase may exist as dimeric molecules under physiological conditions. The optimum pH and temperature for the enzyme were pH 6.86 and 40 degrees C. It showed good stability at pH 6.2 and temperatures below 30 degrees C. At 40 degrees C, the L-sorbose dehydrogenase lost its activity by 37% in the first 1.5 min and then inactivated slowly. The K., value for L-sorbose was 36 mM. The activity of the L-sorbose dehydrogenase was greatly stimulated by Ca2+, while Mn2+, Fe2+, Cu2+, EDTA and citric acid inhibited the activity of the L-sorbose dehydrogenase to different degrees. (c) 2005 Elsevier Inc. All rights reserved.