Purification and characterization of membrane-bound l-sorbose dehydrogenase from Gluconobacter oxydans GO112

Abstract

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 GO112 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 °C. It showed good stability at pH 6.2 and temperatures below 30 °C. At 40 °C, the l-sorbose dehydrogenase lost its activity by 37% in the first 1.5 min and then inactivated slowly. The K m value for l-sorbose was 36 mM. The activity of the l-sorbose dehydrogenase was greatly stimulated by Ca 2+, while Mn 2+, Fe 2+, Cu 2+, EDTA and citric acid inhibited the activity of the l-sorbose dehydrogenase to different degrees.