Abstract

It is well known that Ketogulonicigenium vulgare Y25 could effectively oxidize L-sorbose to 2-keto-L-gulonic acid (2KGA), an industrial precursor of vitamin C. There in, L-sorbose dehydrogenase is one of the key enzymes responsible for the production of 2KGA. From this organism, the coding region of sdh gene was cloned into pET22b plasmid and its transcription product was overexpressed. This procedure allowed purification of L-sorbose dehydrogenase and production of polyclonal antibodies. In Western blot assays, the antibodies gave a positive reaction against bacteria protein extract and purified L-sorbose dehydrogenase. The molecular mass of the enzyme was 60532 Da, and the N-terminal amino acid sequence was determined to be QTAIT. The Native-PAGE and resting-cell reaction assay showed that purified L-sorbose dehydrogenase could convert L-sorbose to 2KGA, and PQQ was found to be indispensable for its activity as prosthetic group. The enzyme showed broad substrates specificity and the K_m value for L-sorbose and 1-propanol was 21.9 mM and 0.13 mM, respectively. The optimum pH of the enzyme activity was 8.0, and the optimum temperature was 35°C. The activity of the L-sorbose dehydrogenase was greatly stimulated by Ca²⁺ and strongly inhibited by Co²⁺ and Cu²⁺. The results obtained from the present study showed that a PQQ-dependent L-sorbose dehydrogenase could oxidize L-sorbose into 2-keto-L-gulonic acid in vitro.

Key words: Ketogulonicigenium vulgare, 2-keto-L-gulonic acid, L-sorbose dehydrogenase, quinoprotein.