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 Km 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.
Highlights
L-ascorbic acid, commonly known as vitamin C, is generally used in pharmaceutical, food, cosmetic and feedstuff industries as an essential nutrient and a powerful antioxidant
L-sorbose dehydrogenase from Ketogulonicigenium vulgare DSM4025 was found as a new quinoprotein dehydrogenase catalyzing the conversion of L-sorbose to L-sorbosone, and that of L-sorbosone to 2-keto-Lgulonic acid (2KGA) in vitro (Asakura et al, 1999). These results suggested that the characterization of Lsorbose dehydrogenase in K. vulgare is obviously different from that described in the sorbosone pathway
We cloned a sorbose dehydrogenase gene (EIO_2658) from K. vulgare Y25 and presented its enzymatic characters based on protein expression and purification
Summary
L-ascorbic acid, commonly known as vitamin C, is generally used in pharmaceutical, food, cosmetic and feedstuff industries as an essential nutrient and a powerful antioxidant. The most popular process for synthesizing vitamin C is Reichstein’s method in the last century (Reichstein et al, 1934). Because of economical and ecological advantages, the use of microbial processes to produce vitamin C became attractive in the recent years. In 1990, Yin et al established a method, converting Lsorbose into 2-keto-L-gulonic acid (2KGA, a precursor of L-ascorbic acid) by an artificial microbial ecosystem consisting of Gluconobacter oxydans and Bacillus sp. (Yin et al, 1990). The co-culture method has been widely used in China. Functions as the companion bacteria to accelerate the growth of G. oxydans, while G. oxydans is responsible Bacillus sp. functions as the companion bacteria to accelerate the growth of G. oxydans, while G. oxydans is responsible
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