African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5233

Full Length Research Paper

Expression, purification and characterization of a quinoprotein L-sorbose dehydrogenase from Ketogulonicigenium vulgare Y25

Xionghua Xiong1#, Xin Ge1#, Yan Zhao2#, Xiaodong Han3, Jianhua Wang1 and Weicai Zhang1*
1Laboratory of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing, China. 2Central Laboratory, The first Affiliated Hospital of Xiamen University, Xiamen 361003, China. 3College of Life Sciences, Nankai University, Tianjin 300071, China.
Email: [email protected]

  •  Accepted: 10 May 2013
  •  Published: 30 June 2013


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 vulgare2-keto-L-gulonic acidL-sorbose dehydrogenase, quinoprotein.