Purification and characterization of L-sorbose dehydrogenase and L-sorbosone dehydrogenase from Ketogulonicigenium vulgare WSH-001

Abstract:

[Objective] We purified and characterized L-sorbose dehydrogenase and L-sorbosone dehydrogenase from Ketogulonicigenium vulgare WSH-001.[Methods] L-sorbose dehydrogenase gene (sdh) and L-sorbosone dehydrogenase gene (sndh) from K. vulgare WSH-001 were amplified by PCR. The amplified fragments were inserted into pET-28a(+) to obtain expression plasmids, namely pET28a-sdh and pET28a-sndh. Escherichia coli BL21(DE3) harboring the above plasmids was used to express SDH and SNDH. Purified SDH and SNDH were obtained by using HisTrap^TM affinity chromatography and gel filtration chromatography.[Results] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and purified. The molecular weight of SDH and SNDH were 64 kDa and 48 kDa on SDS-PAGE, respectively. Colorimetric assay showed that the enzyme activity of SDH and SNDH was 3.15 U/mg and 6.12 U/mg, respectively. The optimum temperature and pH of the purified SDH were 30℃ and 8.0, respectively, whereas the optimum temperature and pH of the purified SNDH were 35℃ and 8.0, respectively. The enzyme activity of SDH and SNDH was extremely low at pH 3.0, 4.0 and 5.0.[Conclusion] SDH and SNDH from K. vulgare WSH-001 were expressed in E. coli BL21(DE3) and characterized. The results could provide essential reference for the achievement of one-step fermentation of 2-KLG.

Key words:Gluconobacter oxydans;2-keto-L-gulonic acid;L-sorbose dehydrogenase;L-sorbosone dehydrogenase