普通生酮基古龙酸菌中山梨糖脱氢酶和山梨酮脱氢酶的纯化及酶学性质分析

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

摘要:

[目的]对源于普通生酮基古龙酸菌（Ketogulonicigenium vulgare WSH-001）的山梨糖脱氢酶（Sorbosedehydrogenase，SDH）和山梨酮脱氢酶（Sorbosone dehydrogenase，SNDH）的酶学性质进行分析。[方法]以K.vulgare WSH-001基因组DNA为模板，PCR扩增得到山梨糖脱氢酶基因（sdh）和山梨酮脱氢酶基因（sndh），构建重组表达质粒pET28a-sdh、pET28a-sndh，并分别转入大肠杆菌BL21（DE3）中。利用镍柱亲和层析和凝胶过滤层析得到纯化的SDH和SNDH。[结果]成功构建产SDH和SNDH的大肠杆菌BL21（DE3）并对目的酶进行纯化。SDS-PAGE分析结果表明，SDH和SNDH的大小分别为64 kDa和48 kDa，与理论预测值一致。显色法测得SDH酶活为3.15 U/mg，最适反应温度为30℃，最适反应pH为8.0左右；SNDH酶活为6.12 U/mg，最适反应温度为35℃，最适反应pH为8.0左右。在pH 3.0、4.0、5.0的偏酸性条件下，2个酶的酶活受到显著影响。[结论]表达并纯化了来源于普通生酮基古龙酸菌来源的SDH、SNDH，并进行了酶学性质分析，为利用SDH、SNDH实现维生素C前体2-酮基-L-古龙酸的一步法发酵生产提供了必要的参考。

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.