粘质沙雷氏菌中果糖-1,6-/景天庚酮糖-1,7-二磷酸两个双功能酶的研究

Study on two bifunctional fructose-1,6-/sedoheptulose-1,7-bisphosphatases in Serratia marcescens

[目的]明确粘质沙雷氏菌中两个FBPases是否同时具有催化果糖-1,6-二磷酸(F-1,6-BP)与景天庚酮糖-1,7-二磷酸(S-1,7-BP)的功能。[方法]PCR构建p ET28a-Sm-FBPase和p ET28a-Sm-Glp X,IPTG诱导表达于大肠杆菌并应用镍柱纯化靶蛋白。应用体外酶学分析Sm-FBPase与Sm-Glp X酶学特性。[结果]SDS-PAGE结果表明Sm-FBPase、Sm-Glp X的分子量是45 k Da、38 k Da。体外酶学分析表明两个酶均可催化F-1,6-BP和S-1,7-BP。Sm-Glp X是Mn2+或Mg2+依赖型酶,而Sm-FBPase的激活需要Mn2+。根据被测试的底物,Sm-FBPase最适p H 7. 0～7. 5、最适温度40℃; Sm-Glp X最适p H 8. 0、最适温度40～45℃。当F-1,6-BP作底物时,Sm-FBPase与Sm-Glp X的Kcat/Km分别为6. 88、7. 55 S-1mmol-1L。然而,当S-1,7-BP作底物时,Sm-FBPase与Sm-Glp X的Kcat/Km分别是3. 17、8. 54 S-1mmol-1L。[结论]Sm-FBPase和Sm-Glp X均可催化F-1,6-BP与S-1,7-BP,Sm-Glp X催化S-1,7-BP的效率是Sm-FBPase的2. 69倍。

[ Objective ] Experiments were performed to determine whether both of Serratia marcescens FBPases would catalyze fructose - 1,6 - bisphophate （ S - 1,6 - BP） and sedoheptulose - 1,7 - bisphosphate （ S - 1,7 - BP）. [ Methods] PCR was used to generate pET28a - Sm - FBPase and pET2$a -Sm - GlpX. And IPTG was employed to induce their expression in E. co- li and target proteins were purified by Ni - Trap columns, enzymatic traits of Sm - FBPase and Sm - GlpX were analyzed by in vitro enzymatic assays. [ Results ] SDS - PAGE results demonstrated that the molecular weight of Sm- FBPase and Sm - GlpX were 45 kDa and 38 kDa,respectively. Results of in vitro enzymatic assays showed that both enzymes could use fructose - 1,6 - bisphosphate （ F - 1,6 - BP） and S - 1,7 - BP as substrate ; Sm - GlpX was Mn^2＋ or Mg^2＋ dependent enzyme, while the enzyme activity of Sm - FBPase required Mn^2＋ ; the optimal pH and temperature for Sm - FBPase was pH 7.0 to 7.5 and 40 ℃, respectively, while that for Sm - GlpX was pH 8.0 and 40 to 45 ℃, depending on the substrate used in the tests; the Kcat/Km of Sm - FBPase and Sm - GlpX were 6.88 and 7.55 S^-1 mmol^-1 L, when F - 1,6 - BP used as substrate ; while the Kcat/Km of Sm - FBPase and Sm - GlpX were 3.17 and 8.54 S^-1 mmol^-1 L,when S - 1,7 - BP used as substrate,respectively. [ Conclu- sion] Both Sm - FBPase and Sm - GlpX could catalyze F - 1,6 - BP and S - 1,7 - BP,however when S - 1,7 - BP as the substrate,the catalytic efficiency of Sm - GlpX was 2.69 times that of Sm - FBPase.