大肠杆菌苹果酸合酶A的酶学和生理功能研究王程;王敖;赵旵军;朱国萍【摘要】Malate synthase is one of the key enzymes in glyoxylate cycle. The aceB gene, encoding malate synthase A ( MSA) , was amplified from the genomic DNA of Escherichia coli MC1655 using PCR with primers designed according to the sequence of E. coli genome. The PCR product was cloned into pET-29b( + ) , resulting the recombinant plasmid pET-MSA. The target protein ( MSA) was overexpressed in E. coli Rosetta ( DE3) under IPTG induction, and then the enzyme was purified to homogeneity. The molecular weight ( MW) of MSA was about 60 kDa. The optimal pH and temperature of MSA were pH 8. 0 and 30 C, respectively. The maximum activity of purified MSA was observed in the presence of Mg2+ . The Km and Vmax values for acetyl-CoA of MSA were 8. 07 μM and 3. 6μM/min, respectively. Furthermore, the mutant strains, MG: : ΔaceB with MSA deletion and MG: : ΔaceBΔglcB with MSA and malate synthase G( MSG) deletion, were constructed, respectively. It was observed that the growth rate of the mutant E. coli strain with MSA deletion was remarkably lower than that of the wild-type strain, indicating that MSA was essential for E. coli growth on acetate. Although MSG was able to partially recover the function of MSA , the glyoxylate bypass containing MSA was the much more effective metabolic pathway of glyoxylate.%苹果酸合酶是乙醛酸循环的关键酶之一.E.coli中苹果酸合酶A(malate synthase A,MSA)由aceB基因编码.根据E.coli基因组序列设计引物,利用PCR技术扩增aceB基因,并将其克隆入pET-29t,(+),构建了重组表达质粒pET-MSA.经IPTG诱导,MSA在E.coli Rosetta(DE3)中获得高效表达.纯化的MSA蛋白的分子量大小约为60 kDa,最适反应pH值和最适温度分别是pH值8.0、30℃.纯化的蛋白质在Mg2+存在时才能发挥最大的活性,其对乙酰辅酶A的Km和Vmax分别是8.07μM和3.6μM/min.此外构建了MSA和苹果酸合酶G(MSG)基因敲除菌株MG::AaceB和MG::AaceBAglcB.研究发现缺少MSA的E.coli突变菌株在乙酸中的生长速率要比野生型菌株慢很多,表明MSA对大肠杆菌在乙酸中的生长起着重要作用.MSG虽然能部分补偿MSA的作用,但是包含MSA的乙醛酸旁路是更有效的乙醛酸代谢途径.【期刊名称】《生物学杂志》【年(卷),期】2011(028)002【总页数】5页(P39-42,46)【关键词】苹果酸合酶A;表达;动力学;生长速率;大肠杆菌【作者】王程;王敖;赵旵军;朱国萍【作者单位】安徽师范大学生命科学学院,芜湖241000;安徽师范大学生命科学学院,芜湖241000;芜湖市第二人民医院检验科,芜湖241000;安徽师范大学生命科学学院,芜湖241000【正文语种】中文【中图分类】Q7SAbstract:Malate synthase is one of the key enzymes in glyoxylatecycle.TheaceBgene,encoding malate synthase A(MSA),was amplified from the genomic DNA ofEscherichia coliMG1655 using PCR with primersdesigned according to the sequence ofE.coligenome.The PCR productwas cloned into pET-29b(+),resulting the recombinant plasmid pET-MSA.The target protein(MSA)was overexpressed inE.coliRosetta(DE3)under IPTG induction,and then the enzyme was purified to homogeneity.The molecular weight(MW)ofMSA was about60 kDa.The optimalpH and temperature ofMSA were pH 8.0 and 30 C,respectively.Themaximum activity of purifiedMSA was observed in the presenceofMg2+.TheKmandVmaxvalues for acetyl-CoA ofMSA were 8.07μM and 3.6 μM/min,respectively.Further more,the mutantstrains,MG::ΔaceBwithMSA deletion andMG::ΔaceBΔglcBwithMSA andmalate synthase G(MSG)deletion,wereconstructed,respectively.Itwasobserved that the growth rate of themutantE.colistrainwithMSA deletion was remarkably lower than that of the wild-type strain,indicating thatMSA was essential forE.coligrowth on acetate.AlthoughMSGwas able to partially recover the function ofMSA,the glyoxylate bypass containingMSA was themuchmore effectivemetabolic pathway of glyoxylate.Keywords:malate synthase A;expression;kinetics;growth rate;Escherichia coli乙醛酸循环又称乙醛酸途径,是三羧酸循环的回补途径,两者之间存在着某些相同的酶类和中间产物。
乙醛酸循环中有两个关键性酶,即异柠檬酸裂解酶(isocitrate lyase,ICL)和苹果酸合酶 (malate synthase,MS),前者催化异柠檬酸生成乙醛酸和琥珀酸,而后者催化乙醛酸和乙酰辅酶 A缩合形成苹果酸和辅酶 A。
乙醛酸循环的终产物将参与糖异生途径以及其它的生物合成途径[1]。
乙醛酸循环在古菌、细菌、真菌、原生动物以及萌发的植物种子中均有发现[1-2]。
此外,一些研究报道在脊椎动物中也发现了 ICL和MS的活性,尽管人们认为这些动物中并不存在乙醛酸循环。
在微生物和高等植物幼苗以 C2化合物为原料合成碳水化合物的过程中,乙醛酸循环起着十分重要的作用[3]。
同时,一些研究也指出在分枝杆菌 (M ycobacteria)中,病菌的持续感染能力与乙醛酸循环直接相关[4]。
苹果酸合酶有两种同工酶,即苹果酸合酶A和苹果酸合酶 G,分别简称为MSA和MSG。
MSG仅存在于细菌中,而 MSA在细菌、真菌和植物中都有发现。
E.coli同时含有这两种苹果酸合酶,其中MSA由 aceB基因编码,参与乙醛酸循环,MSG由glcB基因编码,与乙醇酸盐(glycolate)的利用有关[5]。
本文依据 GenBank中已报道的 E.coli基因组序列,克隆了 aceB基因,实现了高效表达与纯化,并对MSA的酶学性质进行了鉴定。
另一方面,利用染色体同源重组技术敲除了 E.coliMG1655中的 aceB基因和glcB基因,通过测定缺陷型菌株的生长速率,对 MSA的生理功能进行了初步研究。
1.1 材料1.1.1 菌株和质粒E.coliMG1655和DH5α为本实验室保存,质粒pET-29b(+)及宿主菌E.coliRosetta(DE3)由中国科学技术大学合肥微尺度物质科学国家实验室周丛照教授惠赠。
1.1.2 培养基和试剂LB、SOB、SOC和MD培养基的配方见文献[6]。
生长曲线测量时使用的乙酸-MOPS和葡萄糖-MOPS培养基中分别包含 2%的乙酸和 2%的葡萄糖,如文献所述[7]。
DNA聚合酶、限制性内切酶购于 New England Biolabs,T4 DNA连接酶、质粒提取试剂盒(W izard®Plus SVminipreps DNA Purification System)、胶回收试剂盒(W izard®SV Gel and PCR Clean-Up System)购于 Promega公司,高保真PrimeSTAR®HS DNA聚合酶购于TaKaRa公司,标准蛋白分子量购于Fermentas公司,蛋白质纯化试剂盒购于 Clontech公司。