大肠杆菌苹果酸合酶A的酶学和生理功能研究

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-29b(+),构建了重组表达质粒pET-MSA。经IPTG诱导,MSA在E.coliRosetta(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::ΔaceB和MG::ΔaceBΔglcB。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。

Characterization and physiological role of malate synthase A in Escherichia coil

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 MG1655 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 3.6 μM/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.