聚苹果酸聚合途径中苹果酰辅酶A连接酶基因的克隆、表达及酶学性质

Gene cloning，expression and characterization of malate-CoA ligase in the polymerization pathway of polymalic acid from Aureobasidium pullulans

摘要:

摘要:【目的】研究出芽短梗霉聚苹果酸聚合途径中苹果酰辅酶A连接酶基因及其酶学特性。【方法】通过设计兼并引物，采用IPCR技术从出芽短梗霉CCTCC M2012223的基因组中扩增得到苹果酰辅酶A连接酶基因的cDNA全长序列，构建表达载体，通过大肠杆菌异源表达，Ni-NTA柱层析纯化酶蛋白，分析其酶学特性。【结果】获得苹果酰辅酶A连接酶基因序列全长为1498 bp，编码440 aa，含有4个外显子和3个内含子。该重组酶最适反应温度为25℃，最适反应pH值为8.0，高浓度底物ATP明显对酶活性具有抑制作用，单体选择性表明对底物草酸、草酰乙酸、丁酸、丙二酸也具有很好催化活性。【结论】成功从出芽短梗霉CCTCC M2012223中克隆获得聚苹果酸聚合途径的苹果酰辅酶A连接酶基因，为聚苹果酸聚合途径解析及新型可降解材料创制奠定基础。

Abstract:

Abstract:［Objective］To clone and characterize the malate-CoA ligase in the polymalic acid biosynthetic pathway from Aureobasidium pullulans CCTCC M2012223.［Methods］ The malate-CoA ligase gene was cloned into the expression vector pET-Mcl by IPCR technique，and expressed in Escherichia coli BL21 (DE3)．After purified with Ni-NTA column chromatography，the protein was characterized.［Result］The full-length of malate-CoA ligase gene was 1498 bp，and composed with 440 amino acids containing 4 exons and 3 introns. The optimal temperature and pH was 25℃ and 8.0，respectively，but the high substrate concentration of ATP could obviously inhibited the enzyme activity．The monomer selectivity showed that the enzyme catalyzed the substrates of oxalic acid，oxaloacetic acid，butyric acid，and malonic acid.［Conclusion］ The malate-CoA ligase gene in the polymerization pathway of polymalic acid from Aureobasidium pullulans CCTCC M2012223 was successfully cloned，which will be helpful in deeply understanding the polymerization pathway and producing new polymers．