热稳定酯酰辅酶A合成酶的异源表达及酶学特性

Heterologous expression and characterization of a thermostable acylCoA synthetase

摘要:

[目的]为寻找能合成丙酰辅酶A和丁酰辅酶A等聚酮合成前体的生物催化剂，用体外酶学实验对一个酯酰辅酶A合成酶进行了表征。[方法]利用丙二酰辅酶A合成酶作为输入序列，通过BLAST程序在Caldicellulosiruptor owensensis OL的基因组中找到1个酯酰辅酶A合成酶基因。在大肠杆菌中进行了异源表达，并通过亲和层析进行纯化。底物谱、最适反应条件、稳定性和动力学参数通过体外酶学实验进行表征，而定点突变则用于活性中心的氨基酸残基的分析。[结果]该酶具有较好的底物宽泛性，可识别丙酸、丁酸、2-甲基丙酸、戊酸、3-甲基丁酸、2-甲基丁酸以及环己甲酸等一系列单酸。反应最适温度为30℃，最适pH为7.0。70℃保温8 h后仍有45%的活性残留，表明该酶相对比较稳定。通过活性中心3个位点的定点突变可以改变酶的底物特异性。[结论] C. owensensis OL来源的酯酰辅酶A合成酶是潜在的生物催化剂，可以用于聚酮前体的合成。

Abstract:

[Objective] An acyl-CoA synthetase (ACS) was characterized by in vitro biochemical assays to get a potential biocatalyst for the generation of polyketide precursors such as propionyl-CoA and butyryl-CoA.[Methods] An ACS was identified in Caldicellulosiruptor owensensis OL genome by BLAST program using malonyl-CoA synthetase as the query sequence. The enzyme was heterologously expressed in Escherichia coli cells and purified to homogeneity by affinity purification. In vitro biochemical assays were used to reveal the substrate profile, the optimum reaction condition, the stability and the kinetics of the enzyme while the site-directed mutagenesis was used to assay the function of active site residues.[Results] The enzyme was promiscuous enough to accept propionic acid, butyric acid, 2-methyl-propionic acid, pentanoic acid, 2-methyl-butyric acid, 3-methyl-butyric acid and cyclohexanecarboxylic acid as the substrates. It was most active at 30℃ and pH 7.0 and relatively stable because 45% activity remained after 8 hours incubation at 70℃. Interestingly, the substrate specificity of the enzyme could be switched by engineering three residues of active site.[Conclusion] The ACS from C. owensensis OL was a potential biocatalyst for the generation of polyketide precursors.