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【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°C,最适p H为7.0。70°C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。
【Aim】 To search for a biocatalyst capable of synthesizing polyketide precursors such as propionyl-CoA and butyryl-CoA, an ester acyl-CoA synthase was characterized by in vitro enzymatic experiments. 【Method】 Using the malonyl-CoA synthetase as input sequence, one ester acyl-CoA synthetase gene was found in the genome of Caldicellulosiruptor owensensis OL by BLAST. Heterologous expression in E. coli and purification by affinity chromatography. Substrate spectra, optimum reaction conditions, stability and kinetic parameters were characterized by in vitro enzymatic assays, while site-directed mutagenesis was used to analyze amino acid residues in the active site. 【Result】 The results showed that this enzyme has good substrate specificity and can recognize propionic acid, butyric acid, 2-methylpropionic acid, pentanoic acid, 3-methylbutyric acid, 2-methylbutyric acid and cyclohexanecarboxylic acid A series of monoacids. The optimum reaction temperature is 30 ° C, the optimum p H is 7.0.70 ° C incubated for 8 h after 45% residual activity, indicating that the enzyme is relatively stable. Site-directed mutagenesis at three sites in the active site can alter the substrate specificity of the enzyme. 【Conclusion】 The ester acyl-CoA synthetase derived from C.owensensis OL is a potential biocatalyst and can be used for the synthesis of polyketide precursors.