毛果杨中链酰基辅酶A合成酶的克隆及酶学分析

Cloning and enzymatic analysis of medium-chain acyl coenzyme A synthetase in Populus trichocarpa

中链酰基辅酶A合成酶(MACS)属于腺苷酸合成酶超基因家族,催化中链脂肪酸与辅酶A结合形成相应的中链酰基辅酶A。本研究通过同源检索毛果杨基因组数据库中的4CL基因,克隆得到毛果杨Pt MACS1的基因序列(基因编号:est Ext_fgenesh4_pg.c_640066)。通过序列分析可知,Box I和Box II两个在4CL中保守的结构域在该蛋白中并不保守。将Pt MACS1与原核表达载体p ET-30a(+)构建融合表达载体Pt MACS1-p ET-30a(+),转化大肠杆菌BL21(DE3)后诱导重组蛋白进行表达。镍柱纯化重组蛋白后进行酶学活性分析,研究结果表明该蛋白对中链脂肪酸己酸、壬酸、癸酸显示出明显活性:Kcat分别为(130±2.65)、(193±3.46)、(201±5.51)μmol/(min·mg),以该蛋白的最适底物癸酸测得该蛋白的最适反应温度为37℃,最适反应p H为7.0。该结果表明,Pt MACS1属于毛果杨中链酰基辅酶A合成酶家族一员,为后续毛果杨腺苷酸合成酶超基因家族的鉴定及分类分析提供资料。

Medium-chain acyl coenzyme A synthetase（ MACS） family is a subfamily of adenylate-forming enzymes superfamily,catalyzing the medium-chain fatty acids with Co A to produce medium-chain-acylCo A. PtM ACS1（ gene model： estE xt_fgenesh4_pg. c_640066） was cloned via blast in the database JGI of Populus trichocarpa. Sequence analysis showed that the conserved domains Box I and Box II in 4CL were not conserved in the proteins. Expression vector PtM ACS1-p ET-30a（ ＋） was constructed and transformed into E. coli BL21（ DE3） to express the recombinant protein. The recombinant protein was purified by Ni-NTA affinity chromatography,enzymatic analysis showed that the recombinant protein had remarkable catalytic activity to the medium-chain fatty acids,such as hexanoic acid,nonanoic acid and decylic acid,and the Kcat were 130,193 and 201 μmol / L /（ min·mg） respectively. When decylic acid was used as the substrate,the optimum temperature was 37 ℃ and p H was 7. 0. The results demonstrate that PtM ACS1 is one of the MACS family members,and supplies research data in identifying and classifying members from adenylate-forming enzymes superfamily in Populus trichocarpa.