Cloning and enzymatic analysis of medium-chain acyl coenzyme A synthetase in Populus trichocarpa.
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摘要: 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PtMACS1的基因序列(基因编号:estExt_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PtMACS1与原核表达载体 pET-30a(+) 构建融合表达载体PtMACS1- pET-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(1302.65)、(1933.46)、(2015.51) mol/(minmg),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pH为7.0。该结果表明,PtMACS1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。Abstract: Medium-chain acyl coenzyme A synthetase (MACS) family is a subfamily of adenylate-forming enzymes superfamily, catalyzing the medium-chain fatty acids with CoA to produce medium-chain-acyl-CoA. PtMACS1 (gene model:estExt_fgenesh4_pg.c_640066) was cloned via blast in the database JGI of Populus trichocarpa. Sequence analysis showed that the conserved domains BoxI and BoxII in 4CL were not conserved in the proteins. Expression vector PtMACS1-pET-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 mol/L/(minmg) respectively. When decylic acid was used as the substrate, the optimum temperature was 37 ℃ and pH was 7.0. The results demonstrate that PtMACS1 is one of the MACS family members, and supplies research data in identifying and classifying members from adenylate-forming enzymes superfamily in Populus trichocarpa.
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Keywords:
- Populus trichocarpa /
- MACS /
- gene clone /
- prokaryotic expression /
- enzymatic analysis
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