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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (4): 63-69    DOI: 10.13523/j.cb.20180409
技术与方法     
Acyl-ACPs的规模化合成
丁威1,2,冯延宾1,曹旭鹏1,薛松1()
1 中国科学院大连化学物理研究所 大连 116023
2 中国科学院大学 北京 100049
Large-Scale Synthesis of Acyl-ACPs
Wei DING1,2,Yan-bin FENG1,Xu-peng CAO1,Song XUE1()
1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
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摘要：

脂酰-酰基载体蛋白(fatty acyl-acyl carrier protein, acyl-ACP)是多种生物合成途径中的酰基供体。因供给限制,体外研究常用类似物acyl-CoA替代,而CoA部分和ACP有较大差异,限制了相关酶对底物识别的认识。因此稳定获得大量acyl-ACP是体外研究相关酶的催化机制及其代谢途径的关键。研究以holo-ACP和C4~C18链长脂肪酸为底物,在哈氏弧菌acyl-ACP合成酶(Vibrio harveyi acyl-ACP synthetase, VhAasS)催化下合成不同碳链长度的acyl-ACP;通过高效液相色谱(HPLC)方法,确定不同碳链长度acyl-ACP的合成产率。结果表明:碳链为C4~C14的acyl-ACP产率均高于90.0%,16:0-ACP产率为85.9%,18:1-ACP产率仅为25.7%。通过加入Li +优化反应体系,16:0-ACP、18:1-ACP的产率达90.0%。进一步优化扩大反应体系可稳定获得20mg以上acyl-ACP;最后,把合成的acyl-ACP应用到甘油-3-磷酸酰基转移酶催化的反应体系中。不同链长acyl-ACP的规模化合成研究,为体外研究相关酶的催化机制提供重要基础。

关键词： acyl-ACP合成酶;  acyl-ACP;  高效液相色谱    
Abstract:

Acyl-acyl carrier proteins (acyl-ACPs) are substrates for many biosynthesis pathways. However, acyl-ACP is substituted by acyl-CoA for studies in vitro as a result of supply restriction, which causes many questions in enzymatic analysis. Thus, obtaining large scale of acyl-ACP steadily is very important to study the related enzymes and metabolic pathways in vitro. acyl-ACP synthetase catalyze the conversion of holo-ACP using fatty acid as acyl donor in vitro while no productivity has been reported before. Here an acyl-ACP synthetase from Vibrio harveyi was used to catalyze the synthesis of (C4~C18) with holo-ACP, and the yield of acyl-ACP was confirmed by high performance liquid chromatography (HPLC). The results indicated that the yields of medium chains (C4~C14) acyl-ACPs were more than 90.0% while the long chain yields of 16:0-ACP and 18:1-ACP were 85.9% and 25.7%, respectively. Via introducing Li + to the reaction system, the yield of long chain acyl-ACPs were elevated above 90.0%。Then the reaction parameters were optimized in the enlarged reaction system, and more than 20mg acyl-ACPs were steadily obtained. Additionally, two species of holo-ACP were used to validate the versatility of the reaction system. Finally the acyl-ACP activity was conformed using a glycerol-3-phosphate acyltransferase. The synthesis of different chain acyl-ACPs are of great significance for research of the catalysis mechanism of related enzymes.

Key words: acyl-ACP synthetase    acyl-ACP    HPLC
收稿日期: 2018-01-30 出版日期: 2018-05-08
ZTFLH:  Q816  
基金资助: * 国家自然科学基金资助项目(21576253)
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丁威
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引用本文:

丁威,冯延宾,曹旭鹏,薛松. Acyl-ACPs的规模化合成[J]. 中国生物工程杂志, 2018, 38(4): 63-69.

Wei DING,Yan-bin FENG,Xu-peng CAO,Song XUE. Large-Scale Synthesis of Acyl-ACPs. China Biotechnology, 2018, 38(4): 63-69.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180409    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I4/63

酶 参与的反应和代谢过程 参考文献
硫酯酶 催化不同链长acyl-ACP水解;参与脂肪酸、聚酮类抗生素合成 [1]
脂肪酸去饱和酶 催化acyl-ACP去饱和;参与不饱和脂肪酸合成 [9]
甘油-3-磷酸酰基转移酶 催化acyl-ACP和甘油-3-磷酸反应,生成LPA;参与磷脂合成 [2]
聚酮合酶 催化酮脂酰ACP缩合;参与聚酮类抗生素的合成 [3]
UDP-氨基葡萄糖
乙酸酐酰基转移酶		 催化β-羟肉豆蔻酸和UDP-氨基葡萄糖乙酸酐,生成UDP-3-单酰氨基葡萄糖乙酸酐;
参与类脂A的合成		 [4]
表1  以acyl-ACP为底物的酶
图1  acyl-ACP的合成方法
时间
(min)		 流量
(ml/min)		 乙腈
(%)		 水
(1‰三氟乙酸)
0 1 35 65
15 1 55 45
16 1 35 65
20 1 35 65
表2  HPLC流动相的梯度洗脱程序
合成体系中各成分 50μl合成体系
各成分含量1)		 20ml合成体系
各成分含量2)
FA-Na (μmol/L) 150 200
holo-ACP (μmol/L) 50 75
MgCl2 (mmol/L) 10 10
Li2SO4 (mmol/L) 10 10
ATP (mmol/L) 5 10
DTT (mmol/L) 2 2
Tris-HCl (mmol/L) 25 25
温度 (℃) 30 30
时间 (h) 0.5 2
表3  acyl-ACP合成反应体系
图2  HPLC检测apo-ACP和holo-ACP
acyl-ACP 保留时间(min) Aholo-ACP Aacyl-ACP 产率(%)
4:0-ACP 6.63 4 567.56 4 432.46 97.0
6:0-ACP 7.77 6 015.08 5 616.05 93.4
8:0-ACP 8.52 3 513.82 3 460.80 98.5
10:0-ACP 9.75 10 421.60 9 372.87 90.0
12:0-ACP 11.07 6 816.42 6 607.80 96.9
14:0-ACP 12.48 4 073.94 3 676.46 90.2
16:0-ACP 13.57 2 201.83 1 890.69 85.9
18:1-ACP 13.89 2 201.83 565.83 25.7
表4  不同链长acyl-ACP的保留时间及产率
反应体系 保留时间(min) Aholo-ACP Aacyl-ACP 产率(%)
a 18:1-ACP, 0.5% Triton X-100 14.61 2 201.83 785.83 35.8
b 18:1-ACP,10mmol/L Li2SO4 13.89 2 190.19 1 943.85 88.8
c 16:0-ACP,10mmol/L Li2SO4 13.58 2 190.19 2 092.82 95.5
表5  不同反应条件下acyl-ACP的合成效率
图3  Sp6803 ACPs 的高效液相色谱图
反应体系 保留时间(min) Aholo-ACP Aacyl-ACP 产率(%)
a Table 1.2.3b with 5mmol/L ATP 14.61 9 962.66 5 123.59 51.4
b Table 1.2.3b with 10mmol/L ATP 14.61 9 962.66 8 485.48 85.2
表6  18:1-ACP规模化合成的条件优化
图4  acyl-ACP的生物活性检测
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