Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1
Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1
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DOI：
10.1016/s1381-1177(03)00064-x
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发表时间：
2003-10-01
影响因子：
--
通讯作者：
Cao, SG
中科院分区：
文献类型：
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作者：
Gao, RJ;Feng, Y;Cao, SG
将嗜热好氧菌Aeropyrum pernix K1的APE 1547基因克隆到大肠杆菌中，编码582个氨基酸残基。BL 21（DE 3）的表达载体pET 11 a。将APE 1547与A. pernix K1数据库显示，它显示出脂肪酶基序和低同源性与已知的嗜热酯酶。但与几种酰基氨基酸释放酶有高度同源性。重组蛋白经离子交换层析和凝胶过滤层析纯化后，具有酯酶活性和酰基氨基酸释放酶（AARE）活性。酶的最适反应温度为90 ℃，最适反应pH为8.0。该重组蛋白对多种底物具有水解活性，如不同烷基链长度的对硝基苯基链烷酸酯、PNA标记的氨基酸和肽。对底物辛酸对硝基苯基酯观察到最高活性。重组酶非常稳定，并且具有蛋白浓度依赖性。在90 ℃时的半衰期超过160 h。浓度为2.14 mg/ml，这使得这种新的酯酶对于生物技术应用非常有吸引力。(C)2003 Elsevier B. V.保留所有权利。
The gene APE 1547 of the aerobic thermophilic Aeropyrum pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli. BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90 degreesC and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p-nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90 degreesC was over 160 h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications. (C) 2003 Elsevier B.V. All rights reserved.