Jeon Jeong Ho, Lee Hyun Sook, Lee Jung Hun, Koo Bon-Sung, Lee Chang-Muk, Lee Sang Hee, Kang Sung Gyun, Lee Jung-Hyun
Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan, 15627 Republic of Korea ; National Leading Research Laboratory of Drug Resistance Proteomics, Department of Biological Sciences, Myongji University, 116 Myongjiro, Yongin, Gyeonggido, 17058 Republic of Korea.
Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan, 15627 Republic of Korea ; Department of Marine Biotechnology, University of Science and Technology, Daejeon, 34113 Republic of Korea.
Springerplus. 2016 Apr 26;5:525. doi: 10.1186/s40064-016-2172-y. eCollection 2016.
A metagenomic library was constructed from a soil sample of spindle tree-rhizosphere. From this library, one clone with esterase activity was selected. The sequence analysis revealed an open reading frame (EstSTR1) encoded protein of 390 amino acids. EstSTR1 is a family VIII carboxylesterase and retains the S-X-X-K motif conserved in both family VIII carboxylesterases and class C β-lactamases. The estSTR1 gene was overexpressed in E. coli and the recombinant protein was purified by purified by metal chelating affinity chromatography and size-exclusion chromatography. EstSTR1 hydrolysed p-nitrophenyl esters, exhibited the highest activity toward p-nitrophenyl butyrate. Furthermore, EstSTR1 could hydrolyse third- and fourth-generation cephalosporins (cefotaxime and cefepime) as well as first-generation cephalosporin (cephalothin). Site-directed mutagenesis studies revealed that a catalytic residue, Ser71, of EstSTR1 plays an essential role in hydrolysing both antibiotics and p-nitrophenyl esters. We demonstrate that a metagenome-derived carboxylesterase displays β-lactam-hydrolysing activities toward third- and fourth-generation cephalosporins.
从卫矛属植物根际土壤样本构建了一个宏基因组文库。从该文库中筛选出一个具有酯酶活性的克隆。序列分析显示一个开放阅读框（EstSTR1）编码390个氨基酸的蛋白质。EstSTR1是VIII族羧酸酯酶，保留了VIII族羧酸酯酶和C类β-内酰胺酶中保守的S-X-X-K基序。estSTR1基因在大肠杆菌中过表达，重组蛋白通过金属螯合亲和层析和尺寸排阻层析进行纯化。EstSTR1能水解对硝基苯酯，对对硝基苯丁酸酯表现出最高活性。此外，EstSTR1能水解第三代和第四代头孢菌素（头孢噻肟和头孢吡肟）以及第一代头孢菌素（头孢噻吩）。定点突变研究表明，EstSTR1的催化残基Ser71在水解抗生素和对硝基苯酯中起关键作用。我们证明了一种宏基因组来源的羧酸酯酶对第三代和第四代头孢菌素具有β-内酰胺水解活性。
