The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.
The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.
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DOI：
10.1371/journal.pone.0047665
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Streit WR
中科院分区：
文献类型：
--
作者：
Chow J;Kovacic F;Dall Antonia Y;Krauss U;Fersini F;Schmeisser C;Lauinger B;Bongen P;Pietruszka J;Schmidt M;Menyes I;Bornscheuer UT;Eckstein M;Thum O;Liese A;Mueller-Dieckmann J;Jaeger KE;Streit WR
三酰甘油脂肪酶（EC www.example.com）催化水解和合成反应，具有广谱底物，使其特别适合于许多生物技术应用。目前使用的大多数脂肪酶来源于嗜温生物，对热变性敏感，而只有少数具有高耐热性。在这里，我们报告的两个新的热稳定性细菌脂肪酶的功能宏基因组筛选确定的鉴定和表征。从维持在65至75 ° C的富集培养物构建宏基因组文库，并进行筛选，从而鉴定出最初10个具有脂解活性的克隆。随后，两个开放阅读框被确定为编码脂肪酶，LipS和LipT。比较序列分析表明，这两种酶是新的脂肪酶家族的成员。LipS是一种30.2 kDa的蛋白质，在70 ° C下的半衰期为48小时。lipT基因编码多聚体酶，在70 ° C下半衰期为3小时。LipS的最适温度为70 ° C，LipT为75 ° C。这两种酶催化长链（C12和C14）脂肪酸酯的水解，并另外水解了许多工业相关的底物。LipS对（R）-布洛芬-苯酯具有高度特异性，对映体过量（ee）为99%。此外，LipS能够在70 ° C下合成月桂酸1-丙酯和肉豆蔻酸1-十四烷基酯，其速率与来自南极假丝酵母的脂肪酶CalB的速率相似。LipS代表具有显著合成活性的热稳定宏基因组衍生的脂肪酶的第一个实例。其X射线结构的解析度为1.99 μ m，揭示了异常紧凑的盖结构。
Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.
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影响因子：
3.7
作者：
Ebrahimi M;Lakizadeh A;Agha-Golzadeh P;Ebrahimie E;Ebrahimi M
通讯作者：
Ebrahimi M
影响因子：
2.7
作者：
Domínguez, A;Sanromán, A;Longo, MA
通讯作者：
Longo, MA
影响因子：
4.7
作者：
Estrada Gutarra, Melissa Limoeiro;de Godoy, Mateus Gomes;Guimaraes Freire, Denise Maria
通讯作者：
Guimaraes Freire, Denise Maria
影响因子：
3.2
作者：
BROCK, TD;FREEZE, H
通讯作者：
FREEZE, H
DOI：
10.1107/s0907444910007493
发表时间：
2010-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Emsley P;Lohkamp B;Scott WG;Cowtan K
通讯作者：
Cowtan K