Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability
Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability
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DOI：
10.1007/s11010-012-1483-8
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发表时间：
2013-01-01
影响因子：
4.3
通讯作者：
Kaur, Jagdeep
中科院分区：
文献类型：
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作者：
Kumar, Rakesh;Sharma, Monika;Kaur, Jagdeep
在本研究中，我们使用定向进化方法从宏基因组起源工程脂肪酶。生成了一个变体S311C，对其进行了详细的表征，并与野生型进行了比较。野生型和变异型脂肪酶过表达并纯化至均匀性。纯化的脂肪酶（变异型和野生型）的最适温度几乎相同，分别为45℃和50℃。与野生型相比，该变异蛋白在60℃时具有很高的热稳定性（54倍），并且比野生型表现出很高的动力学效率。对野生型和变异型脂肪酶的同源性模型分析表明，取代位点位于蛋白表面。这种取代与邻近的疏水残基一起可能参与了导致活性位点的强疏水通道的形成。本研究确定了疏水相互作用在蛋白质稳定性和酶活性增强中的作用。
In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 degrees C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 degrees C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.