Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx.P. 19046 Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil.
Departamento de Química, Universidade Federal do Paraná, Cx.P. 19081 Centro Politécnico, Curitiba, 81531-980, Paraná, Brazil.
Sci Rep. 2018 Jul 3;8(1):10000. doi: 10.1038/s41598-018-27579-8.
We determined the effect of the His-tag on the structure, activity, stability and immobilization of LipC12, a highly active lipase from a metagenomic library. We purified LipC12 with a N-terminal His-tag and then removed the tag using tobacco etch virus (TEV) protease. Circular dichroism analysis showed that the overall structure of LipC12 was largely unaffected by His-tag removal. The specific hydrolytic activities against natural and artificial substrates were significantly increased by the removal of the His-tag. On the other hand, His-tagged LipC12 was significantly more active and stable in the presence of polar organic solvents than untagged LipC12. The immobilization efficiency on Immobead 150 was 100% for both forms of LipC12 and protein desorption studies confirmed that the His-tag does not participate in the covalent binding of the enzyme. In the case of immobilized LipC12, the His-tag negatively influenced the hydrolytic activity, as it had for the free lipase, however, it positively influenced the esterification activity. These results raise the possibility of tailoring recombinant lipases for different applications, where the His-tag may be retained or removed, as appropriate for the desired activity.
我们确定了 His 标签对 LipC12（一种来自宏基因组文库的高活性脂肪酶）的结构、活性、稳定性和固定化的影响。我们用 N 端 His 标签纯化 LipC12，然后用烟草蚀纹病毒（TEV）蛋白酶去除标签。圆二色性分析表明，His 标签去除对 LipC12 的整体结构影响不大。去除 His 标签后，对天然和人工底物的特异性水解活性显著提高。另一方面，与未标记的 LipC12 相比，带有 His 标签的 LipC12 在极性有机溶剂中具有更高的活性和稳定性。两种形式的 LipC12 在 Immobead 150 上的固定化效率均为 100%，蛋白质解吸研究证实 His 标签不参与酶的共价结合。在固定化 LipC12 的情况下，His 标签对水解活性产生负面影响，就像对游离脂肪酶一样，然而，它对酯化活性产生积极影响。这些结果提出了一种可能性，即可以根据所需的活性，适当地保留或去除重组脂肪酶中的 His 标签，以适应不同的应用。
