Enhanced Thermostability of Lipoxygenase from Anabaena sp PCC 7120 by Site-Directed Mutagenesis Based on Computer-Aided Rational Design
Enhanced Thermostability of Lipoxygenase from Anabaena sp PCC 7120 by Site-Directed Mutagenesis Based on Computer-Aided Rational Design
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DOI：
10.1007/s12010-015-1950-2
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发表时间：
2016-04-01
影响因子：
3
通讯作者：
Lu, Zhaoxin
中科院分区：
文献类型：
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作者：
Diao, Hanwen;Zhang, Chong;Lu, Zhaoxin
鱼腥藻PCC 7120脂肪氧合酶（Ana-LOX）对热不稳定。因此，提高酶的热稳定性是十分必要的。Ana-LOX定点突变的靶位点选择基于计算机辅助合理设计。Ana-LOX的热稳定性和比活性随着在靶位点421和位点40处用丙氨酸取代缬氨酸而提高。与在50 A ℃下具有3.8 min失活半衰期（T（1/2））的野生型酶相比，具有V421 A和V40 A取代的突变酶的T（1/2）分别增加至4.4和7.0 min。双突变体V421 A/V40 A显示出协同效应，T（1/2）值为8.3 min，与原始Ana-LOX相比提高了1.18倍。V421 A、V40 A和V421 A/V40 A也分别获得比活性增加4.83、41.58和80.07%。该研究为酶的分子修饰和计算机辅助合理设计提供了有益的理论参考。
Lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX) was thermally unstable. So, improving the thermostability of the enzyme was quite essential. The target site of Ana-LOX selected for site-directed mutagenesis was based on computer-aided rational design. The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40. Compared to the wild-type enzyme which has a half-life (T (1/2)) of inactivation of 3.8 min at 50 A degrees C, the T (1/2) of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0 min, respectively. The double mutant V421A/V40A showed a synergistic effect with a T (1/2) value of 8.3 min, resulting in a 1.18-fold improvement compared to the original Ana-LOX. V421A, V40A, and V421A/V40A also obtained 4.83, 41.58, and 80.07 % increase in specific activity, respectively. This study provides useful theoretical reference for enzyme molecular modification and computer-aided rational design.