Diao Hanwen, Zhang Chong, Wang Shuicheng, Lu Fengxia, Lu Zhaoxin
Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China.
Appl Biochem Biotechnol. 2016 Apr;178(7):1339-50. doi: 10.1007/s12010-015-1950-2. Epub 2015 Dec 19.
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 °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.
来自鱼腥藻属PCC 7120（Ana-LOX）的脂氧合酶热稳定性较差。因此，提高该酶的热稳定性至关重要。用于定点诱变的Ana-LOX目标位点是基于计算机辅助的合理设计选定的。在目标位点421和位点40处将缬氨酸替换为丙氨酸后，Ana-LOX的热稳定性和比活性得到了提高。与野生型酶相比，野生型酶在50℃下失活的半衰期（T1/2）为3.8分钟，V421A和V40A取代的突变酶的T1/2分别增加到4.4分钟和7.0分钟。双突变体V421A/V40A表现出协同效应，T1/2值为8.3分钟，与原始Ana-LOX相比提高了1.18倍。V421A、V40A和V421A/V40A的比活性也分别提高了4.83%、41.58%和80.07%。该研究为酶分子修饰和计算机辅助合理设计提供了有用的理论参考。
