Site-directed mutagenesis of lipoxygenase from Anabaena sp.PCC 7120 and enzymatic properties of the mutants
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摘要: 重组鱼腥藻脂肪氧合酶（Ana-LOX）基因来自于Anabaena sp.PCC 7120。利用定点突变技术将Ana-LOX的421位和40位的缬氨酸（Val）突变为丙氨酸（Ala）,获得突变体V421A、V40A和V421A/V40A。野生型Ana-LOX在50℃下半衰期为3.8 min。而突变酶V421A和V40A在50℃的半衰期分别为4.4 min和7.0 min。突变酶V421A/V40A的半衰期为8.3 min,与野生型Ana-LOX相比,提高了1.18倍。相对于野生酶,突变酶V421A、V40A和V421A/V40A的比活力分别提高了4.83%、41.58%、80.07%。突变酶的最适反应温度均比野生酶（35℃）高5℃。改造后的突变酶更能适合工业生产的需要,对于实际应用具有重要价值。Abstract: The thermostability and specific activity of lipoxygenase(Ana-LOX) from Anabaena sp. PCC 7120 were improved with replacing valine with alanine by site-directed mutagenesis. Compared to the wild-type enzyme which had a half-life(T1/2) of inactivation of 3.8 min at 50 ℃,the T1/2of mutant enzymes with V421 A and V40 A substitution increased to 4.4 and 7.0 min,respectively. The double mutant V421A/V40 A showed a synergistic effect with a T1/2value of 8.3 min,resulting in a 1.18-fold improvement compared to the original Ana-LOX.V421 A,V40A and V421A/V40 A also obtained 4.83%,41.58% and 80.07% increase in specific activity,respectively.And,the mutant enzymes obtained 5℃ increase in optimum temperature than the wild-type enzyme(35℃).This study provided useful theoretical reference for enzyme molecular modification and application in vitro.
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Keywords:
- lipoxygenase /
- site-directed mutagenesis /
- enzymatic properties
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