重组鱼腥藻脂肪氧合酶定点突变及突变酶酶学性质研究  

Site-directed mutagenesis of lipoxygenase from Anabaena sp.PCC 7120 and enzymatic properties of the mutants

作　　者：刁含文[1] 张充[1] 吕凤霞[1] 别小妹[1] 陆兆新[1] 

机构地区：[1]南京农业大学食品科学与技术学院,江苏南京210095

出　　处：《食品工业科技》2015年第18期160-164,169,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(31470095;31201423);国家863计划(2012AA022207);江苏省科技支撑计划(BE2011390)

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。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.

关 键 词：脂肪氧合酶 定点突变 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]