摘要
精氨酸酶1缺乏症(arginase 1 deficiency,ARG 1-D)是一种罕见的遗传性代谢疾病,导致患者进行性痉挛性瘫痪、认知障碍和癫痫发作.来源于人源的重组人精氨酸酶1(recombinant human arginase 1,rhArg1)是其潜在的治疗药物,但还存在活性低、半衰期短等缺陷限制了其临床应用.本研究采用定向进化的方法,通过易错PCR构建rhArg1的随机突变文库,经高通量筛选获得活性提高的突变体,联合点饱和突变探讨了 R21和V182位点对活性的影响.研究发现,在反应体系中不含Mn2+的条件下,突变体V182D、V182S、V182H和R21N的kcat值相比于rhArg1提高 2.0、1.9、1.7 和 1.3 倍,突变体 V182D、V182S、R21D 和 R21N 的kcat/Km分别是 rhArg1 的 2.1、1.7、1.4 和 1.4倍.突变体R21D和V182L对底物的亲和力有所增强.本研究通过定向进化和点饱和突变获得了 rhArg1活性提高的突变体,从而提升了其在医疗领域的应用前景.
Abstract
Arginase 1 deficiency(ARG1-D)is a rare genetic metabolic disorder that leads to progressive spastic paralysis,cognitive impairment,and seizures.Recombinant human arginase 1(rhArg1)is a potential therapeutic agent for this condition,but its clinical application is limited by low activity and short half-life.In this study,we employed directed evolution to address these issues.A random mutation library of rhArgl was constructed using error-prone PCR,and high-throughput screening was used to identify mutants with enhanced activity.Site-saturation mutagenesis was also performed to investigate the effects of residues R21 and V182 on enzyme activity.Our findings revealed that under reaction conditions devoid of Mn2+,the kcat values of the mutants V182D,V182S,V182H,and R21N increased by 2.0,1.9,1.7,and 1.3 times respectively,compared to rhArg1.The kcat/Km values of mutants V182D,V182S,R21D,and R21N were 2.1,1.7,1.4,and 1.4 times higher than those of rhArg1,respectively.Additionally,mutants R21D and V182L showed enhanced substrate affinity.Through directed evolution and site-saturation mutagenesis,we successfully obtained rhArg1 mutants with improved activity,thereby enhancing its potential for clinical application.
维普
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