Directed evolution to enhance the catalytic activity of human arginase 1
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.
arginase 1 deficiencyrecombinant human arginase 1directed evolutionsite-saturation mutagenesis
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维普
