Directed evolution to enhance the catalytic activity of human arginase 1
10.16438/j.0513-4870.2024-0489
- VernacularTitle:定向进化提高人精氨酸酶1的催化活性
- Author:
Cui-yue FENG
1
;
Chen-yu WANG
1
;
Meng-jia TANG
1
;
Shuai FAN
2
;
Zhao-yong YANG
2
;
Zhi-fei ZHANG
1
Author Information
1. School of Pharmacy, North China University of Science and Technology, Tangshan
2. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing
- Publication Type:Research Article
- Keywords:
arginase 1 deficiency;
recombinant human arginase 1;
irected evolution;
site-saturation mutagenesis
- From:
Acta Pharmaceutica Sinica
2024;59(12):3402-3408
- CountryChina
- Language:Chinese
-
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 rhArg1 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.
