P. G. Department of Biosciences, UGC-Centre of Advanced Studies, Satellite Campus, Sardar Patel University, Sardar Patel Maidan, Bakrol-Vadtal Road, Bakrol, Gujarat 388 315, India.
Synergy Cignpost Diagnostics, 3 Mills Studio, London, E3 3DU, United Kingdom.
Bioresour Technol. 2022 Apr;349:126870. doi: 10.1016/j.biortech.2022.126870. Epub 2022 Feb 19.
Bioremediation systems coupled to efficient microbial enzymes have emerged as an attractive approach for the in-situ removal of hazardous organophosphates (OPs) pesticides from the polluted environment. However, the role of engineered enzymes in OPs-degradation is rarely studied. In this study, the potential OPs-hydrolase (opdH) gene (Arthrobacter sp. HM01) was isolated, cloned, expressed, and purified. The recombinant organophosphate hydrolase (ropdH) was ∼29 kDa; which catalyzed a broad-range of OPs-pesticides in organic-solvent (∼99 % in 30 min), and was found to increase the catalytic efficiency by 10-folds over the native enzyme (k/K: 10 Ms). The degraded metabolites were analyzed using HPLC/GCMS. Through site-directed mutagenesis, it was confirmed that, conserved metal-bridged residue (Lys-127), plays a crucial role in OPs-degradation, which shows ∼18-folds decline in OPs-degradation. Furthermore, the catalytic activity and its stability has been enhanced by >2.0-fold through biochemical optimization. Thus, the study suggests that ropdH has all the required properties for OPs bioremediation.
生物修复系统与高效微生物酶结合，已成为从污染环境中原位去除危险有机磷酸酯 (OPs) 农药的一种有吸引力的方法。然而，工程酶在 OPs 降解中的作用很少被研究。在这项研究中，分离、克隆、表达和纯化了潜在的 OPs 水解酶 (opdH) 基因 (节杆菌 HM01)。重组有机磷水解酶 (ropdH) 约为 29 kDa；它在有机溶剂中催化广泛范围的 OPs 农药 (30 分钟内约 99%)，并被发现比天然酶提高了 10 倍的催化效率 (k/K: 10 Ms)。使用 HPLC/GCMS 分析降解代谢物。通过定点突变，证实保守的金属桥连残基 (Lys-127) 在 OPs 降解中起着关键作用，其 OPs 降解能力下降约 18 倍。此外，通过生化优化，催化活性和稳定性提高了>2.0 倍。因此，该研究表明 ropdH 具有 OPs 生物修复所需的所有特性。
