Current microbiology. 2025 Apr 10;82(6):239. doi: 10.1007/s00284-025-04183-4 Q32.32024

Cloning and Expression of the Isoprocarb Hydrolase Gene cehA from the Newly Isolated Isoprocarb-Degrading Strain Sphingobium sp. R-7

新异丙虫磺降解菌Sphingobium sp. R-7中异丙虫磺水解酶基因cehA的克隆及表达翻译改进

Jing Wu¹, Ao Gao¹, Bohan Wang¹, Yingxin Yang¹, Na Li^{2 3}, Hao Zhang¹

作者单位 +展开

作者单位

¹ College of Life Science, Nanyang Normal University, Nanyang, 473061, Henan, China.

² College of Life Science, Nanyang Normal University, Nanyang, 473061, Henan, China. 20182031@nynu.edu.cn.

³ Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, China. 20182031@nynu.edu.cn.

DOI: 10.1007/s00284-025-04183-4 PMID: 40208354

摘要中英对照阅读

To remove the isoprocarb residues from the environment, a bacterial strain that was capable of degrading isoprocarb was isolated from agricultural soils where isoprocarb has been applied for a long period, and named R-7. On the basis of its cellular morphology and phenotypic features and 16S rRNA phylogenetic analysis, strain R-7 was preliminarily identified as Sphingobium sp. This strain could grow on isoprocarb as a sole carbon source and degrade 98.3% of 0.5 mM of isoprocarb within 16 h in batch liquid culture. The metabolite produced was identified as 2-isopropylphenol by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) analysis. Strain R-7 hydrolysed the ester bond of isoprocarb to generate 2-isopropylphenol, but this metabolite was not further degraded. Upon the genome comparison, the isoprocarb hydrolase gene cehA was cloned from strain R-7 and expressed in Escherichia coli BL21. The purified CehA_R-7 displayed maximal enzymatic activity at 40 °C and pH 7.0. The apparent K_m and k_cat values of CehA_R-7 for isoprocarb were 169.12 ± 7.74 µmol L^-1 and 347 ± 17.73 min^-1, respectively. CehA_R-7 could hydrolyse carbaryl and isoprocarb efficiently, although its ability to hydrolyse carbofuran, oxamyl and methomyl was poor. In conclusion, this study provided an efficient isoprocarb-degrading microorganism, and identified the isoprocarb hydrolase CehA from strain R-7, which has potential applications for microbial remediation of isoprocarb-polluted environments.

Keywords：isoprocarb hydrolase gene; sphingobium sp. r-7; isoprocarb degrading

为了从环境中去除异丙威残留，从长期施用异丙威的农业土壤中分离出一种能够降解异丙威的细菌菌株，并将其命名为R-7。根据细胞形态和表型特征以及16S rRNA系统发育分析，初步鉴定该菌株为鞘氨醇单胞菌（Sphingobium sp.）。该菌株能够在以异丙威作为唯一碳源的液体培养基中生长，并在分批液体培养中于16小时内降解0.5 mM异丙威的98.3%。通过高效液相色谱-串联质谱（HPLC-MS/MS）分析确定产生的代谢物为2-异丙苯酚。菌株R-7水解异丙威中的酯键生成2-异丙苯酚，但该代谢产物不再进一步降解。通过对基因组进行比较，从菌株R-7中克隆了异丙威水解酶基因cehA，并在大肠杆菌BL21中表达。纯化的CehA_R-7在40°C和pH 7.0时显示出最大酶活性。对于异丙威，CehA_R-7的表观K_m值为169.12 ± 7.74 µmol L^-1，k_cat值为347 ± 17.73 min^-1。CehA_R-7能够有效水解异丙威和卡巴呋喃（carbaryl），虽然它对克百威、奥威米和灭多威的水解能力较差。总之，本研究提供了一种高效的异丙威降解微生物，并从菌株R-7中鉴定了异丙威水解酶CehA，该酶在受异丙威污染环境的微生物修复方面具有潜在应用。