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Heterologous expression of bacterial cytochrome P450 from Microbacterium keratanolyticum ZY and its application in dichloromethane dechlorination
Environmental Pollution ( IF 8.9 ) Pub Date : 2021-06-15 , DOI: 10.1016/j.envpol.2021.117597
Jun Hu 1 , Yan Zhang 1 , Yuexin Wu 2 , Jiajun Zheng 1 , Zhiliang Yu 3 , Haifeng Qian 1 , Jianming Yu 1 , Zhuowei Cheng 1 , Jianmeng Chen 1
Affiliation  

Dichloromethane (DCM) is a volatile halogenated hydrocarbon with teratogenic, mutagenic and carcinogenic effects. Biodegradation is generally regarded as an effective and economical approach of pollutant disposal. In this study, a novel strain was isolated and its cytochrome P450 was heterologously expressed for DCM degradation. The isolate, Microbacterium keratanolyticum ZY, was characterized as a Gram-positive, rod-shaped and flagella-existed bacterium without spores (GenBank No. SUB8814364; CCTCC M 2019953). After successive whole-genome sequencing, assembly and annotation, eight identified functional genes (encoding cytochrome P450, monooxygenase, dehalogenase and hydrolase) were successfully cloned and expressed in Escherichia coli BL21 (DE3). The recombinant strain expressing cytochrome P450 presented the highest degradation efficiency (90.6%). Moreover, the specific activity of the recombinant cytochrome P450 was more than 1.2 times that of the recombinant dehalogenase (from Methylobacterium rhodesianum H13) under their optimum conditions. The kinetics of DCM degradation by recombinant cytochrome P450 was well fitted with the Haldane model and the value of maximum specific degradation rate was determined to be 0.7 s−1. The DCM degradation might occur through successive hydroxylation, dehydrohalogenation, dechlorination and oxidation to generate gem-halohydrin, formyl chloride, formaldehyde and formic acid. The study helps to comprehensively understand the DCM dechlorination process under the actions of bacterial functional enzymes (cytochrome P450 and dehalogenase).



中文翻译:

溶角微杆菌ZY细菌细胞色素P450的异源表达及其在二氯甲烷脱氯中的应用

二氯甲烷 (DCM) 是一种挥发性卤代烃,具有致畸、致突变和致癌作用。生物降解通常被认为是一种有效且经济的污染物处理方法。在这项研究中,分离出一种新菌株,其细胞色素 P450 异源表达用于 DCM 降解。分离物,溶角质微杆菌ZY,被表征为革兰氏阳性、杆状和鞭毛存在的细菌,没有孢子(基因库编号 SUB8814364;CCTCC M 2019953)。经过连续的全基因组测序、组装和注释,8个已鉴定的功能基因(编码细胞色素P450、单加氧酶、脱卤酶和水解酶)在大肠杆菌中成功克隆并表达BL21 (DE3)。表达细胞色素 P450 的重组菌株的降解效率最高(90.6%)。此外,在最佳条件下,重组细胞色素 P450 的比活性是重组脱卤酶(来自Methylobacterium rhodesianum H13)的1.2 倍以上。重组细胞色素 P450 降解 DCM 的动力学与 Haldane 模型非常吻合,最大比降解速率的值确定为 0.7 s -1. DCM降解可能通过连续的羟基化、脱卤化氢、脱氯和氧化发生,生成偕卤代醇、甲酰氯、甲醛和甲酸。该研究有助于全面了解细菌功能酶(细胞色素P450和脱卤酶)作用下的DCM脱氯过程。

更新日期:2021-06-15
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