Applied Biochemistry and Biotechnology ( IF 3.1 ) Pub Date : 2021-08-20 , DOI: 10.1007/s12010-021-03645-2 Oladipupo A Aregbesola 1 , Ajit Kumar 1 , Mduduzi P Mokoena 1 , Ademola O Olaniran 1
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The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64 kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30 °C and 7.0, respectively. CpsB showed functional stability between pH 6.0–7.5 and temperature 25–30 °C. The enzyme–substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 μM.s−1, 140 μM, 0.099 s−1 and 7.07 × 10−4 μM−1.s−1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.
Graphical abstract
中文翻译：
来自本土热带芽孢杆菌菌株 AOA-CPS1 的经典五氯酚羟化苯丙氨酸 4-单加氧酶：克隆、过表达、纯化、表征和结构同源性建模
此处检测、扩增（来自热带芽孢杆菌菌株 AOA-CPS1 的基因组）、克隆、过表达、纯化和表征代谢混杂的五氯苯酚 (PCP) 羟基化 Phe4MO（表示为 CpsB）。将1.755kb的基因克隆到pET15b载体中，表达≅64kDa的单体蛋白，经一步亲和层析纯化至均质，总产率为82.1%。发现该酶的最适温度和pH分别为30℃和7.0。 CpsB 在 pH 6.0–7.5 和温度 25–30 °C 之间表现出功能稳定性。酶-底物反应动力学研究显示了酶的变构性质，并使用 NADH 作为共底物遵循预稳态，表观v max 、 K m 、 k cat和k cat / K m值为 0.465 μMs -1 ， 140 μM、0.099 s -1和7.07 × 10 -4 μM -1。 s -1 ，分别用于基板PCP。凝胶内胰蛋白酶消化实验和生物信息学工具证实所报道的酶是具有多个假定保守结构域和金属离子结合位点的 Phe4MO。尽管已报道 Phe4MO 具有多种催化功能，但在此我们首次报道，它通过羟基化 PCP 发挥 PCP 脱卤酶或 PCP-4-单加氧酶的作用。因此，可以进一步探索这种酶在五氯苯酚和其他相关异生物质的生物修复中的用途。
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