土壤细菌新Mn (II)氧化蛋白PomA的异源表达及其酶学特性

Heterologous expression and enzymatic characterization of PomA, a novel Mn(II)-oxidizing protein derived from a soil bacterium

摘要:

【背景】锰元素在生物地球化学循环中发挥着重要作用，在自然界主要以锰氧化物形式存在。细菌介导的Mn (II)氧化作用是地表锰氧化物形成的重要因素。当前关于细菌Mn (II)氧化蛋白研究多集中于水生细菌，而土壤细菌Mn (II)氧化蛋白研究有待加强。【目的】以土壤锰氧化普罗威登斯菌(Providencia manganoxydans) LLDRA6为研究对象，克隆鉴定新的Mn (II)氧化蛋白。【方法】提取菌株LLDRA6总DNA，设计引物克隆目的基因pomA，利用大肠杆菌(Escherichia coli)表达菌株BL21和表达载体pET-32a (+)对其进行异源表达，超声破碎获得粗酶液，使用Ni-NTA琼脂糖蛋白纯化树脂纯化目的蛋白PomA，研究该蛋白体外Mn (II)氧化活性及酶学特性。【结果】成功构建了表达菌株BL21-pET-pomA，分离纯化出PomA蛋白，其分子量大小为59.4 kDa。序列分析表明，PomA具有4个保守的铜离子结合位点，属于多铜氧化酶。液体LB培养基培养发现，BL21-pET-pomA的Mn (II)氧化能力是BL21-pET的3.6倍。体外试验表明，PomA同时具有Mn (II)氧化活性和漆酶活性。其中，PomA的Mn (II)氧化最适反应温度为60℃，最适反应pH值为7.8，酶学参数K_m为(3.11±0.02) mmol/L，V_max为3.931×10^–6 mol/(L·min)，k_cat为2.38 min^–1。PomA的漆酶活性最适反应温度为60℃，最适反应pH值为3.0，K_m为(47.29±0.02)μmol/L，V_max为3.401×10^–6 mol/(L·min)，k_cat为0.23 s^–1。【结论】PomA属于多铜氧化酶，同时具有Mn (II)氧化活性和漆酶活性。由于其氨基酸序列与其它Mn (II)氧化多铜氧化酶存在较大差异，可能导致它们在催化活性上存在差异。

Abstract:

[Background] The element manganese (Mn) plays an essential role in the biogeochemical cycling and predominantly exists in the form of manganese oxides in nature. Bacterium-mediated Mn(II) oxidation plays a significant role in the formation of manganese oxides on the Earth’s surface. Currently, most studies on proteins involved in bacterial Mn(II) oxidation focus on aquatic bacteria, while the further research is needed for understanding the proteins involved in Mn(II) oxidation in soil bacteria. [Objective] To identify a novel Mn(II)-oxidizing enzyme derived from the soil bacterium Providencia manganoxydans LLDRA6. [Methods] PCR was conducted with the genomic DNA of strain LLDRA6 as the template and specific primers to amplify pomA. Subsequently, the gene was heterologously expressed in Escherichia coli strain BL21 via the plasmid pET-32a(+). The target protein PomA was purified by a Ni-NTA agarose protein purification kit. The Mn(II)-oxidizing activity and enzymatic characteristics of this protein were investigated in vitro. [Results] The protein PomA was successfully purified from the strain BL21-pET-pomA, with a molecular weight of 59.4 kDa. The results of the bioinformatics analysis showed that PomA had four conserved copper ion-binding sites, being a multicopper oxidase (MCO). In the liquid LB medium, the Mn(II)-oxidizing activity of BL21-pET-pomA was 3.6 times that of BL21-pET. Furthermore, PomA exhibited both Mn(II)-oxidizing and laccase activities. Specifically, the optimal conditions for PomA to exert the Mn(II)-oxidizing activity were 60 ℃ and pH 7.8, and the enzyme showed the K_m, V_max, and k_cat of (3.11±0.02) mmol/L, 3.931×10^–6 mol/(L·min), and 2.38 min^–1, respectively. The optimal conditions for PomA to exert the laccase activity were 60 ℃ and pH 3.0, and the enzyme showed the K_m, V_max, and k_cat of (47.29±0.02) μmol/L, 3.401×10^–6 mol/(L·min), and 0.23 s^–1, respectively. [Conclusion] PomA is a member of the MCO family, exhibiting both Mn(II)-oxidizing and laccase activities. The amino acid sequence of PomA exhibits significant divergence from other members of the MCO family, which potentially leads to distinct variations in their catalytic activities for Mn(II) oxidation.