宏基因组来源耐Mn2+、热稳定细菌漆酶的分子克隆及酶学特性

Molecular cloning and enzyme characterization of a metagenome- derived bacterial laccase with Mn2+ tolerance and thermostability

摘要:

【背景】漆酶和锰过氧化物酶(Manganese peroxidase，Mnp)是木质素降解的主要酶，二者有协同效应。Mnp活性依赖于Mn2+，而Mn2+是大多数漆酶的抑制剂。【目的】获得耐Mn2+的细菌漆酶用于木质素降解。【方法】构建许昌市某污水河污泥宏基因组文库，通过活性筛选获得细菌漆酶基因lac1542。使用大肠杆菌异源表达Lac1542，研究纯化后的重组蛋白酶学性质并进一步检测了含Lac1542复合酶系降解木质素能力。【结果】测序结果显示lac1542编码一个含513个氨基酸的蛋白。以ABTS为底物Lac1542最适反应pH为4.0，在pH 3.0?6.5范围内酶活性稳定。最适反应温度是75 °C，在70 °C以下酶活性稳定；100 mmol/L的Mn2+仍能提高酶的活性。动力学参数研究发现，该酶的最适底物顺序为：ABTS>丁香醛联氮>儿茶酚>2,6-DMP>愈创木酚。Lac1542/Mnp复合酶系对木质素降解率为47.8%，比单独使用Mnp木质素降解率(22.4%)提高25.4%。Lac1542/Mnp/灰盖鬼伞过氧化物酶(Coprinus cinereus Peroxidase，CIP)复合酶系木质素降解高达71.5%，比Mnp/CIP酶系木质素降解率(48.9%)提高22.6%，加入Lac1542后的复合酶系能明显提高木质素的降解率。【结论】Lac1542的可溶性表达、耐受高浓度Mn2+、热稳定性使得Lac1542可以替代一些经典的真菌漆酶应用于制浆、造纸、纤维素乙醇生产、染料脱色等工业。

Abstract:

[Background] Laccase and manganese peroxidase (Mnp) are major enzymes in lignin biodegradation and they act synergistically. The activity of Mnp depends on the Mn2+, however, Mn2+ is the inhibitor to most laccases. [Objective] The aim of our study is to obtain bacterial laccase with Mn2+ tolerance for lignin degradation. [Methods] We constructed the metagenomic library with environmental DNA isolated from sewage river sediment of Xuchang, and identified a gene encoding a bacteria laccase, lac1542, from the metagenomic library via activity-based functional screening. Then, we overexpressed Lac1542 heterologously as soluble active enzyme in Escherichia coli, and characterized the recombinant enzyme after purification subsequently. Finally, we further investigated the lignin degradation ability of the complex enzyme systems containing Lac1542. [Results] Sequence analysis revealed that lac1542 encoded a protein of 513 amino acids. The purified Lac1542 exhibited maximal activity at 75 °C and pH 4.0 with ABTS as substrate, and this enzyme was stable in the pH range of 3.0–6.5 and at the temperature below 70 °C. Interestingly, the enzymatic activity was increased after addition of 100 mmol/L Mn2+. Also, the optimal substrates were in the order of ABTS>Syringaldazine>catechol>2,6-DMP>guaiacol based on the kinetic parameters. In addition, the degradation percentage of Lac1542/Mnp toward lignin was 47.8%, 25.4% higher than that (22.4%) of Mnp. The degradation percentage of Lac1542/Mnp/Coprinus cinereus Peroxidase (CIP) toward lignin was reached up to 71.5%, 22.6% higher than that (48.9%) of Mnp/CIP. These results suggested that the degradation percentage of the complex enzyme system toward lignin was distinctly improved after addition of Lac1542. [Conclusion] The highly soluble expression, tolerance to high concentration of Mn2+, and thermostability of Lac1542 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and cellulosic ethanol production, and dye decolorization.