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

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.

Key words:Bacterial laccase, Mn2+ tolerance, Enzyme characterization, Metagenome, Lignin degradation