Expression and characterization of thermostable catechol 1,2-dioxygenase from a fecal microbial metagenome of Nycticebus pygmaeus

Abstract:

[Objective] The gene (catPLCgl) coding for catechol 1,2-dioxygenase from a fecal microbial metagenome of Nycticebus pygmaeus was cloned and expressed in Escherichia coli BL21(DE3), and the purified recombinant CatPLCgl was characterized. [Methods] The full-length catPLCgl was obtained based on the metagenomic high-throughput sequencing technology, and its amino acid sequence was analyzed. Then catPLCgl was cloned into the pEASY-E2 vector, and characterized by the heterologous expression in Escherichia coli BL21(DE3). [Results] The gene full-length of catPLCgl is 852 bp with a G+C content of 48% and encodes 283 amino acids, with a theoretical molecular weight of 33.56 kD. Characterization shows that the optimal pH was 7.0, and the enzyme activity remained more than 90% after being processed for 1 h at pH between 7.0 and 10.0. The thermal activity of purified recombinant CatPLCgl was optimal at 40 °C at pH 7.0. The enzyme was stable at 25 °C and 40 °C, retaining nearly 100% activity after pre-incubation for 210 h. The kinetic parameters Km, Vmax and kcat values were 24.9 μmol/L, 8.3 μmol/(min·g) and 13.7 s–1 respectively. Fe2+, Hg2+, Cu2+, Triton X-100, SDS and Ag+ dramatically reduced the enzymatic activity, and other metal ions or organic reagents have less effect. [Conclusion] Recombinant CatPLCgl has good temperature stability and alkali resistance.

Key words:Catechol 1,2-dioxygenase, Fecal microbial metagenome, Heterologous expression, Thermal stability