Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli

Two novel genes (tvms10a, tvmz2a) were identified in the metagenomic DNA of Rusa unicolor and Equus burchelli fecal samples. The amplified PCR product of tvms10a is composed of 917 bp and the gene was found to encode a protein containing 165 amino acids, while the tvmz2a PCR product was 1053 bp long encoding 298 amino acid proteins. The gene has 72% primary sequence identity with Clostridiales sp. These amplified PCR products which can encode FAE were cloned into pGEMT Easy TA cloning vector and then sub-cloned into the EcoRI site of pET32a expression vector to generate pET32-tvms10a and pET32-tvmz2a, which was then transformed into Escherichia coli BL21. The recombinants were grown in LB medium and gene expression was induced with IPTG for 6 h. Purified recombinant Tvms10a and Tvmz2a proteins showed molecular masses of 18.6 and 31.2 kDa respectively, and displayed hydrolytic activity towards substrate ethyl ferulate. The activities of Tvms10a and Tvmz2a produced in E. coli were 15 and 9 U/min respectively, and their specific activities 16.6 and 10.4 U/mg protein respectively. The optimal pH is between 5.0 and 8.0 and the optimal temperature is 37 °C for enzyme reaction. Unusually, these proteins were found to be capable of releasing ferulic acid (FA) and diferulic acid (diFA) from untreated crude plant cell wall materials. The substrate utilization preferences and sequence similarity of these clones place it in the type-D sub-class of FAE.

► Two novel genes were identified in the metagenomic DNA of wild animal fecal samples. ► Tvms10a and Tvmz2a show sequence similarity to xylanase/chitin deacetylase domain. ► Enzyme activity of Tvms10a, Tvmz2a produced in E. coli were 15, 9 U/min respectively. ► The purified protein is releasing FA and diFA from crude plant cell wall materials. ► Substrate utilization and sequence similarity of these clones place it in type-D FAE.