Identification of cellulase gene from the metagenome of Equus burchelli fecal samples and functional characterization of a novel bifunctional cellulolytic enzyme.

The metagenomic approach has been used successfully to isolate novel biocatalyst gene from uncultured microorganisms. The gene encoding exo-1,4-ß-glucanase avicelase was amplified from the metagenome of the Equus burchelli fecal sample and cloned. The gene was found to be of 1,007 bp of nucleotide which encodes a protein of 318 amino acids with a calculated MW of 36 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolases 6 superfamily. The expressed protein was active towards the substrates avicel and carboxymethyl cellulose, indicating that it has bifunctional cellulolytic enzyme activity. The recombinant protein showed an activity of 5.23 U with specific activity of 6.8 U mg(-1) protein with the substrate avicel, while when CMC was used, an activity of 3.0 U with a specific activity of 4.2 U mg(-1) protein was achieved. Its optimum pH was determined to be 7.0 and optimum temperature of 35°C.

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 917bp and the gene was found to encode a protein containing 165 amino acids, while the tvmz2a PCR product was 1053bp 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 6h. Purified recombinant Tvms10a and Tvmz2a proteins showed molecular masses of 18.6 and 31.2kDa respectively, and displayed hydrolytic activity towards substrate ethyl ferulate. The activities of Tvms10a and Tvmz2a produced in E. coli were 15 and 9U/min respectively, and their specific activities 16.6 and 10.4U/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.

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut.

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.