PMID:22441123

Citation	Chandrasekharaiah, M, Thulasi, A, Vijayarani, K, Kumar, DP, Santosh, SS, Palanivel, C, Jose, VL and Sampath, KT (2012) Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli. Gene 500:134-9
Abstract	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.
Links	PubMed Online version:10.1016/j.gene.2012.03.027
Keywords	Animals; Carboxylic Ester Hydrolases/chemistry; Carboxylic Ester Hydrolases/genetics; Carboxylic Ester Hydrolases/isolation & purification; Deer/metabolism; Equidae/metabolism; Feces/enzymology; Metagenomics/methods
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9BACT:F8SUI8		GO:0030600: feruloyl esterase activity	ECO:0000314:		F		Table 1.	complete CACAO 8197
9BACT:F8SUI8	enables	GO:0030600: feruloyl esterase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
9BACT:F8SUI7		GO:0030600: feruloyl esterase activity	ECO:0000314:		F		Table 1.	complete CACAO 8198
9BACT:F8SUI7	enables	GO:0030600: feruloyl esterase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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