Identification, Characterization, and Structural Analysis of Genes Encoding Cellulolytic/Xylanolytic Activity Isolated from Goat Rumen Bacterial Metagenomes
The incorporation of enzymatic synthesis into wide array of eco-friendly bioprocesses such as the hydrolysis of cellulose/hemi-cellulose has become an illustrious tool in deriving well defined bioactive compounds and biodegradable industrial products. The ruminant’s rumen microbial communities are perceived as the most potential biochemical reservoir of inordinately diverse and multi-functional cellulolytic enzymes with peculiar functional adaptation to enhance biotechnological processes. The infancy in understanding about the rumen microbial genetic diversity and a lack of suitable screening culture techniques has limited the exploitation of multiple promising enzymes. In this study, we applied culture independent sequence guided metagenomics approach to identify, isolate, clone and functionally characterize cellulase and xylanase genes. Genomic DNA was extracted from the goat ruminal fluid and converted to a sequencing library using Nextera DNA sample prep kits followed by sequencing analysis on Illumina HiSeq 2000. The metagenomic sequences were assembled and translated into proteins. A pair of primers were designed against the assembled gene sequences, and several genes encoding cellulolytic/xylanolytic activities were further amplified and cloned. Multiple sequence alignment was done to align Sanger Sequencing results with the predicted assembled gene sequences and deposited into the NCBI database with the nucleotide accession number. Four recombinant proteins were over-expressed and purified to their homogeneity with the expected size. Several endoglucanases and xylanase enzymes expressed their cellulolytic properties during the enzymatic plate assay analysis. Bio-informatics tools exploited to predict the 3D structure of Endoglucanase A and Endo 1, 4 beta Xylanase enzymes revealed that Endoglucanase A and Endo 1, 4 beta Xylanase is similar to their homologous enzymes at 56% and 59% identity level respectively. This implies that both the protein sequences have never been identified and characterized to date and thus present novel protein sequences. More importantly, the two glutamic acid residues present within the domain (E189 as a proton donor and E315 as a nucleophile donor) were predicted to function a catalytic role in Endo 1, 4 beta xylanase. Similarly, the two glutamic residues: E170 and E305 were predicted to act as a catalytic residue in case of Endoglucanase A. Understanding the complex interplay of the goat rumen microbiome employing metagenomic technique not only provides potential applicative benefits but allows for deeper insights into microbial ecology and genetic diversity. This could possibly aid in addressing questions associated with optimization of synthetic enzymes catalytic performance.
"Identification, Characterization, and Structural Analysis of Genes Encoding Cellulolytic/Xylanolytic Activity Isolated from Goat Rumen Bacterial Metagenomes"
ETD Collection for Tennessee State University.