• Asian-Australasian Journal of Animal Sciences
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• v.26 no.8
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• pp.1144-1151
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• 2013

In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below $E^{-10}$ using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below $10^{-5}$. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen.

• Journal of agriculture & life science
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• v.46 no.2
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• pp.129-137
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• 2012

A carboxymethyl cellulase gene, cel5B, was cloned, sequenced, and expressed in Escherichia coli. pRCS20 in E. coli was identified from metagenomic cosmid library of cow rumen for cellulase activity on a carboxymethyl cellulose agar plates. Cosmid clone (RCS20) was partially digested with Sau3AI, ligated into BamHI site of pBluescript II SK+ vector, and transformed into E. coli $DH5{\alpha}$. The insert DNA of 1.3 kb was obtained, designated cel5B, which has the activity of hydrolyzation of CMC. The cel5B gene had an open reading frame (ORF) of 1,059 bp encoding 352 amino acids with a signal peptide of 48 amino acids and the conserved region, VIYEIYNEPL, belongs to the glycosyl hydrolase family 5. The molecular mass of Cel5B protein expressed from E. coli $DH5{\alpha}$ exhibited to be about 34 kDa by CMC-SDS-PAGE. The optimal pH was 8.0, and the optimal temperature was about $50^{\circ}C$ for its enzymatic activity.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.1013-1021
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• 2005

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular $\alpha$-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5$\alpha$ and sequenced. The $\alpha$-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PACE. The enzyme was 21 to $59\%$ sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and $40\%$. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5$\alpha$ was enhanced in the presence of $Mg^{2+}$ (20 mM) and $Ca^{2+}$ (30 mM) and inhibited in the presence of $Fe^{2+}$ and $Cu^{2+}$. The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyh is supposed to obtained from unculturable rumen bacterium in cow rumen environment.

• BMB Reports
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• v.44 no.1
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• pp.52-57
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• 2011

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 $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

• Animal Bioscience
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• v.37 no.2_spc
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• pp.346-359
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• 2024

Advanced and innovative breeding and management of meat-producing animals are needed to address the global food security and sustainability challenges. Beef production is an important industry for securing animal protein resources in the world and meat quality significantly contributes to the economic values and human needs. Improvement of cattle feed efficiency has become an urgent task as it can lower the environmental burden of methane gas emissions and the reduce the consumption of human edible cereal grains. Cattle depend on their symbiotic microbiome and its activity in the rumen and gut to maintain growth and health. Recent developments in high-throughput omics analysis (metagenome, metatranscriptome, metabolome, metaproteome and so on) have made it possible to comprehensively analyze microbiome, hosts and their interactions and to define their roles in affecting cattle biology. In this review, we focus on the relationships among gut microbiome and beef meat quality, feed efficiency, methane emission as well as host genetics in beef cattle, aiming to determine the current knowledge gaps for the development of the strategies to improve the sustainability of beef production.

• Journal of agriculture & life science
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• v.46 no.3
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• pp.109-118
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• 2012

The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est2R) was 2,120 bp in length, encoding a protein of 516 amino acid residues with a calculated molecular weight of 57,286 Da. The molecular weight of the enzyme was estimated to be 57,000 Da by SDS-PAGE. Est2R shared 35.6% amino acid identity with esterase (CAH19079) of uncultured prokaryote. The Est2R was most active at $20-40^{\circ}C$, and showed optimum at $30^{\circ}C$ and pH 8.0. The most activity of Est2R for the different chain length of p-nitrophenyl ester group as substrate was p-nitrophenyl acetate. Moreover, the enzyme was found to be most active without organic solvent, followed by 98% active with ethanol, and the enzyme activity was highly affected by the acetonitrile. The enzyme was significantly inhibited by $Zn^{2+}$ but stimulated by $Ca^{2+}$. So, novel esterase gene est2R is likely to obtain from cow rumen metagenome and supposed to use for industrial purpose.

• Journal of Life Science
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• v.22 no.4
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• pp.437-446
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• 2012

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$ $coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$ $coli$ $DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$ $coli$ $DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.5
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• pp.738-747
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• 2018

Objective: In a previous study, analysis of Illumina sequenced metagenomic DNA data of bacteria in Vietnamese goats' rumen showed a high diversity of putative lignocellulolytic genes. In this study, taxonomy speculation of microbial community and lignocellulolytic bacteria population in the rumen was conducted to elucidate a role of bacterial structure for effective degradation of plant materials. Methods: The metagenomic data had been subjected into Basic Local Alignment Search Tool (BLASTX) algorithm and the National Center for Biotechnology Information non-redundant sequence database. Here the BLASTX hits were further processed by the Metagenome Analyzer program to statistically analyze the abundance of taxa. Results: Microbial community in the rumen is defined by dominance of Bacteroidetes compared to Firmicutes. The ratio of Firmicutes versus Bacteroidetes was 0.36:1. An abundance of Synergistetes was uniquely identified in the goat microbiome may be formed by host genotype. With regard to bacterial lignocellulose degraders, the ratio of lignocellulolytic genes affiliated with Firmicutes compared to the genes linked to Bacteroidetes was 0.11:1, in which the genes encoding putative hemicellulases, carbohydrate esterases, polysaccharide lyases originated from Bacteroidetes were 14 to 20 times higher than from Firmicutes. Firmicutes seem to possess more cellulose hydrolysis capacity showing a Firmicutes/Bacteroidetes ratio of 0.35:1. Analysis of lignocellulolytic potential degraders shows that four species belonged to Bacteroidetes phylum, while two species belonged to Firmicutes phylum harbouring at least 12 different catalytic domains for all lignocellulose pretreatment, cellulose, as well as hemicellulose saccharification. Conclusion: Based on these findings, we speculate that increasing the members of Bacteroidetes to keep a low ratio of Firmicutes versus Bacteroidetes in goat rumen has resulted most likely in an increased lignocellulose digestion.

• Journal of Life Science
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• v.20 no.9
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• pp.1306-1313
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• 2010

The gene encoding esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est1R) was 2,465 bp in length, encoding a protein of 366 amino acid residues, and the molecular weight of the enzyme was 61,166 Da. Est1R of rumen cosmid library shared 5.9% amino acid identity with Est1R (P37967) of PNB carboxylesterase, 6.1% with Est1R (1EEAA) of acetylcholinesterase and 6.1% with Est1R (1H23A) of chain A. BlastP in NCBI database analysis of Est1R revealed that it was not homologous to previous known lipases and esterases. Est1R showed optimum activity at pH 7.0 and $40^{\circ}C$. On the other hand, the enzyme was found to be most active without organic solvent, followed by 95% activity with methanol, and the enzyme activity was highly affected by hexane (lost 51% activity). Therefore, the novel esterase gene est1R is likely obtainable from cow rumen metagenome and may be utilized for industrial purposes.