This article introduces the steps of constructing and using probe to exploit the gene encoding pectinesterase from metagenome DNA sequencing data by next generation gene sequencing tools. Probe was used to exploit and select the gene encoding for pectinesterase from the metagenome DNA sequences of bacteria in goat rumen and thereby select a sequence to express in E. coli. According to the CAZy classification system, pectinesterase belongs to the family of carbohydrates esterases CE8 is an enzyme that has many applications in the food processing industry, environmental treatment, animal feed processing and medicine. As the results, 3 sequences of CE8 was retrieved from CAZy database and one probe was designed, this probe length was 367 amino acids contained all the conserved amino acid residues: 200 conserved residues in all sequence, 72 residues similar in almost sequences and residues conserved in many sequences and homologus; choosed highest alkalinity index. Using the probe designed, we filtered four coding sequences for pectinesterase from metagenome DNA sequencing data of bacteria in goat rumen. Spatial structure estimation with Phyre2 has only one sequencing (code 46301) with 100% sequence identity and 90% query coverage with pectinesterase. A artificial gene were synthesized and inserted into the vector pET22b (+) at the NcoI, XhoI to co-express with chaperone pG-KJE8 in E. coli. The recombinant pectinesterase enzyme is expressed in soluble form and has a pectin substrate biodegradation activity. The results demonstrate that using probe for gene extraction is feasible.

Cellulase is an important enzyme that plays a role in cleaving β-1,4 glucoside on cellulose to release glucose, which is of economic value and can be applied in many different fields. The 1545 bp endoglucanase gene mined from goat rumen's bacterial metagenomic data was expressed in Escherichia coli Rosetta2. In this study, the recombinant endoglucanase was purified by his-tag affinity chromatography with differrent processes, such as using phosphate buffer with or without sodium cloride, pretreatment of samples with ammonium sulphate before supplying into affinity column, using various concentration of imidazole for washing... Finally the endoglucanse was sucessfully purified by his-tag affinity column using sodium chloride-free phosphate buffer of which 150 mM and 400 mM imidazole were used for washing and enzyme elution, respectively. The resulting enzyme showed its high purity of 99%. CMC plate assay confirmed that although less active than commercial cellulase (Sigma), the recombinant cellulase hydrolyzed CMC to form a clear zone (halo) around the well. The purified enzyme is capable of using as material for further analysis.

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A gene coding for GH5 endoglucanase exploited from metagnomic DNA data of bacteria in Vietnamese goats’ rumen was modularity structure including a catalytic module, a fibronectin-3 like module and an X module. The recombinant enzyme was sucessfully expressed in E. coli and purified.  To study the effect of some metal ions and chemicals on enzyme activity, in this study, we used some tools including Swiss-Prot, ProFunc, COFACTOR for prediction of enzyme structure and ligands interaction. The obtained results indicated that the most similar structure with enzyme had two conserved residues (Asp-190 và Asp-192) linked with Mn2+ within a radius of ~ 3.5 Å from the center of ion Mn2+ and enzyme molecule contained a disulphide bond. Experimental results for essessment of the effect of some metal ions (Ca2 +, Mn2 +, Mg2+, Ni2+, K+, Co2+, Cu2+, Zn2+, Fe3+) at the final concentration of 10 mM and of six common chemicals including SDS (1%), urea (1 µM), 2-mercaptoethanol (1 µM), EDTA (1 µM), tween 80 (1mM), triton X-100 (1 µM) showed that only Mn2+ increased enzyme activity slightly at concentration of 10 mM and two times at the concentration of 40 mM Mn2+. The Mn2+ has been identified as a specific binding agent may increase the stability and activity of endoglucanase GH5.