Metabolic products obtaining from microorganisms of geothermal ecologies often show special characteristics which help their cells to survive, grow and develop under extreme conditions. Exploiting the microbial gene resource of those environments demands a new approach via uncultured methods. Thanks to the development of metagenomics and bioinformatic softwares, we can exploit novel genes from environment directly. Based on Binh Chau hotspring’s DNA metagenome sequencing, ORF [denovogenes]_32768 encoding for β-glucosidase is selected for expression into pET17b vector because it shown a low similartity of amino acid sequence as compared to others in Genbank, a high alkali and Tm predicted values. To improve the expression efficiency of β-glucosidase, some factors (host strains, medium culture, IPTG concentration, aeration…) are investigated. The results showed that the recombinant E. coli C43(DE3) reached the highest dried biomass at 8.26 g/L and the maximum enzymatic activity at 0.34 U/mL in shaking condition (TB medium plus 0.25 mM IPTG with the ratio of cultured /flask volume is 20%, 42-48 hours, 30°C). This study demonstrates the capacity of mining a novel gene encoded for enzyme from DNA metagenome of Vietnam hot spring as well as produces recombinant enzyme for biomass conversion.
Expression of microbial target genes in Escherichia coli is broadly used due to its advantages namely: well established system, easy to manipulate, a huge biomass, high level productivity, safe and inexpensive to grow. Metagenomic technique has been applying in Vietnam recently for effective mining of uncultured gene resources, especially in endemic mini-ecologies such as hot springs where the cell densities are low. DNA metagenome of Binh Chau hot spring was isolated and sequenced by Illumia Hiseq TM . Based on analyses of databases of cellulase-encoded genes, denovogenes 18736 gene sequence for thermal endoglucanase was selected for expression in E. coli. In this paper, some factors for expression of endoglucanase have been investigated. The results show that appropriate gene expression conditions are: Expression performed in E. coli C43 (DE3) on TB medium at 30 o C with 0.25 mM of IPTG as inducer, the culture volume of 20% compared with the bottle volume and the expression time is 42-48 hours. In this condition, the biomass production and soluble enzyme activity can reached up to 5.54-5.58 g /L and 1.92-1.98 U/mL, respectively. Our results show the prospect of exploiting microbial genes without culture. , 2019. The effect of some factors on expression of gene encoding endoglucanase from DNA metagenome of Binh Chau hot spring in Escherichia coli. Tap chi Sinh hoc, 41(2): 111-118. https://doi.
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