The effect of some factors on expression of gene encoding endoglucanase from dna metagenome of Binh Chau hot spring in Escherichia coli

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 HiseqTM. 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 30oC 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

A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 ◦C and its apparent half-life at 60 ◦C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale