4929
Ahmed Abdel-Rahman Abd-Allah Radwan
Genetic Improvement of Desulfurizing Bacteria
Biodesulfurization, Dibenzothiophene, dsz genes, Recombinants, Gibb’s assay and HPLC analysis.
Desulfurization activity of eight local strains, isolated previously and characterized as a DBT-metabolizing bacteria, was investigated to select biodesulfurization (BDS) strain for cloning the dsz genes in E. coil to alleviate the sulfur repression mechanism to enhance the biocatalysts mass production for BDS commercial application. PCR-based detection of dsz genes using specific primers targeting the conserved regions ARTLERG and RYDRADEFL in dszA gene showed that four strains (HN1, NK1, Sh42 and HN2 strains) out of the eight are BDS strains. They demonstrated positive amplification at the corresponding size as R. erythropolis IGTS8, standard BDS strain. Furthermore, sequence analysis of amplified fragments (≈ 370 bp) revealed that the dszA sequences of these four strains had high homology with already known dsz sequences in GenBank database, indicating that these strains carry the dsz genes and follow the 4S pathway when grow on organosulfur compounds. Moreover, amplification of complete dsz genes from detected BDS strains was led to selection of dsz genes of R. erythropolis HN2 strain for cloning. The promoterless fragments (dszAB and dszC) of HN2 strain were constructed in plasmid pET 29a(+) under T7 promoter control. Recombinant plasmids pET29a-AB and pET29a-C were introduced into E. coli DH5ɑ for multiplication, then to E. coli BL21(DE3)pLysS for protein expression. Expression of dsz genes were tested on SDS-PAGE and the activity were estimated by Gibb’s assay and HPLC analysis. Results of HPLC were showed improvement in desulfurization activity of the recombinants about two times in comparison with the activity of the wild type (HN2). In addition to, expression of dsz genes in foreign microorganisms (E. coil BL21) under sulfate non-repressible promoter (T7 promoter) would help in limitingproblems of BDS commercial application.
2015
M.Sc
Al-Azhar
Science