Inhibition of coenzyme Qs accumulation in engineered escherichia coli by high concentration of farnesyl diphosphate

Coenzyme Q[10] [CoQ[10]] is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase [FPPS] is a responsible enzyme for biosynthesis of farnesyl diphosphate [FPP], a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ[10]-producing Escherichia coli [E. coli]. Two CoQ[10]-producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS [ispA] under the control of either the trc or P[BAD] promoters. Over-expression of ispA under the control of P[BAD] promoter led to a relative increase in CoQ[10] production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ[10] production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ[10] production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629 +/- 40 to 30 +/- 13 and 564 +/- 28 to 80 +/- 14 micro g/g Dried Cell Weight [DCW], respectively. The results showed high level of FPP reduces endogenous CoQ8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. The reduction in CoQ[10] production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ[10]

Isolation and identification of a new bacillus cereus strain and characterization of its neopullulanase

Identification and use of more efficient enzymes in the food and pharmaceutical industries is the focus of many researchers. The aim of this study was to search for a new bacterial strain capable of producing high levels of pullulanase applicable to biotechnology, the starch bioprocessing and food industries. A new pullulan hydrolyzing Bacillus strain was isolated and designated SDK2. Morphological and biochemical tests identified the strain as a putative Bacillus cereus strain, which was further characterized and confirmed through 16s rRNA sequencing, and was submitted to GeneBank, under the accession number FR6864500. Quantative analysis of the strain's pullulanase activity was carried out by the Dintrosalicyclic [DNS] acid-based assay. Thin layer chromatography [TLC] of the culture supernatant, identified the extracellular pullulanase as neopullulanase. Effects of temperature and pH on pullulanase activity were also studied. The optimum conditions for enzyme activity, as represented by 60°C and a pH of 7, resulted in an activity of 13.43 U/ml, which is much higher than some of the previously reported activities. However, growth of B. cereus SDK2 was also observed at a pH range of 5 to 10, and temperatures of 30°C to 50°C. The effect of metal ions and reagents, such as Mg[+2], Ca[+2], Zn[+2], Cu[+2], Fe[+2], Ni[+2] on enzyme activity showed that Ca[+2] ions increased pullulan activity, whereas the other ions and reagents inhibited pullulanase activity. The ability of B. cereus SDK2 to produce high levels of neopullulanase stable at 60°C that can generate panose from pullulan, make this newly isolated strain a valuable source of debranching enzyme for biotechnology, the starch bioprocess and medical industries

Decontamination of tomato, red cabbage, carrot, fresh parsley and fresh green onion inoculated with Shigella sonnet and Shigella flexneri by some essential oils [in vitro conditions]

Essential oils and their major constituents are useful sources of antimicrobial compounds. There are a few reports on the decontamination and antimicrobial activity of essential oils towards Shigella spp. In this study, the antimicrobial and decontamination potentials of essential oils at different concentrations, belonging to plants such as Thymus vulgaris, Saturiea hortensis, Mentha polegium, Cuminum cyminum, Lavandula officinalis and Mentha viridis L. [spearmint], towards Shigella sonnei and Shigella flexneri were investigated. The disk diffusion method demonstrated the antimicrobial potential of the essential oils. The ability of essential oils to decontaminate vegetables such as, tomato, red cabbage, carrot, fresh parsley and fresh green onion that were previously inoculated with Shigella spp. was determined. Inhibitory effects of essential oils towards Shigella spp. were noted in the disk diffusion method. There was a reduction in Shigella population following inoculation of cultures with 0.5% and 0.1% [v/v] essential oils. This study confirmed that essential oils have the potential to be used for decontamination of vegetables

Optimizing primary recovery and refolding of human interferon-beta from escherichia coli inclusion bodies

The refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. The purpose of this study was optimization of recombinant human interferon-beta purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity. Triton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-beta inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, beta-mercaptoethanol and dithiothritol. The best recovery was obtained in the presence of 0.5% TritonX-100 [v/v]. Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon- beta. Successful refolding was achieved by gradient elution [decreasing the guanidine-hydrochloride concentration] in the presence of L-arginine. Partial purification was also achieved continuously, and recombinant human interferon-p was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon. In this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg

Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences

Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli, the lipase gene [btl2] was cloned downstream of the native Bacillus signal peptide and also in fusion with the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors [pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA] were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies

In silico genome-wide screening for TnrA-regulated genes of Bacillus clausii

Bacillus clausii TnrA transcription factor is required for global nitrogen regulation. In order to obtain an overview of gene regulation by TnrA in B. clausii KSM-K16, the entire genome of B. clausii was screened for the consensus sequence, 5'-TGTNAN7TNACA-3' known as the TnrA box, and 13 transcription units were found containing a putative TnrA box. The TnrA targets identified in this study were tnrA, glnA, nrgA, nasFDEB, puc genes, licT, the two operons of the oligopeptide ABC transporter, lytR, transcriptional regulator of the Lrp/AsnC family, sodium-dependent transporter of SNF family, hyu genes and a biochemically uncharacterized protein