In this study, partially puriWed lysozyme was incorporated into zein Wlms in combination with chickpea albumin extract (CPAE), bovine serum albumin (BSA) and disodium EDTA. The zein Wlms showed an inherent free radical scavenging activity. Incorporation of lysozyme did not contribute to soluble free radical scavenging activity of zein Wlms. However, the incorporation of lysozyme in combination with CPAE increased the soluble and immobilized free radical scavenging activity of zein Wlms 17% to 25% and almost 84%, respectively. The incorporation of CPAE also improved the distribution of partially puriWed lysozyme preparation in zein Wlms and enabled the controlled release of lysozyme by reducing its release rate from zein Wlms between 1.5-and 3.5-fold, depending on the concentration of incorporated CPAE. In contrast, the BSA incorporation made distribution of lysozyme more heterogeneous and it did not contribute to the free radical scavenging activity of Wlms signiWcantly. The combinational incorporation of partially puriWed lysozyme with disodium EDTA · 2H 2 O or CPAE and disodium EDTA · 2H 2 O gave zein Wlms eVective on Escherichia coli and Bacillus subtilis. This study clearly showed the beneWts of using functional protein extracts to control lysozyme distribution and release rate and to improve antioxidant activity in zein Wlms.
An extracellular lipase producing bacterium was isolated from a soil sample, and identified as a strain of Pseudomonas fluorescens by 16S rRNA gene sequencing. It was named Pseudomonas fluorescens KE38. KE38 showed psychrotolerant properties with an optimum growth temperature of 25 °C. The lipase enzyme secreted by KE38 was purified 41.13-fold with an overall yield of 54.99%, and a specific activity of 337.3 U/mg. The molecular mass of purified lipase was estimated to be approximately 43 kDa by SDS-PAGE. Although the lipase was active at a temperature range of 15-65 °C, it exhibited maximum activity at 45 °C, at pH 8.0. The enzyme exhibited high stability retaining 100% and 70% of its activity after an incubation period of 45 and 100 min at 45 °C and pH 8.0 respectively. It also showed a broad substrate specificity acting on p-nitrophenyl esters with C8-C18 acyl groups as substrates and was activated by Ca2+ and Ni2+ at 1 mM. While the enzyme retained its activity levels in the presence of a variety of organic solvents, DMSO and dimethylformamide enhanced this. High stability, broad substrate specificity and activity at cold temperatures in the presence of organic solvents, and metal ions make the extracellular lipase of KE38 a candidate for industrial applications.State Planning Agency of Turke
A combination of culture-dependent and independent methods was used in an attempt to identify the bacteria present in kefir grains and kefir liquid. Culture-independent methods involved direct extraction of DNA by mechanical means from either grains or liquid, followed by PCR amplification and sequencing of 16S rRNA genes. Culture-dependent methods were performed by inoculating samples from both the kefir grains and the kefir liquid to solid media followed by incubation under either aerobic or anerobic conditions in order to selectively enrich aerobic and anaerobic bacteria. Pure cultures were isolated from enriched bacteria and their DNA was extracted for the amplification and sequencing of 16S rRNA genes. Results indicate that kefir grains had a different bacterial composition compared to the kefir liquid. While Lactobacillus kefiranofaciens, Lactobacillus kefiri, Enterococcus faecium and Acetobacter syzygii were found only in the kefir grains, Lactobacillus helveticus was found only in the kefir liquid. On the other hand, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides and Acetobacter lovaniensis were found to be present in both the grains and the liquid. To the best of our knowledge, this work is the first to report the presence of A.lovaniensis, A. syzygii and Enterococcus faecium in kefir.
No abstract
African monkeys are resistant to HIV-1 infection due to intrinsic restriction mechanisms found in their cells. However, although they can be infected by monkey-adapted modified HIV-1 particles that are designed to overcome known restriction factors, virus numbers drop to undetectable levels in immunocompetent animals. These results indicate the possibility of the presence of yet unidentified factor(s) that restrict HIV-1 in old-world monkey (OWM) cells after integration of the viral genome into the host cell chromosome. In the light of these findings, we hypothesized that OWMs might have evolved resistance mechanism(s) against HIV-1 by switching specific gene(s) on in response to the synthesis of viral proteins in infected cells. In an attempt to mimic post-infection status, we expressed HIV-1 Tat gene in African green monkey cells and compared the whole proteome with normal cells and identified secretory leukocyte protease inhibitor (SLPI), a protein with known extracellular anti-HIV-1 activity, as an over-expressed protein in the presence of HIV-1 Tat protein by 2D-PAGE and mass spectrometry analysis. We also showed that overexpression of SLPI in the presence of HIV-1 Tat was specific to monkey cells. Our results also suggest that SLPI had a previously undiscovered intracellular anti-HIV activity in addition to its extracellular activity.
A novel cold-active true lipase from Pseudomonas sp. KE38 was cloned, sequencing and expressed in E. coli by degenerate PCR and genome walking technique. The open reading frame of the cloned gene encoded a polypeptide chain of 617 amino acids with a confirmed molecular weight of 64 kD. Phylogenetic analysis of the deduced amino acid sequence of the lipase indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases. Recombinant lipase was purified in denatured form as inclusion bodies, which were then renatured by urea followed by dialysis. Lipase activity was determined titrimetrically using olive oil as substrate. The enzyme showed optimal activity at 25 °C, pH 8.5 and was highly stable in the presence of various metal ions and organic solvents. Low optimal temperature and high activity in the presence of methanol and ethanol make this lipase a potential candidate for transesterification reactions and biodiesel production.
The gas‐phase fragmentation reactions of the a7 ions derived from glutamine (Q) containing model heptapeptides have been studied in detail with low‐energy collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS). Specifically, the positional effect of the Q residue has been investigated on the fragmentation reactions of a7 ions. The study involves two sets of permuted isomers of the Q containing model heptapeptides. The first set contains the QAAAAAA sequence, and the second set involves of QYAGFLV sequence, where the position of the Q residue is changed from N‐ to C‐terminal gradually for both peptide series. An intense loss of ammonia from the a7 ions followed by internal amino acid eliminations strongly supports forming the imine‐amides structure via cyclization/rearrangement reaction for all studied a7 ions. This is in agreement with the pioneering study reported by Bythell et al. (2010, 10.1021/ja101556g). A novel rearrangement reaction is detected upon fragmentation of imine‐amide structure, which yields a protonated C‐terminal amidated hexapeptide excluding the Q residue. A possible fragmentation mechanism was proposed to form the protonated C‐terminal amidated hexapeptide, assisted via nucleophilic attack of the side chain amide nitrogen of the Q residue on its N‐protonated imine carbon atom of the rearranged imine‐amide structure. Highlights The gas‐phase fragmentation reactions of a7 ions obtained from protonated model peptides containing glutamine residue were studied by ESI‐MS/MS. A rearranged imine‐amide structure is the predominant even for a7 ions. Novel rearrangement reaction is observed which forms a protonated C‐terminal amidated hexapeptide excluding Q residue upon fragmentation of the imine‐amide structure.
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