Capillary Electrophoresis Separation of Protein Composition of γ-Irradiated Food Pathogens Listeria monocytogenes and Staphylococcus aureus

which were previously treated at different irradiation doses., one protein (50 S ribosomal protein) with the MW of 16.3 kDa was significantly decreased at a low dose of irradiation treatment and the other protein (transcriptional regulator CtsR) with the MW of 17.7 kDa was increased significantly (P≤0.05) at all doses of irradiation treatment compared to control.. The research further confirmed that capillary electrophoresis is a useful method to separate and analyse proteins expression which may be related to the resistance or sensitivity of food pathogens to γ-irradiation

Isolation and Characterization of EstC, a New Cold-Active Esterase from Streptomyces coelicolor A3(2)

The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5–9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6–11. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C12), displaying optimal activity with the valerate (C5) ester (kcat/Km = 737±77 s−1 mM−1). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors

Adaptation of the Highly Productive T7 Expression System to Streptomyces lividans▿ †

Streptomyces lividans is a Gram-positive bacterium known for its remarkable secretion efficiency and low extracellular protease activity. In the present work, we adapted the highly productive T7 expression system to S. lividans. A codon-optimized T7 RNA polymerase gene was chromosomally integrated, and a bifunctional T7 expression vector was constructed

Short-Chain Flavor Ester Synthesis in Organic Media by an <i>E. coli</i> Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase

<div><p>Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to ‘naturally’ produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into <i>E. coli</i> BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.</p></div

Effect of γ-irradiation on gene expression of heat shock proteins in the foodborne pathogen Escherichia coli O157:H7.

International audienceThe expression levels of seven genes (clpB, dnaK, groES, grpE, htpG, htpX and ibpB) encoding heat shock proteins (HSP) in Escherichia coli O157:H7 (E. coli) gamma irradiated was investigated. Timing impact of post-irradiated RNA extraction on the expression levels of these seven genes was also studied at a dose damaging the bacterial cells (0.4 kGy). Bacterial samples were γ-irradiated at 0.4 kGy and at a lethal dose of 1.3 kGy. RNA was extracted at 0 min post irradiation for both irradiation doses and at 15, 30, 60, 90 or 120 min post-irradiation at the dose damaging the cells. Quantification of the gene expression was performed using quantitative real-time polymerase chain reaction (q-RT-PCR). The expression of genes encoding HSP was a very dynamic process evolving rapidly when E. coli cells were irradiated at 0.4 kGy. Notably, groES, grpE and ibpB were more up- regulated at 1.3 kGy than those at 0.4 kGy. For the seven genes studied there were more damaged proteins during irradiation at the lethal dose and this dose causes increased expression in HSP which contributes to damage reparation. Expression patterns of genes encoding HSP in E. coli treated by γ-irradiation are different from those treated by heat shock

Conversion rates of LipIAF5-2 WCB transesterification and esterification toward acyl donors and acceptors.

<p>A) The full bars represent the conversion of triglyceride substrates and the shaded gray bars the conversion of equivalent free fatty acids (FFA). The substrates employed are glyceryl triacetate and acetic acid (C2∶0), glyceryl tributyrate and tributyric acid (C4∶0), glyceryl trihexanoate and hexanoic acid (C6∶0) and glyceryl trioctanoate and octanoic acid (C8∶0). The results are expressed as the molar conversion of 1 mmol of each substrate after 6 hours of incubation at 40°C with 4 molar equivalents of methanol and 10 mg of LipIAF5-2 WCB. B) Impact of acyl acceptors on transesterification by LipIAF5-2 WCB. Transesterification of 1 mmol of glyceryl tributyrate was done using 4 molar equivalents of each primary alcohols for 3 hours at 40°C using 10 mg of LipIAF5-2 WCB and 10% (w/w) water. Results are averaged over two independent experiments performed in triplicate.</p

Sequence alignment of EstC with homologous lipases.

<p>NP_629313, EstC from <i>S. coelicolor</i> A3(2); EDY57684, putative hydrolase from <i>S. sviceus</i> ATCC 29083; EFL34682, putative hydrolase from <i>S. viridochromogenes</i> DSM 40736; BAC70810, putative hydrolase from <i>S. avermitilis</i> MA-4680; YP_288574, putative hydrolase from <i>Thermobifida fusca</i> strain YX; P24640, lipase 3 from <i>Moraxella</i> sp.; ADB11055, lipase from <i>Psychrobacter</i> sp. G; AAK81864, lipase from <i>Streptococcus</i> sp. (N1). The conserved Gly-X_aa-Ser-X_aa-Gly pentapeptide and the proposed oxyanion hole are boxed. Residues of the catalytic triad are shown by arrows. Sequence alignment was performed with TCoffee <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032041#pone.0032041-Notredame1" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032041#pone.0032041-Poirot1" target="_blank">[53]</a>.</p