The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Chris W. Michiels - One of the best experts on this subject based on the ideXlab platform.
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role of porins in sensitivity of escherichia coli to antibacterial activity of the Lactoperoxidase enzyme system
Applied and Environmental Microbiology, 2005Co-Authors: Philipp De Spiegeleer, Jan Sermon, Kristof Vanoirbeek, Abram Aertsen, Chris W. MichielsAbstract:Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN−) from thiocyanate (SCN−), using H2O2 as an oxidant. To gain insight into the antibacterial mode of action of the Lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the Lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the Lactoperoxidase enzyme system because of a reduced uptake of OSCN−.
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Unique stress response to the Lactoperoxidase-thiocyanate enzyme system in Escherichia coli
Research in Microbiology, 2004Co-Authors: Jan Sermon, Kristof Vanoirbeek, Philipp De Spiegeleer, Rob Van Houdt, Abram Aertsen, Chris W. MichielsAbstract:Abstract Using a differential fluorescence induction approach, we screened a promoter trap library constructed in a vector with a promoterless gfp gene for Escherichia coli MG1655 promoters that are induced upon challenge with the antimicrobial Lactoperoxidase-thiocyanate enzyme system. None of the thirteen identified Lactoperoxidase-inducible open reading frames was inducible by H2O2 or by the superoxide generator plumbagin. However, analysis of specific promoters of known stress genes showed some of these, including recA, dnaK and sodA, to be inducible by the Lactoperoxidase-thiocyanate enzyme system. The results show that the Lactoperoxidase-thiocyanate enzyme system elicits a distinct stress response different from but partly overlapping other oxidative stress responses. Several of the induced genes or pathways may be involved in bacterial defense against the toxic effects of the Lactoperoxidase-thiocyanate enzyme system.
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high sucrose concentration protects e coli against high pressure inactivation but not against high pressure sensitization to the Lactoperoxidase system
International Journal of Food Microbiology, 2003Co-Authors: Isabelle Van Opstal, Suzy C M Vanmuysen, Chris W. MichielsAbstract:Abstract The inactivation of Escherichia coli by high hydrostatic pressure treatment at up to 550 MPa and 20 °C was studied in potassium phosphate buffer containing high concentrations of sucrose. E. coli strain MG1655 was pressure-sensitive in the absence of sucrose, but became highly pressure resistant in the presence of 10% to 50% (w/v) sucrose. The pressure resistance of E. coli strain LMM1010, a previously described derivative of MG1655 that is pressure resistant in the absence of sucrose, was further increased in the presence of sucrose, to a similar level as for strain MG1655 in the presence of sucrose. When cell suspensions of either strain were stored after pressure treatment for 24 h at 20 °C, a further reduction of the plate counts indicative of pressure induced sublethal injury was observed, that was positively correlated with pressure intensity and negatively with sucrose concentration. Addition of the Lactoperoxidase system to the cell suspensions strongly enhanced high pressure inactivation of E. coli at high sucrose concentrations. Using a pressure intensity of only 250 MPa, both E. coli strains were sensitized for the Lactoperoxidase system in up to 30% (w/v) sucrose, resulting in at least 106-fold inactivation within 24 h or less after pressure treatment. For comparison, a pressure treatment at 250 MPa in the absence of the Lactoperoxidase system did not cause any inactivation of either strain even in the absence of sucrose. At sucrose concentrations above 30% (w/v), no or very little inactivation occurred even in the presence of the Lactoperoxidase system.
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inactivation of escherichia coli and listeria innocua in milk by combined treatment with high hydrostatic pressure and the Lactoperoxidase system
Applied and Environmental Microbiology, 2000Co-Authors: Cristina Garciagraells, Caroline Valckx, Chris W. MichielsAbstract:We have studied inactivation of four strains each of Escherichia coli and Listeria innocua in milk by the combined use of high hydrostatic pressure and the Lactoperoxidase-thiocyanate-hydrogen peroxide system as a potential mild food preservation method. The Lactoperoxidase system alone exerted a bacteriostatic effect on both species for at least 24 h at room temperature, but none of the strains was inactivated. Upon high-pressure treatment in the presence of the Lactoperoxidase system, different results were obtained for E. coli and L. innocua. For none of the E. coli strains did the Lactoperoxidase system increase the inactivation compared to a treatment with high pressure alone. However, a strong synergistic interaction of both treatments was observed for L. innocua. Inactivation exceeding 7 decades was achieved for all strains with a mild treatment (400 MPa, 15 min, 20°C), which in the absence of the Lactoperoxidase system caused only 2 to 5 decades of inactivation depending on the strain. Milk as a substrate was found to have a considerable effect protecting E. coli and L. innocua against pressure inactivation and reducing the effectiveness of the Lactoperoxidase system under pressure on L. innocua. Time course experiments showed that L. innocua counts continued to decrease in the first hours after pressure treatment in the presence of the Lactoperoxidase system. E. coli counts remained constant for at least 24 h, except after treatment at the highest pressure level (600 MPa, 15 min, 20°C), in which case, in the presence of the Lactoperoxidase system, a transient decrease was observed, indicating sublethal injury rather than true inactivation.
Hasan Ozdemir - One of the best experts on this subject based on the ideXlab platform.
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impact of some avermectins on Lactoperoxidase in bovine milk
International Journal of Food Properties, 2016Co-Authors: Zeynep Koksal, Ilhami Gulcin, Hasan Ozdemir, Ramazan Kalin, Ali AtaseverAbstract:Many macrocyclic lactones, including avermectins, are known to be used as a veterinary drug, agricultural pesticides, and insecticides. Lactoperoxidase (EC 1.11.1.7) is one of the peroxidases found in milk. Lactoperoxidase has a natural host defense system against micro-organisms and a natural antimicrobial system. In this study, some macrocyclic lactones, including emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin were investigated for in vitro inhibitory effects on the bovine Lactoperoxidase enzyme, which was purified using amberlite CG-50 H+ resin and sepharose 4B-L-tyrosine-sulphanamide affinity chromatography 344.6-fold, with a yield of 61.1% and a specific activity of 39.11 EU/mg protein. Emamectin-benzoate, doramectin, eprinomectin, abamectin, moxidectin-vetranal, and ivermectin are also known strong antiparasitary properties. In this study, we demonstrated that avermectins have strong Lactoperoxidase inhibitory effects. Of these, the emamectin-benzoate wa...
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the inhibitory effects of l adrenaline on Lactoperoxidase enzyme purified from bovine milk
International Journal of Food Properties, 2012Co-Authors: Melda şisecioglu, Ilhami Gulcin, Murat Cankaya, Hasan OzdemirAbstract:L-Adrenaline belongs to a group of compounds known as catecholamines, which play an important role in the regulation of the physiological process in living organisms. In the present study, the inhibitory effect of L-adrenaline on Lactoperoxidase was examined. Lactoperoxidase (E.C.1.11.1.7) was purified from bovine milk with three consecutive steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange, and Sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified with a yield of 42.18%, a specific activity of 30.33 EU/mg proteins, and 20.77 purification fold. Enzyme purity was determined with SDS-PAGE, where a single band was observed. The Rz (A412/A280) value for Lactoperoxidase was 0.9. The effect of L-adrenaline on Lactoperoxidase was determined using ABTS as a chromogenic substrate. The half maximal inhibitory concentration (IC50) value and an inhibition constant (Ki) values for L-adrenaline were 34.5 and 2.26 μM, respectively. L-Adrenaline was found to be a non-competitive inhibitor.
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The effects of norepinephrine on Lactoperoxidase enzyme (LPO)
Scientific Research and Essays, 2010Co-Authors: Melda şisecioglu, Ilhami Gulcin, Murat Cankaya, Ali Atasever, Hasan OzdemirAbstract:Norepinephrine, a hormone and a neurotransmitter, was known as catecholamine. In the present study, we examined the inhibitory effect of norepinephrine on Lactoperoxidase enzyme purified from bovine milk. Lactoperoxidase (LPO; E.C.1.11.1.7) was purified from bovine milk with three purification steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange chromatography and Sephadex G-100 gel filtration chromatography, respectively. LPO was purified with a yield of 31.5%, a specific activity of 30.33 EU/mg proteins and 20.77 purification fold. To determine enzyme purity, SDS-PAGE was performed and single band was observed. The Rz (A412/A280) value for LPO was 0.9. The effect of norepinephrine on Lactoperoxidase was determined using ABTS as a chromogenic substrate. The half maximal inhibitory concentration (IC50) value norepinephrine was found to be 67.2 µM. Also, inhibition constant (Ki) for norepinephrine was found to be 62.0 µM. Norepinephrine was found as non-competitive inhibitor. Key words: Noradrenaline, norepinephrine, Lactoperoxidase, LPO, enzyme purification, inhibition.
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the inhibitory effect of propofol on bovine Lactoperoxidase
Protein and Peptide Letters, 2009Co-Authors: Melda şisecioglu, Ilhami Gulcin, Murat Cankaya, Hasan OzdemirAbstract:Propofol (2,6-diisopropylphenol) is a hypnotic intravenous agent with in vivo antioxidant properties. This study was undertaken to examine the in vitro effect of propofol on Lactoperoxidase (LPO; E.C. 1.11.1.7) obtained from bovine milk. Lactoperoxidase was purified with three purification steps: Amberlite CG-50 resin, CM-Sephadex C-50 ionexchange chromatography and Sephadex G-100 gel filtration chromatography, respectively. Lactoperoxidase was purified with a yield of 21.6%, a specific activity of 34 EU/mg proteins and 14.7-fold purification. One enzyme unit is defined as the oxidation of 1 μmol ABTS per min under the assay condition (25oC, pH: 6.0). To determine enzyme purity, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The effect of propofol on Lactoperoxidase were determined using 2,2-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a chromogenic substrate. The IC50 value of propofol was found as 15.97 μM. Also, Ki constant for propofol was 3.72 μM and propofol was found as competitive inhibitor.
Mertxe De Renobales - One of the best experts on this subject based on the ideXlab platform.
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Alkaline Phosphatase, Acid Phosphatase, Lactoperoxidase, and Lipoprotein Lipase Activities in Industrial Ewe's Milk and Cheese
Journal of Agricultural and Food Chemistry, 1998Co-Authors: F. Chávarri, M. Virto, A. Santisteban, Mertxe De RenobalesAbstract:Alkaline phosphatase activity in raw, industrial ewe's milk increased steadily > 2-fold between January [1.7 units (U)/mL] and June (3.75 U/mL), whereas acid phosphatase increased 4-fold in January and February (17 mU/mL) and then remained constant until the end of lactation. By contrast, lipoprotein lipase exhibited a downward trend and Lactoperoxidase decreased 2-fold during lactation. When assayed at cheese-ripening temperatures, acid phosphatase retained 16% of its activity at 37 °C, whereas Lactoperoxidase retained between 30 and 45% of its activity at 20 °C. The rate of hydrolysis of model triacylglycerols by lipoprotein lipase was highest for tricaprylin. Although alkaline phosphatase in raw milk cheeses was variable from 1 to 180 days of ripening, no apparent reactivation was observed. The activity of acid phosphatase increased 2-fold during the 180 days of ripening in the cheeses made in summer, whereas in winter and spring much smaller increases were observed. Both raw milk cheeses made in summer and all pasteurized milk cheeses had very low levels of Lactoperoxidase throughout ripening.
Amita Chand - One of the best experts on this subject based on the ideXlab platform.
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capture of lactoferrin and Lactoperoxidase from raw whole milk by cation exchange chromatography
Separation and Purification Technology, 2006Co-Authors: Amita ChandAbstract:Abstract The extraction of high-value dairy proteins such as lactoferrin and Lactoperoxidase normally requires extensive pre-treatments of milk to remove fat and caseins by centrifugation, precipitation, Ca 2+ chelation and/or filtration. Similarly, fat and caseins are normally removed prior to capture of recombinant proteins from the milk of transgenic animals. Such pre-treatments can result in significant loss of protein yield and/or activity. In this paper, we demonstrate that it is possible to pass significant quantities of raw, untreated milk through a 5 cm high XK16 chromatography column (volume 10 mL) packed with SP Sepharose Big Beads™ (GE Healthcare, Uppsala, Sweden) without exceeding the maximum allowable backpressure, provided that the processing temperature is kept nominally around milking temperature (35–37 °C). Results show that more than 100 column volumes of raw milk could be loaded at 300 cm/h before breakthrough of Lactoperoxidase occurred. The dynamic capacity for adsorbing lactoferrin and Lactoperoxidase simultaneously under these conditions was approximately 48.6 mg/mL of packed resin. Minor leakage (4.6% of the feed concentration) of lactoferrin occurred throughout the loading process but major breakthrough occurred only after approximately 100 column volumes were loaded.
Bo Lonnerdal - One of the best experts on this subject based on the ideXlab platform.
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purification and quantification of Lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human Lactoperoxidase
The American Journal of Clinical Nutrition, 2001Co-Authors: Kouichirou Shin, Hirotoshi Hayasawa, Bo LonnerdalAbstract:Background: Two heme-containing peroxidases, secretory Lactoperoxidase and leukocyte-derived myeloperoxidase, which play host defense roles through antimicrobial activity, were previously identified in human colostrum. Within several days after the start of lactation, the relative contribution of myeloperoxidase to the peroxidase activity in milk was shown to decline as the number of milk leukocytes decreased. Objective: Our knowledge of Lactoperoxidase in human milk is still limited. The objective of this study was to use specific antibodies as a means of simplifying the purification and quantification of Lactoperoxidase. Design: Polyclonal antibodies were raised against recombinant human Lactoperoxidase. Immunoglobulin G (IgG) was isolated by means of a protein A column and was characterized by immunoblotting. For the purification of Lactoperoxidase from whey, a cation-exchange column and an immunoaffinity column with coupled IgG were used. The concentration of Lactoperoxidase was determined by a sandwich enzyme-linked immunosorbent assay by using purified native Lactoperoxidase as a standard. Native and biotinylated IgG were used as capture and detector antibodies, respectively. Results: Two bands with molecular masses of 80 and 100 kDa were detected in an immunoblot of human whey. Similar heterogeneity was observed in the sodium dodecyl sulfate-polyacrylamide gel electophoresis profile of purified Lactoperoxidase. The mean (±SD) concentration of Lactoperoxidase in 26 whey samples was estimated to be 0.77 ± 0.38 mg/L. The concentrations were positively correlated with the peroxidase activity detected in these samples. Conclusion: Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.
