Home About us Contact
|
Home About us Contact | ||
|
|
||
Residual Activity (residual + activity)
Selected AbstractsCHARACTERIZATION OF POLYPHENOL OXIDASE FROM ROOSTER POTATO (SOLANUM TUBEROSUM CV ROOSTER)JOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2010D. NI EIDHIN ABSTRACT The isolation and purification of polyphenol oxidase from potatoes (Solanum tuberosum cv. Rooster) is described. A 64-fold purified preparation has been obtained with 10% yield by a procedure involving (NH4)2SO4 precipitation, phenyl sepharose chromatography, ion exchange chromatography and hydroxyapatite chromatography. The partially purified enzyme has both cresolase and catecholase activity. Activity was lower toward monophenols than diphenols. Enzyme activity was optimal at pH 6.0,6.5 and at 30C. Greater than 50% activity was retained during storage for 72 h at pH 6.0,7.5. Residual activity was greater than 50% after incubation at 20C for 72 h, 30C for 48 h, 40C for 24 h, 50C for 2 h and 60C for 15 min. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid. Sodium dodecyl sulphate appeared to activate the enzyme. The enzyme was capable of cross-linking casein but did not increase gel-strengths in acidified milk gels. PRACTICAL APPLICATIONS Rooster is the most important potato cultivar grown in Ireland and data on its isolation and characterization has not been reported previously. This work describes a method to isolate polyphenol oxidase and characterization of the enzyme. Information on characterization of the enzyme could be valuable in relation to control of enzymatic browning during current processing and in minimum processing. There is potential for use of the enzyme in the emerging cross-linking area, as the results show some success and there may be potential of more cross-linking as the field develops and as interest in natural methods of cross-linking for food texture grows. This could lead to an important use for potato waste. Food product applications are given. [source] Thermal Inactivation Kinetics of Peroxidase and Lipoxygenase from Broccoli, Green Asparagus and CarrotsJOURNAL OF FOOD SCIENCE, Issue 1 2002E.F. Morales-Blancas ABSTRACT: Thewermal inactivation curves for peroxidase (POD) and lipoxygenase (LOX) in broccoli (florets), green asparagus (tip and stem), and carrots (cortex and core) extracts were determined in the range of 70 to 95 °C for 0 to 600 s. The capillary tube method was used to obtain quasi-isothermal conditions. The kinetics of both enzymes showed a biphasic first-order model, while at 70 °C, LOX in asparagus showed a monophasic first-order behavior. LOX activity was not detected for carrots. Kinetic parameters, k and Ea, were determined for heat-labile and heatresistant isoenzyme fractions. Additionally, initial and residual activities for both enzymes within tissue sections showed a different distribution and heat stability. [source] Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysisBIOTECHNOLOGY PROGRESS, Issue 3 2010Shivcharan Prasad Abstract Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent-denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Insulin analogues: have they changed insulin treatment and improved glycaemic control?DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue S1 2002Sten Madsbad Abstract To improve insulin therapy, new insulin analogues have been developed. Two fast-acting analogues with a more rapid onset of effect and a shorter duration of action combined with a low day-to-day variation in absorption rate are now available. Despite this favourable time,action profile most studies have not been able to show any improvement in overall glycaemic control with the fast-acting analogues. A reduced post-prandial increase in blood glucose has been found in all studies, whereas between 3 and 5,h after the meal and during the night an increased blood glucose level is the normal course. This is probably the main explanation for the absence of improvement in overall glycaemic control when compared with regular human insulin. A tendency to a reduction in hypoglycaemic events during treatment with fast-acting analogues has been observed in most studies. Recent studies have indicated that NPH insulin administered several times daily at mealtimes can improve glycaemic control without increasing the risk of hypoglycaemia. The fast-acting analogues are now also available as insulin mixed with NPH. Insulin glargine is a new long-acting insulin which is soluble and precipitates after injection, resulting in a long half-life with a residual activity of about 50% 24,h after injection. Insulin glargine is a peakless insulin and studies in both type 1 and type 2 diabetic patients indicate that glargine improves fasting blood glucose control and reduces the incidence of nocturnal hypoglycaemia. Surprisingly, the new fast,acting analogues have not achieved the expected commercial success, which emphasises the need for new strategies for basal insulin supplementation, exercise, diet and blood glucose monitoring. Copyright © 2002 John Wiley & Sons, Ltd. [source] Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamideFEBS JOURNAL, Issue 17 2006Marion B. Ansorge-Schumacher In two cycles of an error-prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4-fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method. [source] Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensisFEBS JOURNAL, Issue 3 2002Evert Bokma Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the ,1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with ,,2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower kcat and an approximately twofold higher Km than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N -acetyl group of the ,1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125,and Tyr183 contribute to catalysis by positioning the,carbonyl oxygen of the N -acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis. [source] Identification of catalytically important residues in the active site of Escherichia coli transaldolaseFEBS JOURNAL, Issue 8 2001Ulrich Schörken The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB -deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon,carbon bond cleavage and stabilization of the carbanion/enamine intermediate. [source] Functional and structural analysis of five mutations identified in methylmalonic aciduria cbIB type,HUMAN MUTATION, Issue 9 2010Ana Jorge-Finnigan Abstract ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a KM for ATP and KD for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations. Hum Mutat 31:1033,1042, 2010. © 2010 Wiley-Liss, Inc. [source] Mutational spectrum and genotype,phenotype correlations in mevalonate kinase deficiency,HUMAN MUTATION, Issue 8 2006Saskia H.L. Mandey Abstract Mevalonate kinase deficiency (MKD) is an autosomal recessive autoinflammatory disorder caused by mutations in the MVK gene resulting in deficient activity of mevalonate kinase (MK). Depending on the clinical severity, MKD may present as hyper-IgD and periodic fever syndrome (HIDS) or the more severe mevalonic aciduria (MA). We analyzed the MVK gene in 57 patients with MKD and found 39 different mutations including 15 novel mutations, expanding the total mutational spectrum of MKD to 63 mutations. To get more insight into the genotype,phenotype correlation in MKD, we studied the effect of selected missense mutations on MK protein stability and activity in various patient fibroblast cell lines. All MKD cell lines showed markedly decreased MK activities that correlated well with the clinical severity and, for most of the cell lines, with the amount of MK protein. When fibroblasts of MKD patients were cultured under conditions known to promote a more controlled protein folding, all cell lines of patients with the HIDS phenotype and few cell lines of patients with the MA phenotype showed an increase in the residual MK activity. This increase in enzyme activity correlates well with an increase in the MK protein levels in these cell lines, indicating that most of the mutations in MKD affect stability and/or folding of the MK protein rather than affecting the catalytic properties of the enzyme. The finding that the residual activity in MKD can be manipulated by environmental conditions may offer therapeutic options to alleviate or prevent the clinical symptoms associated with MKD. Hum Mutat 27(8), 796,802, 2006. © 2006 Wiley-Liss, Inc. [source] Mutations in the holocarboxylase synthetase gene HLCS,HUMAN MUTATION, Issue 4 2005Yoichi Suzuki Abstract Holocarboxylase synthetase (HLCS) deficiency is an autosomal recessive disorder. HLCS is an enzyme that catalyzes biotin incorporation into carboxylases and histones. Since the first report of the cDNA sequence, 30 mutations in the HLCS gene have been reported. Mutations occur throughout the entire coding region except exons 6 and 10. The types of mutations are one single amino acid deletion, five single nucleotide insertions/deletions, 22 missense mutations, and two nonsense mutations. The only intronic mutation identified thus far is c.1519+5G>A (also designated IVS10+5G>A), which causes a splice error. Several lines of evidence suggest that c.1519+5G>A is a founder mutation in Scandinavian patients. Prevalence of this mutation is about 10 times higher in the Faroe Islands than in the rest of the world. The mutations p.L237P and c.780delG are predominant only in Japanese patients. These are probably founder mutations in this population. Mutations p.R508W and p.V550M are identified in several ethic groups and accompanied with various haplotypes, suggesting that these are recurrent mutations. There is a good relationship between clinical biotin responsiveness and the residual activity of HLCS. A combination of a null mutation and a point mutation that shows less than a few percent of the normal activity results in neonatal onset. Patients who have mutant HLCS with higher residual activity develop symptom after the neonatal period and show a good clinical response to biotin therapy. Hum Mutat 26(4), 285,290, 2005. © 2005 Wiley-Liss, Inc. [source] Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A,HUMAN MUTATION, Issue 3 2005Nadja Bogdanova Abstract Hemophilia A is the most frequently occurring X-linked bleeding disorder, affecting one to two out of 10,000 males worldwide. Various types of mutations in the F8 gene are causative for this condition. It is well known that the most common mutation in severely affected patients is the intron 22 inversion, which accounts for about 45% of cases with F8 residual activity of less than 1%. Therefore, the aim of the present study was to determine the spectrum and distribution of mutations in the F8 gene in a large group of patients with severe hemophilia A who previously tested negative for the common intron 22 inversion. Here we report on a mutation analysis of 86 patients collected under the above-mentioned criterion. The pathogenic molecular defect was identified in all patients, and thus our detection rate was virtually 100%. Thirty-four of the identified mutations are described for the first time. The newly detected amino acid substitutions were scored for potential gross or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling. Hum Mutat Res 26(3), 249,254, 2005. © 2005 Wiley-Liss, Inc. [source] Influence of residual milk-clotting enzyme and proteolysis on melting properties of soft cheeseINTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 3 2007M C CANDIOTI In this work, we assessed the influence of coagulant residual activity and primary proteolysis on Cremoso Argentino cheese melting properties. For that purpose, we made Cremoso soft cheeses using different amounts of coagulant, and also obtained samples in which milk-clotting enzyme was inactivated. Primary proteolysis correlated with residual activity of coagulant in early stages of cheese ripening; however, it was similar in all cheeses after 30 days. The hydrolysis of caseins did not significantly affect the melting ability of the cheeses, expressed as the area increase after heating samples under standardized conditions. Samples with similar proximate composition showed some changes in meltability; those seemed related to pH evolution during ripening. [source] Second Generation Sol-Gel Encapsulated Lipases: Robust Heterogeneous BiocatalystsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6-7 2003Manfred Abstract The original procedure for the encapsulation of lipases in sol-gel materials produced by the fluoride-catalyzed hydrolysis of mixtures of RSi(OCH3)3 and Si(OCH3)4 has been improved considerably. This involves higher enzyme loading, variation of the alkylsilane precursor, and the use of additives such as isopropyl alcohol, 18-crown-6, Tween,80®, methyl-,-cyclodextrin and/or KCl. A dramatic increase in enzyme activity is observed. The sol-gel lipase immobilizates are also excellent catalysts in the kinetic resolution of chiral alcohols and amines, recycling without any substantial loss in enantioselectivity and a residual activity of 70% being possible even after 20 reaction cycles. [source] Aged pesticide residues are detrimental to agrobiont spiders (Araneae)JOURNAL OF APPLIED ENTOMOLOGY, Issue 8 2008S. Pekár Abstract Spiders are among the most abundant arthropods in agroecosystems, playing an important role as natural enemies of various pests. In this study we evaluated residual activity of selected pesticides on the mortality and behaviour of four spider species (Dictyna uncinata, Pardosa palustris, Philodromus cespitum and Theridion impressum). We used three pesticides: a herbicide Command (clomazone), and insecticides Decis (deltamethrin) and Nurelle (chlorpyrifos and cypermethrin). Mortality was recorded after exposure of spiders to fresh (2-h), 5, 10, 15 and 20-day-old residues. For each residue mortality was evaluated after 24,72 h. Residual effect differed between preparations and, in some cases, between spiders. All of the Nurelle residues (fresh to 20-day-old) caused 100% mortality in all spider species. Residues of Command were rather harmless (causing <20% mortality) to all spider species as the herbicide activity declined with age. Residues of Decis had species-specific effects as the mortality varied between 0 and 90%. In Dictyna the mortality gradually declined with the age of residues, while in Pardosa the mortality increased. In Philodromus and Theridion the mortality declined up to 10-day-old residues and then increased so that 20-day-old residues caused almost as high mortality as the new ones. Exposure to pesticide residues immediately affected the movement of Pardosa spiders. Residues of Decis and Nurelle decreased spider locomotion, while those of Command increased locomotion in comparison with the control. In another experiment, we studied repellence of fresh pesticide residues to Pardosa spiders. In comparison with the control, spiders stayed a similar time on the surface treated with Command, but four times less on Decis and nine times less on Nurelle-treated surfaces, respectively. In conclusion, aged insecticide residues possess a high activity and can cause long-term decline in the abundance and prolonged behavioural disturbance of spiders in agroecosystems. [source] PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN FROM WEISSELLA PARAMESENTEROIDES DFR-8, AN ISOLATE FROM CUCUMBER (CUCUMIS SATIVUS)JOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2010AJAY PAL ABSTRACT Bacteriocin from Weissella paramesenteroides DFR-8 isolated from cucumber (Cucumis sativus) was purified by using only two steps, viz., pH-mediated cell adsorption,desorption method and gel permeation chromatography. A single peak observed in the purity check by analytical Reverse Phase-High Performance Liquid Chromatography (Waters 600 analytical HPLC system, Milford, MA) and a single band (molecular weight,3.74 kDa) shown on SDS-PAGE analysis strongly indicated the homogeneity of the bacteriocin preparation. Treatment with proteolytic enzymes abolished the antimicrobial activity indicating the proteinaceous nature of bacteriocin. The purified bacteriocin exhibited a broad inhibitory spectrum against foodborne pathogens and spoilage microorganisms, including gram-negative bacteria such as Salmonella typhimurium, Vibrio parahaemolyticus, Aeromonas hydrophila and Listeria monocytogenes. Response surface methodology was employed to study the interactive effect of temperature and pH on bacteriocin activity, and a regression equation was developed. The bacteriocin retained full activity after storage at,20C for 90 days, while partial and complete activity loss was observed when stored at 4 and 37C, respectively. PRACTICAL APPLICATION In recent years, bacteriocins of lactic acid bacteria have gained much attention as food biopreservatives because of their origin from generally regarded as safe organisms. In spite of various bacteriocins studied worldwide, studies on bacteriocins of Weissella paramesenteroides remain rare. The present work involves the purification of bacteriocin up to absolute homogeneity from W. paramesenteroides, an isolate first time reported from cucumber (Cucumis sativus). The purified bacteriocin (molecular weight ,3.74 kDa) was found to inhibit a large number of foodborne pathogens, including Listeria monocytogenes, which is resistant to commercially available bacteriocin, i.e., nisin. The application of central composite rotatable design enabled us to design a regression equation from which the residual activity of bacteriocin can be predicted at any given conditions of temperature and pH within the experimental domain. The broad inhibitory spectrum and thermostability of bacteriocin suggest its potential application in food preservation. [source] STABILITY AND PROPERTIES OF A THERMOSTABLE ,-GALACTOSIDASE IMMOBILIZED ON CHTTINJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2000JADWIGA MACIU ABSTRACT Thermostable ,-galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized ,-galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively. [source] Polyphenol Oxidase from Apple (Malus domestica Borkh. cv Bramley's Seedling): Purification Strategies and CharacterizationJOURNAL OF FOOD SCIENCE, Issue 1 2006Deirdre M. Ni Eidhin ABSTRACT Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7-fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30 °C and the enzyme had residual activity of greater than 50% during storage for 72 h at 20 °C to 30 °C and for 24 h at 40 °C to 50 °C. Of the substrates tested, activity was greatest with 4-methylcatechol followed by catechol, pyrogallol, and (,)epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid. [source] Heat-moisture Treatments of Cowpea Flour and Their Effects on Phytase InactivationJOURNAL OF FOOD SCIENCE, Issue 2 2005Nicole S. Affrifah ABSTRACT: Samples of finely ground cowpea flour with moisture content adjusted to 10%, 25%, 35% (dry basis) were heated in sealed retort pouches at 70 to 95°C for periods of 2 to 32 min. Phytase showed a high thermal resistance with residual activity ranging between 50% and 95%. Thermal inactivation of cowpea phytase was adequately described by a fractional conversion model based on a 1st-order rate equation. Overall, increasing temperature and initial moisture content resulted in increased enzyme inactivation. Estimated activation energies between 70 and 95°C were 33.3, 37.9, and 43.4 kJ/mol at 10%, 25%, and 35% moisture, respectively. The kinetic models generated were successfully used to predict phytase activity in cowpea flour. [source] High-Pressure Processing of Orange Juice: Kinetics of Pectinmethylesterase InactivationJOURNAL OF FOOD SCIENCE, Issue 2 2001U. Nienaber ABSTRACT: A kinetic study of pectinmethylesterase (PME) inactivation in orange juice was conducted. Juice samples were subjected to combinations of high pressure (400, 500, 600 MPa) and thermal (25, 37.5, 50 °C) treatments for various time periods. PME inactivation followed a first-order kinetic model with a residual activity of pressure-resistant enzyme remaining. Calculated D-values ranged from 4.6 min to 117.5 min at 600 MPa/50 °C and 400 MPa/25 °C, respectively. Pressures in excess of 500 MPa resulted in sufficiently fast inactivation rates for economic viability of the process. [source] Impact of Supercritical Carbon Dioxide and High Pressure on Lipoxygenase and Peroxidase ActivityJOURNAL OF FOOD SCIENCE, Issue 8 2000W. TEDJO ABSTRACT: The effects of supercritical carbon dioxide (ScCO2) treatment and high hydrostatic pressure treatment on the activities of lipoxygenase (LOX) and peroxidase (POD) were studied. Hydrostatic pressure treatment (240 MPa, 55 °C, 15 min) of LOX and POD in 30% sucrose solutions without buffer led to approximately 80% and approximately 50% residual activity, respectively. Application of ScCO2 (35.2 MPa, 40 °C, 15 min for LOX and 62.1 MPa, 55 °C, 15 min for POD) achieved approximately 35% LOX and approximately 65% POD inactivity in 30 % sucrose solutions. Total inactivation of LOX (10.3 MPa, 50 °C and 15 min) and of POD (62.1 MPa, 55 °C and 15 min) could be achieved through ScCO2 treatment of unbuffered solution. Increasing the concentration of sucrose and buffering (pH range 4 to 9) of enzyme solutions resulted in increased resistance of the enzymes to ScCO2 treatment. [source] Effects of tea polyphenols on the activities of soybean trypsin inhibitors and trypsinJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2004Huihua Huang Abstract Tea polyphenols (TPs) and other materials were extracted from Chinese green tea, and their effects on trypsin inhibitors and trypsin were analysed. TPs were found to have a deactivation effect on both Kunitz trypsin inhibitor (KTI) and Bowman,Birk trypsin inhibitor (BBTI). KTI was more easily deactivated than BBTI by complexing with TPs. The deactivation effect of TPs on KTI and BBTI reached a maximum at a TP/KTI ratio of 25 and a TP/BBTI ratio of 16. However, the deactivation effect of TPs on KTI and BBTI was reduced dramatically when KTI and BBTI were already complexed with trypsin. TPs were also found to inhibit trypsin. The inhibitory activity of TPs, KTI and BBTI on trypsin was found to decrease in the order BBTI > gtTI > gtPs. Complete inhibition of trypsin by TPs could not be achieved. When the TP concentration was increased to about 17 µg ml,1, the residual activity of trypsin was maintained at 400 TU mg,1, equivalent to 32% of the initial trypsin activity. In TP inhibition the KM value for trypsin remained unchanged at 5.88 × 10,4 mol l,1 and Vmax decreased when benzoyl- DL -arginine- p -nitroanilide (BAPNA) was used as substrate. The pattern of trypsin inhibition by TPs is non-competitive. Copyright © 2004 Society of Chemical Industry [source] Resistance of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), larvae in Michigan to insecticides with different modes of action and the impact on field residual activityPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 9 2008David Mota-Sanchez Abstract BACKGROUND: The codling moth is one of the principal pests of apple in the world. Resistance monitoring is crucial to the effective management of resistance in codling moth. Three populations of codling moth in neonate larvae were evaluated for resistance to seven insecticides via diet bioassays, and compared with a susceptible population. In addition, apple plots were treated with labeled field rate doses of four insecticides. Treated fruit were exposed to neonate larvae of two populations from commercial orchards. RESULTS: Two populations of codling moth expressed two- and fivefold resistance to azinphos-methyl, seven- and eightfold resistance to phosmet, six- and tenfold resistance to lambda-cyhalothrin, 14- and 16-fold resistance to methoxyfenozide and sixfold resistance to indoxacarb, but no resistance to acetamiprid and spinosad. The impact of the resistance to azinphos-methyl, measured as fruit damage, increased as the insecticide residues aged in the field. In contrast, fruit damage in methoxyfenozide- and lambda-cyhalothrin-treated fruit was observed earlier for resistant codling moth. No differences in efficacy were found for acetamiprid. CONCLUSIONS: Broad-spectrum insecticide resistance was detected for codling moth. Resistance to azinphos-methyl, lambda-cyhalothrin and methoxyfenozide was associated with reduced residual activity in the field. Broad-spectrum resistance presents serious problems for management of the codling moth in Michigan. Copyright © 2008 Society of Chemical Industry [source] Insecticidal activity of the enantiomers of fipronilPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 12 2003Harald B Teicher Abstract The two enantiomers of the insecticide fipronil were made by preparative HPLC. The insecticidal activities of the racemic mixture and the two enantiomers against selected agricultural or household pests (cotton stainer, Dysdercus cingulatus F; grain weevil, Sitophilus granarius L and house fly, Musca domestica L) were determined. There was no significant difference in acute or residual activity between the racemic mixture and the enantiomers of fipronil, indicating that there is no preferred chiral form of the compound in these key species of important insects. This observation clearly suggests that there is no major scope for marketing the insecticide in a one-enantiomer form. Copyright © 2003 Society of Chemical Industry [source] ,-enolase deficiency, a new metabolic myopathy of distal glycolysisANNALS OF NEUROLOGY, Issue 2 2001Giacomo P. Comi MD A severe muscle enolase deficiency, with 5% of residual activity, was detected in a 47-year-old man affected with exercise intolerance and myalgias. No rise of serum lactate was observed with the ischemic forearm exercise. Ultrastructural analysis showed focal sarcoplasmic accumulation of glycogen , particles. The enzyme enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the ,-enolase subunit, the protein product of the ENO3 gene. The ,-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while ,-enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues: a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant ,-enolase. Muscle ,-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis. [source] Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-riboseACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2000Karine Thomazeau Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps require a divalent cation and the first step has a strong preference for magnesium. Manganese ions are highly unfavourable to the reaction: only 3% residual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn2+ and NADPH. The 1.6,Å resolution electron-density map showed the reaction product (2,3-dihydroxy-3-methylvalerate, DHMV) and a density corresponding to (phospho)-ADP-ribose instead of the whole NADP+. This is one of the few structures of an enzyme complexed with its reaction product. The structure of this complex was refined to an R factor of 19.3% and an Rfree of 22.5%. The overall structure of the enzyme is very similar to that of the complex with the reaction-intermediate analogue IpOHA [N -hydroxy- N -isopropyloxamate; Biou et al. (1997), EMBO J.16, 3405,3415]. However, the active site shows some differences: the nicotinamide is cleaved and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to the end of the reaction allowed the proposal of a reaction scheme. Taking this result into account, the enzyme was crystallized with Ni2+ and Zn2+, for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/NADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover, mass-spectrometry measurements confirmed the cleavage of nicotinamide. [source] The first structure of a cold-adapted superoxide dismutase (SOD): biochemical and structural characterization of iron SOD from Aliivibrio salmonicidaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Hege Lynum Pedersen Superoxide dismutases (SODs) are metalloenzymes that catalyse the dismutation of the superoxide radical anion into O2 and H2O2 in a two-step reaction. The crystal structure of the iron superoxide dismutase from the cold-adapted and fish-pathogenic bacterium Aliivibrio salmonicida (asFeSOD) has been determined and refined to 1.7,Å resolution. The protein has been characterized and compared with the closely related homologous iron superoxide dismutase from the mesophilic Escherichia coli (ecFeSOD) in an attempt to rationalize its environmental adaptation. ecFeSOD shares 75% identity with asFeSOD. Compared with the mesophilic FeSOD, the psychrophilic FeSOD has distinct temperature differences in residual activity and thermostability that do not seem to be related to structural differences such as intramolecular or intermolecular ion bonds, hydrogen bonds or cavity sizes. However, an increased net negative charge on the surface of asFeSOD may explain its lower thermostability compared with ecFeSOD. Activity measurements and differential scanning calorimetry measurements revealed that the psychrophilic asFeSOD had a thermostability that was significantly higher than the optimal growth temperature of the host organism. [source] Investigating the potential of Bacillus subtilis ,-amylase as a pressure-temperature-time indicator for high hydrostatic pressure pasteurization processesBIOTECHNOLOGY PROGRESS, Issue 4 2009Tara Grauwet Abstract The potential of Bacillus subtilis ,-amylase (BSA) as a pressure-temperature-time indicator (pTTI) for high pressure pasteurization processing (400,600 MPa; Ti 10,40°C; 1,15 min) was investigated. A stepwise approach was followed for the development of an enzyme-based, extrinsic, isolated pTTI. First, based on literature data on the pressure stability, BSA was selected as a candidate indicator. Next to the accuracy and ease of the measurement of the indicator's response (residual activity) to the pressure treatment, the storage and handling stability of BSA at atmospheric pressure was verified. Second, the stability of BSA at a constant temperature (T) and time in function of pressure (p) was investigated. Solvent engineering was used to shift the inactivation window of BSA in the processing range of interest. Third, the enzyme (1 g/L BSA,MES 0.05 M pH 5.0) was kinetically calibrated under isobaric-isothermal conditions. Time dependent changes in activity could be modeled best by a first-order model. Except for low pressures and high temperatures, a synergistic effect between pressure and temperature could be observed. Based on the model selected to describe the combined p,T-dependency of the inactivation rate constant, an elliptically shaped isorate contour plot could be constructed, illustrating the processing range where BSA can be used to demonstrate temperature gradients. Fourth, the validity of the kinetic model was tested successfully under dynamic conditions similar to those used in food industry. Finally, the indicator was found suitable to demonstrate nonuniformity in two-sectional planes of a vertical, single vessel system. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009 [source] Deactivation of Formate Dehydrogenase (FDH) in Solution and at Gas-Liquid InterfacesBIOTECHNOLOGY PROGRESS, Issue 6 2005Andreas S. Bommarius Enzymes, increasingly important in the synthesis of fine chemicals and pharmaceutical intermediates, are often insufficiently stable under reacting conditions. We have investigated the stability, in homogeneous aqueous solution and at gas-liquid interfaces, of formate dehydrogenase (FDH), important for cofactor regeneration, from Candida boidinii and overexpressed in E. coli. When exposed to mechanical stress, residual activity, [E]t/[E]0, and residual protein were found to scale proportionally with gas-liquid surface area in the bubble column, verifying a surface-driven process, and with time and total throughput in a gear pump, but did not seem to be influenced much by shear in a Couette viscometer. All FDH variants are deactivated by chaotropes but not kosmotropes: the first-order deactivation constant kd correlates well with the Jones-Dole coefficient B but not well with the surface tension increment ,, of various concentrated ammonium salt solutions. This finding might provide guidance for focusing the search for quantitative theories of Hofmeister effects. [source] Outcome of exudative aged-related macular degeneration (ARMD) after 3 intravitreal injections of bevacizumabACTA OPHTHALMOLOGICA, Issue 2009C CREUZOT Purpose To evaluate the efficacy of intravitreal injections of bevacizumab in exudative ARMD. Methods Retrospective study including naive patients suffering from exudative ARMD whatever the type of neovascularisation. All the participants were treated with three monthly 1.25 mg intravitreal injections of bevacizumab. The primary objective was far and near visual acuity (VA) 1, 3 and 6 months after the third injection. The secondary objective was the residual activity of neovascularisation assessed with fluorescein and ICG angiography and retinal thickness evaluated with OCT3. Results 71 eyes of 66 patients were included. Neovascularisation was occult, visible or combined in 65%, 20% and 15% of the cases, respectively. A statistically significant improvement between pre and post-injection VA (LogMAR) was observed one month after the third injection, 0.88±0.57 and 0.77±0.60, respectively, p=0.001). An active neovascularisation was still present at that time in 57.7% of the eyes and 34% at 6 months needing further bevacizumab injections (3.85±0.96 per eye). VA was similar at 1, 3 and 6 months (F71,2=1,54 ; p=0,46). A complication occurred in 3 eyes. Conclusion Three bevacizumab intravitreal injections led to a significant VA improvement. However, more than half of the eyes had an active neovascularisation after these 3 injections. [source] Correlation between severity of mucopolysaccharidoses and combination of the residual enzyme activity and efficiency of glycosaminoglycan synthesisACTA PAEDIATRICA, Issue 4 2009Ewa Piotrowska Abstract Aim: To develop a method for prediction of severity and clinical course of mucopolysaccharidoses (MPS), a group of inherited metabolic diseases. Methods: Various biochemical and clinical parameters (including estimation of the level of clinical severity, presence of specific mutations, residual enzyme activity, urinary glycosaminoglycan (GAG) excretion, storage of GAG in fibroblasts and efficiency of GAG synthesis) of patients suffering from MPS types II, IIIA and IIIB were determined. Correlations between genetic, biochemical and clinical parameters were tested. Results: We found that efficiency of GAG synthesis may contribute to the level of severity of MPS. It appears that (i) combination of low or average efficiency of GAG synthesis and the presence of residual activity of the enzyme is responsible for an attenuated phenotype, (ii) a lack of detectable residual enzyme activity causes a severe phenotype, irrespective of the efficiency of GAG synthesis and (iii) high efficiency of GAG synthesis leads to a severe phenotype, even if residual enzyme activity is detected. This correlation was found to be valid in 15 out of 17 patients tested. Conclusion: Analysis of efficiency of GAG synthesis and residual activity of the enzyme may be considered for prediction of severity of MPS patients' clinical phenotypes. [source] | ||||||||||||||||||||||||||||