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Hydrolytic Activity (hydrolytic + activity)
Selected AbstractsDiel variation in surface and subsurface microbial activity along a gradient of drying in an Australian sand-bed streamFRESHWATER BIOLOGY, Issue 10 2003Cecile Claret Summary 1. Microbes play key roles in nutrient transformation and organic matter mineralisation in the hyporheic zone but their short-term responses to diel variations in discharge and temperature are unknown. Rates of microbial esterase activity were hypothesised to vary vertically and along a gradient of moisture in a drying sand-bed stream where discharge fluctuated daily in response to evapotranspiration. 2. At ,fully saturated', ,moist' and ,dry' locations in three sites along a drying Australian sand-bed stream, microbial activity at three depths (surface, 10 and 30 cm) was assessed using fluorescein diacetate hydrolysis. Samples were collected in mid-summer in the late afternoon and again at dawn to assess diel variation in hydrolytic activity at each site and depth. Data loggers tracked diel variations in temperature at each depth. 3. Hydrolytic activity was up to 10-fold greater in the surface sediments in late afternoon than at dawn in all habitats, and was correlated with surface sediment temperature. Diel differences in activity were not detected at 10 cm, although daily thermal cycles were evident at this depth. Unexpectedly, activity was marginally higher at dawn at 30 cm in all habitats, perhaps reflecting lags in temperature at that depth. 4. Overall, microbial activity declined with depth, strongly correlated with vertical trends in total organic matter and concentrations of dissolved phosphorus. Particulate organic matter, probably buried during a flood 35 days earlier, appeared largely responsible for these vertical trends. On the other hand, there was little evidence for hydrological exchange between much of the hyporheic zone and the surface stream, implying that processes in the subsurface zone of this stream are effectively isolated during baseflow in mid-summer. 5. Diel cycles of wetting and drying in the moist habitats did not enhance esterase activity relative to the dry or fully saturated habitats. Sediment moisture was not correlated with microbial activity, and mats of senescent algae appeared to inhibit water loss from surface sediments in the moist habitat. In this sand-bed stream, local diel fluctuations in water level appear to have less influence on microbial activity and mineralisation of organic matter in the sediments than occasional floods that bury leaf litter and renew many hyporheic zone functions. Subreach-scale processes seem to be the major driving force of microbial processes and nutrient cycling in this sand-bed river. [source] Polysaccharide hydrolysis in aggregates and free enzyme activity in aggregate-free seawater from the north-eastern Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Kai Ziervogel Summary Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico. [source] Xylanases, xylanase families and extremophilic xylanasesFEMS MICROBIOLOGY REVIEWS, Issue 1 2005Tony Collins Abstract Xylanases are hydrolytic enzymes which randomly cleave the , 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented. [source] Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9JOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2003A. Cherif Abstract Aims: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. Methods and Results:Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121°C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12·4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. Conclusions: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. Significance and Impact of the Study: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry. [source] GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Tatsuru Togo Abstract The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe-4-methylcoumaryl-7-amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol-specific phospholipase C (PI-PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)-anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti-c-Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe-MCA, and by PI-PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10-dimethyl-1,2-benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI-anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm. Mol. Reprod. Dev. 67: 465,471, 2004. © 2004 Wiley-Liss, Inc. [source] Probing Enzyme Promiscuity of SGNH HydrolasesCHEMBIOCHEM, Issue 15 2010Dr. Ivana Le Abstract Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstAN,EstPC and EstPN,EstAC were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p -nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities. [source] Polysaccharide hydrolysis in aggregates and free enzyme activity in aggregate-free seawater from the north-eastern Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Kai Ziervogel Summary Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico. [source] Properties of the hatching enzyme from Xenopus laevisFEBS JOURNAL, Issue 18 2001Ting-Jun Fan Using an anti-(glutathione S -transferase,UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 µmol·L,1 at 30 °C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N -,-tosyl- l -lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific ,HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease. [source] Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulisFEBS JOURNAL, Issue 16 2000Bingze Xu A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source] Diel variation in surface and subsurface microbial activity along a gradient of drying in an Australian sand-bed streamFRESHWATER BIOLOGY, Issue 10 2003Cecile Claret Summary 1. Microbes play key roles in nutrient transformation and organic matter mineralisation in the hyporheic zone but their short-term responses to diel variations in discharge and temperature are unknown. Rates of microbial esterase activity were hypothesised to vary vertically and along a gradient of moisture in a drying sand-bed stream where discharge fluctuated daily in response to evapotranspiration. 2. At ,fully saturated', ,moist' and ,dry' locations in three sites along a drying Australian sand-bed stream, microbial activity at three depths (surface, 10 and 30 cm) was assessed using fluorescein diacetate hydrolysis. Samples were collected in mid-summer in the late afternoon and again at dawn to assess diel variation in hydrolytic activity at each site and depth. Data loggers tracked diel variations in temperature at each depth. 3. Hydrolytic activity was up to 10-fold greater in the surface sediments in late afternoon than at dawn in all habitats, and was correlated with surface sediment temperature. Diel differences in activity were not detected at 10 cm, although daily thermal cycles were evident at this depth. Unexpectedly, activity was marginally higher at dawn at 30 cm in all habitats, perhaps reflecting lags in temperature at that depth. 4. Overall, microbial activity declined with depth, strongly correlated with vertical trends in total organic matter and concentrations of dissolved phosphorus. Particulate organic matter, probably buried during a flood 35 days earlier, appeared largely responsible for these vertical trends. On the other hand, there was little evidence for hydrological exchange between much of the hyporheic zone and the surface stream, implying that processes in the subsurface zone of this stream are effectively isolated during baseflow in mid-summer. 5. Diel cycles of wetting and drying in the moist habitats did not enhance esterase activity relative to the dry or fully saturated habitats. Sediment moisture was not correlated with microbial activity, and mats of senescent algae appeared to inhibit water loss from surface sediments in the moist habitat. In this sand-bed stream, local diel fluctuations in water level appear to have less influence on microbial activity and mineralisation of organic matter in the sediments than occasional floods that bury leaf litter and renew many hyporheic zone functions. Subreach-scale processes seem to be the major driving force of microbial processes and nutrient cycling in this sand-bed river. [source] Identification of isopeptidase activity in the midgut of insects: Purification, properties and nutritional ecology of a Hofmannophila pseudospretella (Lepidoptera: Oecophoridae) larval enzymeINSECT SCIENCE, Issue 4 2010Robert M. Simpson Abstract, A ,-glutamyl transpeptidase (isopeptidase) has been purified 580-fold to homogeneity from the midgut of keratinophagous larvae of Hofmannophila pseudospretella. The enzyme is a single polypeptide of molecular mass 80 kDa. The enzyme was identified by its hydrolytic activity against the synthetic substrate, ,-glutamyl-AMC, its molecular mass and inhibition profile compared to other ,-glutamyl transpeptidases. The enzyme is low or absent from most other insect digestive systems apart from other keratinophagous lepidopteran larvae and predatory carabids. While isopeptide bonds are present in high levels of the proteins in the diet of keratinophages, their presence in the diet of predatory beetles has not been established. [source] Enzymatic Production of l -Menthol by a High Substrate Concentration Tolerable Esterase from Newly Isolated Bacillus subtilis ECU0554ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 3 2009Gao-Wei Zheng Abstract Enzymatic preparation of l -menthol has been attracting much attention in the flavor and fragrance industry. A new ideal strain, Bacillus subtilis ECU0554, which exhibited high hydrolytic activity and excellent enantioselectivity towards l -menthyl ester, has been successfully isolated from soil samples through enrichment culture and identified as Bacillus subtilis by 16S rDNA gene sequencing. The esterase extracted from B. subtilis ECU0554 (BSE) showed the best catalytic properties (E>200) for dl -menthyl acetate among the five menthyl esters examined. Enantioselective hydrolysis of 100,mM dl -menthyl acetate at 30°C and pH,7.0, using crude BSE as biocatalyst and 10% ethanol (v/v) as cosolvent, resulted in 49.0% conversion (3,h) and 98.0% ee for the l -menthol produced, which were much better than those using commercial enzymes tested. Moreover, BSE exhibited strong tolerance against high substrate concentration (up to 500,mM), and the concentration of l -menthol produced could reach as high as 182,mM, and more importantly, the optical purity of l -menthol produced was kept above 97% ee, which were not found in previous reports. These results imply that BSE is a potentially promising biocatalyst for the large-scale enzymatic preparation of l -menthol. Using this excellent biocatalyst, the enzymatic production of l -menthol will become a mild, efficient, inexpensive and easy-to-use "green chemistry" methodology. [source] Properties and application of poly(methacrylic acid- co -dodecyl methacrylate- cl - N,N -methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetateJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2008Madan Lal Verma Abstract A range of fatty acid esters is now being produced commercially with immobilized microbial lipases (glycerol ester hydrolases; EC) in nonaqueous solvents. In this study, a synthetic hydrogel was prepared by the copolymerization of methacrylic acid and dodecyl methacrylate in the presence of a crosslinker, N,N -methylene bisacrylamide. A purified alkaline thermotolerant bacterial lipase from Bacilluscereus MTCC 8372 was immobilized on a poly(methacrylic acid- co -dodecyl methacrylate- cl - N,N -methylene bisacrylamide) hydrogel by an adsorption method. The hydrogel showed a 95% binding efficiency for the lipase. The bound lipase was evaluated for its hydrolytic potential toward various p -nitrophenyl acyl esters with various C chain lengths. The bound lipase showed optimal hydrolytic activity toward p -nitrophenyl palmitate at a pH of 8.5 and a temperature of 55°C. The hydrolytic activity of the hydrogel-bound lipase was enhanced by Hg2+, Fe3+, and NH ions at a concentration of 1 mM. The hydrogel-bound lipase was used to synthesize geranyl acetate from geraniol and acetic acid in n -heptane. The optimization of the reaction conditions, such as catalyst loading, effect of substrate concentration, solvent (n -pentane, n -hexane, n -heptane, n -octane, and n -nonane), reaction time, temperature, molecular sieve (3 Å × 1.5 mm) and scale up (at 50-mL level), was studied. The immobilized lipase (25 mg/mL) was used to perform an esterification in n -alkane(s) that resulted in the synthesis of approximately 82.8 mM geranyl acetate at 55°C in n -heptane under continuous shaking (160 rpm) after 15 h when geraniol and acetic acid were used in a ratio of 100 : 100 mM. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 40 mg/mL in reaction volume (2 mL) resulted in an increase in the conversion of reactants into geranyl acetate (90.0 mM). During the repetitive esterification under optimum conditions, the hydrogel-bound lipase produced ester (37.0 mM) after the eighth cycle of reuse. When the reaction volume was scaled up to 50 mL, the ester synthesized was 58.7 mM under optimized conditions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Properties of poly(AAc- co -HPMA- cl -EGDMA) hydrogel-bound lipase of Pseudomonas aeruginosa MTCC-4713 and its use in synthesis of methyl acrylateJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2007Shamsher Singh Kanwar Abstract Microbial lipases (E.C. 3.1.1.3) are preferred biocatalysts for the synthesis of esters in organic solvents. Various extracellular thermoalkaliphilic lipases have been reported from Pseudomonas sp. In the present study, a purified alkaline thermoalkalophilic extracellular lipase of Pseudomonas aeruginosa MTCC-4713 was efficiently immobilized onto a synthetic poly(AAc- co -HPMA- cl -EGDMA) hydrogel by adsorption and the bound lipase was evaluated for its hydrolytic potential towards various p -nitrophenyl acyl esters varying in their C-chain lengths. The bound lipase showed optimal hydrolytic activity towards p -nitrophenyl palmitate (p -NPP) at pH 8.5 and temperature 45°C. The hydrolytic activity of the hydrogel-bound lipase was markedly enhanced by the presence of Hg2+, Fe3+, and NH salt ions in that order. The hydrogel-immobilized lipase (25 mg) was used to perform esterification in various n -alkane(s) that resulted in , 84.9 mM of methyl acrylate at 45°C in n -heptane under shaking (120 rpm) after 6 h, when methanol and acrylic acid were used in a ratio of 100 mM:100 mM, respectively. Addition of a molecular sieve (3Å × 1.5 mm) to the reaction system at a concentration of 100 mg/reaction vol (1 mL) resulted in a moderate enhancement in conversion of reactants into methyl acrylate (85.6 mM). During the repetitive esterification under optimum conditions, the hydrogel-bound lipase produced 71.3 mM of ester after 10th cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 183,191, 2007 [source] Does tissue transglutaminase play a role in Huntington's disease?JOURNAL OF NEUROCHEMISTRY, Issue 2002G. V. W. Johnson Tissue transglutaminase (tTG) catalyzes the incorporation of polyamines into substrates, or the formation of isopeptide bonds. tTG also binds and hydrolyzes GTP/ATP. Huntington's disease (HD) is caused by a pathological expansion of the polyglutamine domain in the protein huntingtin (htt). Because a polypeptide bound Gln residue is the primary determining factor for a tTG substrate, it has been hypothesized that due to the increase in Gln content, mutant htt may modified by tTG and this event may contribute to the pathogenesis of HD, possibly by facilitating the formation of htt aggregates. tTG is increased in HD, suggesting involvement in the pathogenic process. However, tTG is not required for aggregate formation. Further, tTG is excluded from htt aggregates and increasing or decreasing tTG has no effect on the frequency or localization of the aggregates. Considering these and other data, tTG is unlikely to play a major role in the formation of htt inclusions in HD brain. tTG may play a role in modulating neuronal cell death in response to specific stressors. If a stress increases the transamidating activity of tTG (e.g. increases in Ca++ levels), then tTG facilitates the cell death process. In contrast, if a stress does not result in an increase in the transamidating activity of tTG, then tTG protects against cell death. The protective effects of tTG are independent of its transamidating and hence likely dependent on its GTP/ATP binding and hydrolytic activity. Therefore the increase in tTG levels in HD brain could either be helpful or harmful depending on the cellular mechanisms that contribute to neuronal death. Acknowledgements:, Supported by NIH grant AG12396. [source] Decline in leaf growth under salt stress is due to an inhibition of H+ -pumping activity and increase in apoplastic pH of maize leavesJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 4 2009Britta Pitann Abstract In this study, salt-induced changes in the growth rate of maize (Zea mays L.) were investigated during the first phase of salt stress. Leaf growth was reduced in the presence of 100 mM NaCl, and effects were more pronounced for the salt-sensitive cv. Pioneer 3906 in comparison to the hybrid SR03. While hydrolytic activity of plasma membrane remained unaffected, H+ -pumping activity was reduced by 47% in Pioneer 3906, but was unchanged in SR03. Changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein isothiocyanate-dextran (50 mM). Pioneer 3906 responded with an increase of 0.2 pH units in contrast to SR03 for which no apoplastic alkalization was found. With respect to the hypothesis that the apoplastic pH is influenced by salinity, it is suggested that salt resistance is partly achieved due to efficient H+ -ATPase proton pumping, which results in cell-wall acidification and loosening. [source] Does H+ pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?JOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 4 2005Christian Zörb Abstract In the first phase of salt stress, growth of plants is impaired mainly by osmotic stress. To elucidate the effect of NaCl salinity on elongation growth of maize leaves in the first phase of salt stress, we investigated the effect of NaCl on gene expression and activity of the plasmalemma H+ ATPase of elongating leaves of maize (Zea mays L.). Treatment of maize plants with 125 mM NaCl for 3 d decreased leaf growth relative to control plants (1 mM NaCl). Whereas H+ ATPase hydrolytic activity was unaffected, the ability of the H+ ATPase to establish a pH gradient was strongly reduced. Total mRNA of plasmalemma H+ ATPase was slightly increased. However, mRNA of the ATPase isoform MHA1 was significantly reduced and ATPase isoform MHA4 was strongly increased at the mRNA level. Synthesis of total H+ ATPase protein was unchanged as revealed by western blot. The results indicate that reduced pumping of H+ ATPase in leaf plasmalemma under salt stress may be caused by a switch to gene expression of the specific isoform MHA4, which shows inferior H+ -pumping efficiency in comparison to isoforms expressed under control conditions. We propose that reduced H+ pumping of plasmalemma H+ ATPase is involved in the reduction of leaf growth of maize during the first phase of salt stress. Limitiert die H+ -Pumpaktivität der Plasmalemma-ATPase das Blattwachstum von Mais (Zea mays) während der ersten Phase eines Salzstresses? In der ersten Phase eines Salzstresses wird das Pflanzenwachstum hauptsächlich durch osmotischen Stress beeinträchtigt. Zur Klärung des Einflusses der NaCl-Salinität auf das Streckungswachstum von Maisblättern in der ersten Phase eines Salzstresses wurde der Einfluss von NaCl auf die Genexpression und die Aktivität der H+ -ATPase im Plasmalemma von unter Salzstress wachsenden Maisblättern (Zea mays L.) untersucht. Dreitägige Behandlung von Maispflanzen mit 125 mM NaCl verminderte im Vergleich zu Kontrollpflanzen (1 mM NaCl) das Blattwachstum. Während die hydrolytische H+ -ATPase-Aktivität unbeeinflusst blieb, wurde die Fähigkeit, einen pH-Gradienten aufzubauen, stark reduziert. Die Gesamt-mRNA der H+ -ATPase war unverändert. Während jedoch die ATPase-Isoform MHA1 signifikant vermindert war, wurde die Isoform MHA4 auf dem mRNA-Niveau stark erhöht. Die Western-Blot-Analyse zeigte keine Veränderung der Menge des Gesamtproteins der H+ -ATPase. Unsere Ergebnisse sprechen dafür, dass die reduzierte Fähigkeit der H+ -ATPase im Plasmalemma von unter Salzstress gewachsenen Blättern einen pH-Gradienten aufzubauen, durch Genexpression der spezifischen Isoform MHA4 verursacht wird. Diese Isoform weist eine geringere H+ -Pumpeffizienz im Vergleich zu Isoformen auf, die unter Kontrollbedingungen gebildet werden. Wir schlagen daher vor, dass die reduzierte Fähigkeit der H+ -ATPase einen pH-Gradienten aufzubauen, an der Verminderung des Blattwachstums von Mais in der ersten Phase des Salzstresses beteiligt ist. [source] Immobilization of Candida antarctica lipase B on Polystyrene NanoparticlesMACROMOLECULAR RAPID COMMUNICATIONS, Issue 1 2010Nemanja Mileti Abstract Polystyrene (PS) nanoparticles were prepared via a nanoprecipitation process. The influence of the pH of the buffer solution used during the immobilization process on the loading of Candida antarctica lipase B (Cal-B) and on the hydrolytic activity (hydrolysis of p -nitrophenyl acetate) of the immobilized Cal-B was studied. The pH of the buffer solution has no influence on enzyme loading, while immobilized enzyme activity is very dependent on the pH of adsorption. Cal-B immobilized on PS nanoparticles in buffer solution pH 6.8 performed higher hydrolytic activity than crude enzyme powder and Novozyme 435. [source] Hydrolytic enzymes as virulence factors of Candida albicansMYCOSES, Issue 6 2005Martin Schaller Summary Candida albicans is a facultative pathogenic micro-organism that has developed several virulence traits enabling invasion of host tissues and avoidance of host defence mechanisms. Virulence factors that contribute to this process are the hydrolytic enzymes. Most of them are extracellularly secreted by the fungus. The most discussed hydrolytic enzymes produced by C. albicans are secreted aspartic proteinases (Saps). The role of these Saps for C. albicans infections was carefully evaluated in numerous studies, whereas only little is known about the physiological role of the secreted phospholipases (PL) and almost nothing about the involvement of lipases (Lip) in virulence. They may play an important role in the pathogenicity of candidosis and their hydrolytic activity probably has a number of possible functions in addition to the simple role of digesting molecules for nutrition. Saps as the best-studied member of this group of hydrolytic enzymes contribute to host tissue invasion by digesting or destroying cell membranes and by degrading host surface molecules. There is also some evidence that hydrolytic enzymes are able to attack cells and molecules of the host immune system to avoid or resist antimicrobial activity. High hydrolytic activity with broad substrate specificity has been found in several Candida species, most notably in C. albicans. This activity is attributed to multigene families with at least 10 members for Saps and Lips and several members for PL B. Distinct members of these gene families are differentially regulated in various Candida infections. In future, prevention and control of Candida infections might be achieved by pharmacological or immunological tools specifically modulated to inhibit virulence factors, e.g. the family of Saps. [source] Detection of the Sm31 antigen in sera of Schistosoma mansoni, infected patients from a low endemic areaPARASITE IMMUNOLOGY, Issue 1 2010G. S. SULBARÁN Summary Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14,18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113,122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83·3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis. [source] Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leavesPHYSIOLOGIA PLANTARUM, Issue 4 2003Irma N. Roberts A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg,1 h,1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65,75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process. [source] Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a ,-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolasePROTEIN SCIENCE, Issue 8 2009Taku Ohki Abstract Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a ,-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s,1) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the AldCOOH and Tyr370, a hydrogen-bonding network from Ser187 to AldNH, and interaction between AldNH and Gln27-O, derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with AldNH (Kawashima et al., FEBS J 2009; 276:2547,2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a ,-lactamase fold. [source] The importance of starch and sucrose digestion in nutritive biology of synanthropic acaridid mites: ,-Amylases and ,-glucosidases are suitable targets for inhibitor-based strategies of mite control,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Tomas Erban Abstract The adaptation of nine species of mites that infest stored products for starch utilization was tested by (1) enzymatic analysis using feces and whole mite extracts, (2) biotests, and (3) inhibition experiments. Acarus siro, Aleuroglyphus ovatus, and Tyroborus lini were associated with the starch-type substrates and maltose, with higher enzymatic activities observed in whole mite extracts. Lepidoglyphus destructor was associated with the same substrates but had higher activities in feces. Dermatophagoides farinae, Chortoglyphus arcuatus, and Caloglyphus redickorzevi were associated with sucrose. Tyrophagus putrescentiae and Carpoglyphus lactis had low or intermediate enzymatic activity on the tested substrates. Biotests on starch additive diets showed accelerated growth of species associated with the starch-type substrates. The inhibitor acarbose suppressed starch hydrolysis and growth of the mites. We suggest that the species with higher starch hydrolytic activity in feces were more tolerant to acarbose, and ,-amylase and ,-glucosidase of synanthropic mites are suitable targets for inhibitor-based strategies of mite control. © 2009 Wiley Periodicals, Inc. [source] Crystallization and preliminary X-ray crystallographic analysis of the laminarinase endo-,-1,3-glucanase from Pyrococcus furiosusACTA CRYSTALLOGRAPHICA SECTION D, Issue 12-2 2004Andrea Ilari Laminarinase endo-,-1,3 glucanase (LamA) from Pyrococcus furiosus is an enzyme which displays its main hydrolytic activity on the ,-1,3-glucose polymer laminarin. This laminarinase is remarkably resistant to denaturation: its secondary structure is unchanged in 8,M guanidinium chloride. This protein belongs to the family 16 glycosyl hydrolases, which are enzymes that are widely distributed among bacteria, fungi and higher plants. Single crystals of P. furiosus LamA have been obtained by the hanging-drop vapour-diffusion method using 2-methyl-2,4-pentanediol as a precipitant agent. A complete data set has been collected under cryocooling at a synchrotron source. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 44.36, b = 84.76, c = 69.23,Å, , = 90, , = 104.97, , = 90°, and diffract to 2.15,Å resolution. [source] Improvement of low-temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site-directed mutagenesisBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009Chuan-Qi Zhong Abstract By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15,25°C, a down-shift of optimum temperature by ,15°C, as well as a decrease in thermostability, indicating it follows the general principle of trade-off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat-treatment at 60°C for 2,h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc-AAPF-pNA, due to an increase in Km and a decrease in kcat, in contrast to other artificially cold-adapted subtilases with increased low-temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold-adapted variants occurred within or near the substrate-binding region. None of these mutations, however, match the corresponding sites in naturally psychrophilic and other artificially cold-adapted subtilases, implying there are multiple routes to cold adaptation. Homology modeling and structural analysis demonstrated that these mutations led to an increase in mobility of substrate-binding region and a modulation of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures. Our study may provide valuable information needed to develop enzymes coupling high stability and high low-temperature activity, which are highly desired for industrial use. Biotechnol. Bioeng. 2009; 104: 862,870. © 2009 Wiley Periodicals, Inc. [source] Kinetics and Efficacy of an Organophosphorus Hydrolase in a Rodent Model of Methyl-parathion PoisoningACADEMIC EMERGENCY MEDICINE, Issue 7 2010Chip Gresham MD ACADEMIC EMERGENCY MEDICINE 2010; 17:736,740 © 2010 by the Society for Academic Emergency Medicine Abstract Objectives:, Organophosphorus (OP) pesticides exert a tremendous health burden, particularly in the developing world. Limited resources, the severity of intentional OP ingestions, and a paucity of beneficial therapies all contribute to the morbidity and mortality of this broad class of chemicals. A novel theoretical treatment for OP poisoning is the use of an enzyme to degrade the parent OP in the circulation after poisoning. The aims of this study were to determine the pharmacokinetics and efficacy of an OP hydrolase (OpdA) in a rodent model of severe methyl-parathion poisoning. Methods:, Two animal models were used. First, Wistar rats were administered two different doses of the hydrolase (0.15 and 1.5 mg/kg), and the ex vivo hydrolytic activity of plasma was determined by a fluorometric method. Second, an oral methyl-parathion animal poisoning model was developed to mimic severe human poisoning, and the efficacy of postpoisoning OpdA (as measured by survival to 4 and 24 hours) was determined. Results:, The half-life of OpdA in the Wistar rat was dependent on the dose administered and ranged between 45.0 and 57.9 minutes. The poisoning model of three times the lethal dose to 50% of the population (3 × LD50) of methyl-parathion resulted in 88% lethality at 4 and 24 hours. Using a single dose of 0.15 mg/kg OpdA 10 minutes after poisoning resulted in 100% survival at 4 hours (p = 0.001 vs. placebo), but 0% at 24 hours postpoisoning (p = NS vs. placebo). Conclusions:, The OP hydrolase OpdA exhibits pharmacokinetics suitable for repeated dosing and increases short-term survival after severe methyl-parathion poisoning. [source] Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005Ramelito C. Agapay The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8,Å using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8,Å, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters. [source] Understanding the Plasticity of the ,/, Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic PropertiesCHEMBIOCHEM, Issue 3 2009Michael Skjøt Dr. Abstract The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix ,5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices ,5 and ,10. The simulation also confirmed the function of helix ,5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C, branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family. [source] | |||||||||||||||||||