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Sample records for specific enzyme digestion

  1. Specific starch digestion of maize alpha-limit dextrins by recombinant mucosal glucosidase enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch digestion requires two luminal enzymes, salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities from the N- and C-terminals of maltase-glucoamylase (MGAM) and sucrose-isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose b...

  2. Digestive Enzyme Supplementation in Gastrointestinal Diseases

    PubMed Central

    Ianiro, Gianluca; Pecere, Silvia; Giorgio, Valentina; Gasbarrini, Antonio; Cammarota, Giovanni

    2016-01-01

    Background: Digestive enzymes are able to break down proteins and carbohydrates and lipids, and their supplementation may play a role in the management of digestive disorders, from lactose intolerance to cystic fibrosis. To date, several formulations of digestive enzymes are available on the market, being different each other in terms of enzyme type, source and origin, and dosage. Methods: This review, performed through a non-systematic search of the available literature, will provide an overview of the current knowledge of digestive enzyme supplementation in gastrointestinal disorders, discussion of the use of pancreatic enzymes, lactase (β-galactosidase) and conjugated bile acids, and also exploring the future perspective of digestive enzyme supplementation. Results: Currently, the animal-derived enzymes represent an established standard of care, however the growing study of plant-based and microbe-derived enzymes offers great promise in the advancement of digestive enzyme therapy. Conclusion: New frontiers of enzyme replacement are being evaluated also in the treatment of diseases not specifically related to enzyme deficiency, whereas the combination of different enzymes might constitute an intriguing therapeutic option in the future. PMID:26806042

  3. Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: Production of well-defined hexasaccharides

    PubMed Central

    Pomin, Vitor H; Park, Younghee; Huang, Rongrong; Heiss, Christian; Sharp, Joshua S; Azadi, Parastoo; Prestegard, James H

    2012-01-01

    Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein–GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays. PMID:22345629

  4. Characterizations of substrate and enzyme specificity of glucoamylase assays of mucosal starch digestion with determinations of group and single biopsy reference values

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...

  5. Preliminary characterization of digestive enzymes in freshwater mussels

    USGS Publications Warehouse

    Sauey, Blake W.; Amberg, Jon J.; Cooper, Scott T.; Grunwald, Sandra K.; Newton, Teresa J.; Haro, Roger J.

    2015-01-01

    Resource managers lack an effective chemical tool to control the invasive zebra mussel Dreissena polymorpha. Zebra mussels clog water intakes for hydroelectric companies, harm unionid mussel species, and are believed to be a reservoir of avian botulism. Little is known about the digestive physiology of zebra mussels and unionid mussels. The enzymatic profile of the digestive glands of zebra mussels and native threeridge (Amblema plicata) and plain pocketbook mussels (Lampsilis cardium) are characterized using a commercial enzyme kit, api ZYM, and validated the kit with reagent-grade enzymes. A linear correlation was shown for only one of nineteen enzymes, tested between the api ZYM kit and a specific enzyme kit. Thus, the api ZYM kit should only be used to make general comparisons of enzyme presence and to observe trends in enzyme activities. Enzymatic trends were seen in the unionid mussel species, but not in zebra mussels sampled 32 days apart from the same location. Enzymatic classes, based on substrate, showed different trends, with proteolytic and phospholytic enzymes having the most change in relative enzyme activity.

  6. Susceptibility of sweetpotato (Ipomoea batatas) peel proteins to digestive enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sweet potato proteins have been shown to possess antioxidant and antidiabetic properties in vivo. The ability of a protein to exhibit systemic effects is somewhat unusual as proteins are typically susceptible to digestive enzymes. This study was undertaken to better understand how digestive enzymes ...

  7. Monoterpenes as inhibitors of digestive enzymes and counter-adaptations in a specialist avian herbivore.

    PubMed

    Kohl, Kevin D; Pitman, Elizabeth; Robb, Brecken C; Connelly, John W; Dearing, M Denise; Forbey, Jennifer Sorensen

    2015-05-01

    Many plants produce plant secondary metabolites (PSM) that inhibit digestive enzymes of herbivores, thus limiting nutrient availability. In response, some specialist herbivores have evolved digestive enzymes that are resistant to inhibition. Monoterpenes, a class of PSMs, have not been investigated with respect to the interference of specific digestive enzymes, nor have such interactions been studied in avian herbivores. We investigated this interaction in the Greater Sage-Grouse (Phasianidae: Centrocercus urophasianus), which specializes on monoterpene-rich sagebrush species (Artemisia spp.). We first measured the monoterpene concentrations in gut contents of free-ranging sage-grouse. Next, we compared the ability of seven individual monoterpenes present in sagebrush to inhibit a protein-digesting enzyme, aminopeptidase-N. We also measured the inhibitory effects of PSM extracts from two sagebrush species. Inhibition of aminopeptidase-N in sage-grouse was compared to inhibition in chickens (Gallus gallus). We predicted that sage-grouse enzymes would retain higher activity when incubated with isolated monoterpenes or sagebrush extracts than chicken enzymes. We detected unchanged monoterpenes in the gut contents of free-ranging sage-grouse. We found that three isolated oxygenated monoterpenes (borneol, camphor, and 1,8-cineole) inhibited digestive enzymes of both bird species. Camphor and 1,8-cineole inhibited enzymes from chickens more than from sage-grouse. Extracts from both species of sagebrush had similar inhibition of chicken enzymes, but did not inhibit sage-grouse enzymes. These results suggest that specific monoterpenes may limit the protein digestibility of plant material by avian herbivores. Further, this work presents additional evidence that adaptations of digestive enzymes to plant defensive compounds may be a trait of specialist herbivores. PMID:25652583

  8. Digestive enzyme activities in larvae of sharpsnout seabream (Diplodus puntazzo).

    PubMed

    Suzer, Cüneyt; Aktülün, Sevim; Coban, Deniz; Okan Kamaci, H; Saka, Sahin; Firat, Kürşat; Alpbaz, Atilla

    2007-10-01

    The ontogenesis and specific activities of pancreatic and intestinal enzymes were investigated in sharpsnout sea bream, Diplodus puntazzo, during larval development until the end of weaning on day 50. The green-water technique was carried out for larval rearing in triplicate. Trypsin was first detected as early as hatching and sharply increased related to age and exogenous feeding until day 25, but a sharp decrease was observed towards the end of the experiment. Amylase was determined 2 days after hatching (DAH) and sharply increased to 10 DAH. Afterwards, slight decreases were found between 10 and 20 DAH and then slow alterations were continued until end of the experiment. Lipase was measured for the first time on day 4, and then slight increase was found to 25 DAH. After this date, slow variations were maintained until end of the experiment. Pepsin was firstly assayed 32 DAH related with stomach formation and sharply increased to 40 DAH. Then it was fluctuated until end of the experiment. Enzymes of brush border membranes, alkaline phosphatase and aminopeptidase N, showed similar pattern on specific activities during the first 10 days. Thereafter, while specific activity of alkaline phosphatase slightly decreased to 15 DAH and fluctuated until 20 DAH, aminopeptidase N activity slowly declined to 20 DAH. Afterwards, activity of alkaline phosphatase and aminopeptidase N were sharply increased to 30 DAH, showing maturation of the intestinal digestive process and also these activities continued to slight increase until end of the experiment. The specific activity of cytosolic peptidase, leucine-alanine peptidase sharply increased to on day 8, then suddenly declined to 12 DAH and further decreased until 20 DAH. After this date, in contrast to enzymes of brush border membranes, it sharply decreased to 25 DAH and continued to gradually decline until the end of the experiment. These converse expressions were indicative of a maturation of enterocytes and the transition to

  9. Digestive enzymes in juvenile green abalone, Haliotis fulgens, fed natural food.

    PubMed

    García-Carreño, F L; Navarrete del Toro, M A; Serviere-Zaragoza, E

    2003-01-01

    Enzymes responsible for the digestion of food protein by juvenile green abalone (Haliotis fulgens) were studied when fed algae or a sea grass (Phyllospadix torreyi) naturally occurring in the habitat. The effect of food on the composition and activity of the enzymes was also evaluated. Acid, serine proteinases and aminopeptidases, as confirmed by pH profile of activity, specific inhibition and synthetic substrate hydrolysis were found in the digestive organs of juvenile green abalone. Algae and sea grass differentially affected the digestive system in abalone. PMID:12524042

  10. Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

    PubMed

    Tamaki, Fábio K; Pimentel, André C; Dias, Alcides B; Cardoso, Christiane; Ribeiro, Alberto F; Ferreira, Clélia; Terra, Walter R

    2014-11-01

    Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three β-glucosidases, one β-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none β-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single β-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two β-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of β-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion

  11. Cysteine cathepsins as digestive enzymes in the spider Nephilengys cruentata.

    PubMed

    Fuzita, Felipe J; Pinkse, Martijn W H; Verhaert, Peter D E M; Lopes, Adriana R

    2015-05-01

    Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles. PMID:25818482

  12. Digestive enzymes from workers and soldiers of termite Nasutitermes corniger.

    PubMed

    Lima, Thâmarah de Albuquerque; Pontual, Emmanuel Viana; Dornelles, Leonardo Prezzi; Amorim, Poliana Karla; Sá, Roberto Araújo; Coelho, Luana Cassandra Breitenbach Barroso; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes

    2014-10-01

    The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-l-arabinofuranosidase, and β-d-xylanase), α-amylase, and proteases (trypsin-like, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and β-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed. PMID:25026598

  13. The complexities of hydrolytic enzymes from the termite digestive system.

    PubMed

    Saadeddin, Anas

    2014-06-01

    The main challenge in second generation bioethanol production is the efficient breakdown of cellulose to sugar monomers (hydrolysis). Due to the recalcitrant character of cellulose, feedstock pretreatment and adapted hydrolysis steps are needed to obtain fermentable sugar monomers. The conventional industrial production process of second-generation bioethanol from biomass comprises several steps: thermochemical pretreatment, enzymatic hydrolysis and sugar fermentation. This process is undergoing continuous optimization in order to increase the bioethanol yield and reduce the economic cost. Therefore, the discovery of new enzymes with high lignocellulytic activity or new strategies is extremely important. In nature, wood-feeding termites have developed a sophisticated and efficient cellulose degrading system in terms of the rate and extent of cellulose hydrolysis and exploitation. This system, which represents a model for digestive symbiosis has attracted the attention of biofuel researchers. This review describes the termite digestive system, gut symbionts, termite enzyme resources, in vitro studies of isolated enzymes and lignin degradation in termites. PMID:23036053

  14. The Effect of Enzyme Addition on Anaerobic Digestion of Jose Tall Wheat Grass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of the addition of enzyme products containing cellulase, hemicellulase, and Beta-glucosidase to anaerobic digestion systems were studied. Anaerobic digestion tests were performed using batch reactors operated at 35°C. The application of enzyme products in three digestion configurations w...

  15. Protein digestibility of the same protein preparations by human and rat assays and by in vitro enzymic digestion methods.

    PubMed

    Bodwell, C E; Satterlee, L D; Hackler, L R

    1980-03-01

    The apparent and true digestibilities of the same preparations of six proteins (spray dried whole egg, cottage cheese, canned tuna, peanut flour, soy isolate, and wheat gluten) were estimated in four to five men and in rats and compared to estimates of digestibility from three different in vitro enzymic digestion procedures. For all six proteins, the correlation coefficient was 0.46 between true digestibility in humans and in rats; with values for tuna excluded, r = 0.96. With all six proteins, none of the in vitro values was significantly correlated with values from humans or rats. However, with either the three animal proteins alone or the three plant proteins alone, correlations were high (r greater than 0.90) between one or more of the in vitro estimates and the observed true or apparent human and rat digestibilities. The differences in the relationship between enzymic digestion estimates and the human digestibility estimates for plant or animal proteins suggest that for accurate prediction of protein digestibility in humans by these enzymic methods, different equations would have to be used for plant and animal proteins. For protein sources containing both plant and animal protein, use of the in vitro enzymic procedures would give only an approximate estimate of digestibility in humans. PMID:6986763

  16. Sequencing of oligosaccharides using enzyme array digestion with electrochemical and fluorescent detections

    SciTech Connect

    Sun, M.; Lee, C.S.

    1997-12-31

    The objective of this study is to develop a rapid and sensitive method for oligosaccharide sequencing. The oligosaccharides are subjected to the enzyme array digestion with exoglycosidases of known and well-defined specificities. The enzyme array method involves the division of oligosaccharide sample into aliquots, and the incubation of each aliquot with a precisely defined mixture of exoglycosidases. In the enzyme array method, the presence of a specific linkage anywhere in the oligosaccharide is determined by the inability of an enzyme mixture lacking a given enzyme to cleave that linkage ( a stop point) and the ability of the other enzymes to cleave the linkage up to that point. The direct quantification of released monosaccharides from the enzyme array can be achieved by using pulsed amperometric detection (PAD) or by fluorescent derivatization with a fluorophoric agent. The measured monosaccharide concentrations in combination with the enzyme array analysis provide detail characterization of oligosaccharides with their sugar composition, configuration, and linkage information, The released monosaccharides are further quantified by anion exchange chromatography and capillary electrophoresis for the comparison with the results obtained from PAD and fluorescence measurements. Our enzyme array-electrochemical (or fluorescent) detection method does not require any separation procedure and any prior labeling of oligosaccharide and have several practical advantages over the current carbohydrate sequencing techniques including simplicity, speed, and the ability to use small amounts of starting material.

  17. Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes.

    PubMed

    Eriksen, Ellen K; Holm, Halvor; Jensen, Einar; Aaboe, Ragnhild; Devold, Tove G; Jacobsen, Morten; Vegarud, Gerd E

    2010-08-01

    The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37 degrees C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, beta-lactoglobulin (beta-LG) and alpha-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine beta-LG was digested and the peptide profiles obtained were compared with those of the caprine beta-LG in the digested whey. The bovine beta-LG seemed to be more extensively cleaved than the caprine beta-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred. PMID:20307348

  18. Working with Enzymes - Where Is Lactose Digested? An Enzyme Assay for Nutritional Biochemistry Laboratories

    NASA Astrophysics Data System (ADS)

    Pope, Sandi R.; Tolleson, Tonya D.; Williams, R. Jill; Underhill, Russell D.; Deal, S. Todd

    1998-06-01

    At Georgia Southern University, we offer a sophomore-level introductory biochemistry course that is aimed at nutrition and chemistry education majors. The laboratory portion of this course has long lacked an experimental introduction to enzymes. We have developed a simple enzyme assay utilizing lactase enzyme from crushed LactAid tablets and a 5% lactose solution ("synthetic milk"). In the experiment, the students assay the activity of the enzyme on the "synthetic milk" at pHs of approximately 1, 6, and 8 with the stated goal of determining where lactose functions in the digestive tract. The activity of the lactase may be followed chromatographically or spectrophotometrically. The experiment, which is actually a simple pH assay, is easily implemented in allied health chemistry laboratory courses and readily lends itself to adaptation for more complex kinetic assays in upper-level biochemistry laboratory courses. The experimental details, including a list of required supplies and hints for implementation, are provided.

  19. [The synthesis of specific enzyme inhibitors].

    PubMed

    Iakovleva, G M

    1987-04-01

    The review deals with directed synthesis of specific enzyme inhibitors. They are classified within the framework of the mechanistic approach, namely, stable analogues of substrates, which form enzyme complexes mimicking the Michaelis complex or those which influence the chemical stages of enzyme catalysis; conformational inhibitors; substrate analogues participating in enzyme reactions and producing modified products; suicide inhibitors; stage inhibitors (inhibitors influencing certain stages of enzyme reaction); transition state analogues; multisubstrate analogues and collected substrates. Types of chemical modification used in synthesis of the specific inhibitors are discussed. Some possibilities of the quantity structure-activity relationship methods, computer modelling and molecular graphics in designing the optimal structure of inhibitors are mentioned. PMID:3300658

  20. Subclinical zinc deficiency impairs pancreatic digestive enzyme activity and digestive capacity of weaned piglets.

    PubMed

    Brugger, Daniel; Windisch, Wilhelm M

    2016-08-01

    This study investigated the effects of short-term subclinical Zn deficiency on exocrine pancreatic activity and changes in digestive capacity. A total of forty-eight weaned piglets were fed ad libitum a basal diet (maize and soyabean meal) with adequate Zn supply (88 mg Zn/kg diet) during a 2-week acclimatisation phase. Animals were then assigned to eight dietary treatment groups (n 6) according to a complete randomised block design considering litter, live weight and sex. All pigs were fed restrictively (450 g diet/d) the basal diet but with varying ZnSO4.7H2O additions, resulting in 28·1, 33·6, 38·8, 42·7, 47·5, 58·2, 67·8 and 88·0 mg Zn/kg diet for a total experimental period of 8 d. Pancreatic Zn concentrations and pancreatic activities of trypsin, chymotrypsin, carboxypeptidase A and B, elastase and α-amylase exhibited a broken-line response to stepwise reduction in dietary Zn by declining beneath thresholds of 39·0, 58·0, 58·0, 41·2, 47·5, 57·7 and 58·0 mg Zn/kg diet, respectively. Furthermore, carboxypeptidase B and α-amylase activities were significantly lower in samples with reduced pancreatic Zn contents. Coefficients of faecal digestibility of DM, crude protein, total lipids and crude ash responded similarly to pancreatic enzyme activities by declining below dietary thresholds of 54·7, 45·0, 46·9 and 58·2 mg Zn/kg diet, respectively. In conclusion, (1) subclinical Zn deficiency impaired pancreatic exocrine enzymes, (2) this response was connected to pancreatic Zn metabolism and (3) the decline in catalytic activity impaired faecal digestibility already after 1 week of insufficient alimentary Zn supply and very early before clinical deficiency symptoms arise. PMID:27230230

  1. A Trypsin Inhibitor from Clitoria fairchildiana Cotyledons is Active Against Digestive Enzymes of Aedes aegypti Larvae.

    PubMed

    de Oliveira, Lucilene O; Fernandes, Kátia V S; Pádua, Dayanni de Souza; Carvalho, André de O; Lemos, Francisco J A; Gomes, Valdirene M; Oliveira, Antônia E A; Ferreira, André T da Silva; Perales, Jonas

    2015-01-01

    Aedes aegypti, the principal mosquito vector of yellow fever, dengue fever and chikungunya fever virus-transmitted diseases, is an insect closely associated with humans and their housing habitats. As there is no commercially available vaccine, prevention is the most suggested form of avoiding disease spreading and a number of studies are being developed in order to give support to vector control operations. The present study reports on the identification of a trypsin inhibitor isolated from cotyledons of the Clitoria fairchildiana amazonic tree seeds, which was able to reduce by 87.93 % the activity of digestive enzymes of fourth instar A. aegypti larva. A partial amino acid sequence showed strong similarity with sequences from several trypsin inhibitors already reported in the literature. The 13,000 Da isolated inhibitor was seen to be active solely against trypsin-like enzymes, neither acting on papain, α-amylase nor on other serine proteases, such as elastase, chymotrypsin or subtilisin. At least six from seven active digestive proteases from A. aegypti larvae, visualized by zymography, were severely affected soon after exposed to the inhibitor. The strong and specific action of the isolated inhibitor against trypsin digestive enzymes of this insect vector led us to believe that this protein may be a good candidate for a prospective alternative biocontrol method. PMID:26156641

  2. Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae).

    PubMed

    Mehrabadi, Mohammad; Bandani, Ali Reza; Dastranj, Mehdi

    2014-06-01

    The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/β-mannosidases) were little in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed. PMID:24961557

  3. Sturgeon hatching enzyme and the mechanism of egg envelope digestion: Insight into changes in the mechanism of egg envelope digestion during the evolution of ray-finned fish.

    PubMed

    Nagasawa, Tatsuki; Kawaguchi, Mari; Sano, Kaori; Yasumasu, Shigeki

    2015-12-01

    We investigated the evolution of the hatching enzyme gene using bester sturgeon (hybrid of Acipencer ruthenus and Huso huso), a basal member of ray-finned fishes. We purified the bester hatching enzyme from hatching liquid, yielding a single band on SDS-PAGE, then isolated its cDNA from embryos by PCR. The sturgeon hatching enzyme consists of an astacin family protease domain and a CUB domain. The CUB domains are present in frog and bird hatching enzymes, but not in teleostei, suggesting that the domain structure of sturgeon hatching enzyme is the tetrapod type. The purified hatching enzyme swelled the egg envelope, and selectively cleaved one of five egg envelope proteins, ZPAX. Xenopus hatching enzyme preferentially digests ZPAX, thus, the egg envelope digestion process is conserved between amphibians and basal ray-finned fish. Teleostei hatching enzymes cleave the repeat sequences at the N-terminal region of ZPB and ZPC, suggesting that the targets of the teleostei hatching enzymes differ from those of amphibians and sturgeons. Such repeat sequences were not found in the N-terminal region of ZPB and ZPC of amphibians and sturgeons. Our results suggest that the change in substrates of the hatching enzymes was accompanied by the mutation of the amino acid sequence of N-terminal regions of ZPB and ZPC. We conclude that the changes in the mechanism of egg envelope digestion, including the change in the domain structure of the hatching enzymes and the switch in substrate, occurred during the evolution of teleostei, likely triggered by the teleost-specific third whole genome duplication. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 720-732, 2015. © 2015 Wiley Periodicals, Inc. PMID:26514945

  4. The effect of enzyme addition on anaerobic digestion of JoseTall Wheat Grass.

    PubMed

    Romano, Rowena T; Zhang, Ruihong; Teter, Sarah; McGarvey, Jeffery A

    2009-10-01

    The effects of the addition of enzyme products containing cellulase, hemicellulase, and beta-glucosidase to anaerobic digestion systems were studied using JoseTall Wheat Grass (wheat grass) as a model substrate. Anaerobic digestion tests were performed using batch reactors operated at 50 degrees C. The application of enzyme products in three digestion configurations were simulated and investigated: (1) enzyme addition to a single-stage digester, (2) pre-treatment of wheat grass with enzymes followed by a single-stage anaerobic digestion, and (3) enzyme addition to the first stage (hydrolysis and acidification) of a two-stage digestion system. The enzyme products showed positive effects on the solubilization of wheat grass when used alone to treat the wheat grass. However, no significant differences in biogas and methane yields, and volatile solids reduction resulted when the enzyme products were tested in the anaerobic digestion systems. This reveals that the microorganisms present in the inoculum were effective in carrying out the digestion of wheat grass. The types of microorganisms present in the inoculum were identified using 16S rRNA sequence analysis. A comparison of the sequences between the different inocula revealed that the prevalent operational taxonomic units were similar, but that the acidified inoculum contained a higher percentage of the species Thermotogae. PMID:19467589

  5. Digestive Enzymes of the Crustaceans Munida and Their Application in Cheese Manufacturing: A Review

    PubMed Central

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-01-01

    Crustaceans Munida (fam. Galatheideae, ord. Decapodi) were fished in the Southern Adriatic Sea and their proteolytic activities were characterized and tested for potential application in cheese manufacturing. Enzymes extracted from whole crustaceans, mainly serine proteases, showed high caseinolytic and moderate clotting activities. Analysis by 2D zymography of the digestive enzymes extracted from Munida hepatopancreas, showed the presence of several isotrypsin- and isochymotrypsin-like enzymes in the range of 20–34 kDa and 4.1–5.8 pI. Moreover, specific enzymatic assays showed the presence of aminopeptidases and carboxypeptidases A and B. Overall, optimum activity was achieved at pH 7.5 and 40–45 °C. Caseinolytic activity, determined both spectrophotometrically and by SDS gel electrophoresis, indicated higher activity on β-casein than on α-casein. Miniature cheddar-type cheeses and Pecorino-type cheeses were manufactured by adding starter, rennet and Munida extracts to milk. Reverse-phase HPLC and MALDI-ToF mass spectrometry showed a more complex pattern of proteolytic products in cheeses made using Munida instead of chymosin. Munida extracts were found to degrade the chymosin-derived β-casein fragment f193–209, one of the peptides associated with bitterness in cheese. In conclusion, Munida digestive enzymes represent a promising tool for development of new cheese products and shorten cheese ripening when used either alone or in addition to calf rennet. PMID:21822412

  6. Main factors providing specificity of repair enzymes.

    PubMed

    Nevinsky, G A

    2011-01-01

    Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity. It is shown that high affinity of these enzymes to any DNA (10(-4)-10(-8) M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less degree with bases of nucleotide links "covered" by protein globules. Enzyme interactions with specific DNA links are comparable in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10(-1)-10(-2) M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting enzyme atoms and DNA orbitals with accuracy up to 10-15° and, as a result, for high reaction rate. Upon transition from nonspecific to specific DNA, reaction rate (k(cat)) increases by 4-8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are the basis of specificity of repair enzymes. PMID:21568843

  7. An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.

    PubMed

    Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun

    2016-01-01

    Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p < 0.05) by 5.0 and 2.6 percentage unit, respectively. The IVTTD of DM for corn increased (p < 0.05) by 3.1 percentage unit with enzyme complex addition. As the effect of enzyme complex was the greatest in corn digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p < 0.05) by 16.0, 2.8 and 1.2 percentage unit, respectively. The IVTTD of DM for corn starch and hull also increased (p < 0.05) by 8.6 and 0.9 percentage unit, respectively, with enzyme complex addition. In conclusion, the enzyme complex increases in vitro DM digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch. PMID:27247894

  8. Development study on some digestive enzymes of Takifugu rubripes larvae and juvenile

    NASA Astrophysics Data System (ADS)

    Wan, Zhenzhen; Gao, Tianxiang; Zhang, Xiumei; Chen, Chao

    2004-10-01

    The activities of some digestive enzymes are studied for Takifugu rubripes larvae and juvenile from the first feeding to 27d after hatching at selected stages of development. The homogenate of whole larvae body is used for enzymatic determination. Activity of acid protease decreases notably during the beginning days after the commencement of completely exogenous feeding and the days before the beginning of the juvenile stage. Alkaline protease specific activity also decreases at metamorphosis. The activities are associated with the morphology of the developing digestive tract. Amylase activity increases before the first feeding, followed by a decreasing and then a rather constant level. Lipase activity remains low during the larvae and juvenile periods. Alkaline phosphatase activity increases gradually. This reflects the development of brush border membranes of enterocytes.

  9. Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion.

    PubMed

    Bibo-Verdugo, Betsaida; O'Donoghue, Anthony J; Rojo-Arreola, Liliana; Craik, Charles S; García-Carreño, Fernando

    2016-04-01

    Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen. PMID:26613762

  10. A Randomized, Placebo-controlled Trial of Digestive Enzymes in Children with Autism Spectrum Disorders

    PubMed Central

    Saad, Khaled; Eltayeb, Azza A.; Mohamad, Ismail L.; Al-Atram, Abdulrahman A.; Elserogy, Yasser; Bjørklund, Geir; El-Houfey, Amira A.; Nicholson, Bubba

    2015-01-01

    Objective There is growing evidence for a gut-brain connection associated with autism spectrum disorders (ASDs). This suggests a potential benefit from introduced digestive enzymes for children with ASD. Methods We performed a double-blind, randomized clinical trial on 101 children with ASD (82 boys and 19 girls) aged from 3 to 9 years. ASD patients were diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition, text revision (DSM-IV-TR) diagnostic criteria. Structured interviews of at least one hour each both with the parents and the child were performed. Later on, another two hours-session was conducted applying the Childhood Autism Rating Scale (CARS). ASD patients were randomized to receive digestive enzymes or placebo. Results The ASD group receiving digestive enzyme therapy for 3 months had significant improvement in emotional response, general impression autistic score, general behavior and gastrointestinal symptoms. Our study demonstrated the usefulness of digestive enzyme in our population of ASD patients. Conclusion Digestive enzymes are inexpensive, readily available, have an excellent safety profile, and have mildly beneficial effects in ASD patients. Depending on the parameter measured in our study, we propose digestive enzymes for managing symptoms of ASD. Digestive enzyme therapy may be a possible option in treatment protocols for ASD in the future. PMID:26243847

  11. EFFECTS OF FIVE DIVERSE LIGNOCELLULOSIC DIETS ON DIGESTIVE ENZYME BIOCHEMISTRY IN THE TERMITE Reticulitermes flavipes.

    PubMed

    Karl, Zachary J; Scharf, Michael E

    2015-10-01

    Termites have recently drawn much attention as models for biomass processing, mainly due to their lignocellulose digestion capabilities and mutualisms with cellulolytic gut symbionts. This research used the lower termite Reticulitermes flavipes to investigate gut enzyme activity changes in response to feeding on five diverse lignocellulosic diets (cellulose filter paper [FP], pine wood [PW], beech wood xylan [X], corn stover [CS], and soybean residue [SB]). Our objectives were to compare whole-gut digestive enzyme activity and host versus symbiont contributions to enzyme activity after feeding on these diets. Our hypothesis was that enzyme activities would vary among diets as an adaptive mechanism enabling termites and symbiota to optimally utilize variable resources. Results support our "diet-adaptation" hypothesis and further indicate that, in most cases, host contributions are greater than those of symbionts with respect to the enzymes and activities studied. The results obtained thus provide indications as to which types of transcriptomic resources, termite or symbiont, are most relevant for developing recombinant enzyme cocktails tailored to specific feedstocks. With regard to the agricultural feedstocks tested (CS and SB), our results suggest endoglucanase and exoglucanase (cellobiohydrolase) activities are most relevant for CS breakdown; whereas endoglucanase and xylosidase activities are relevant for SB breakdown. However, other unexplored activities than those tested may also be important for breakdown of these two feedstocks. These findings provide new protein-level insights into diet adaptation by termites, and also complement host-symbiont metatranscriptomic studies that have been completed for R. flavipes after FP, PW, CS, and SB feeding. PMID:25980379

  12. Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

    PubMed

    Zhang, Yun-Long; Wu, Qiao-Wan; Hu, Wei-Hua; Wang, Fan; Zhao, Zhong-Bo; He, Hui; Shao, Wei-Han; Fan, Qi-Xue

    2015-12-01

    The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning. PMID:26232086

  13. Digestive enzyme activities in the guts of bonnethead sharks (Sphyrna tiburo) provide insight into their digestive strategy and evidence for microbial digestion in their hindguts.

    PubMed

    Jhaveri, Parth; Papastamatiou, Yannis P; German, Donovan P

    2015-11-01

    Few investigations have studied digestive enzyme activities in the alimentary tracts of sharks to gain insight into how these organisms digest their meals. In this study, we examined the activity levels of proteases, carbohydrases, and lipase in the pancreas, and along the anterior intestine, spiral intestine, and colon of the bonnethead shark, Sphyrna tiburo. We then interpreted our data in the context of a rate-yield continuum to discern this shark's digestive strategy. Our data show anticipated decreasing patterns in the activities of pancreatic enzymes moving posteriorly along the gut, but also show mid spiral intestine peaks in aminopeptidase and lipase activities, which support the spiral intestine as the main site of absorption in bonnetheads. Interestingly, we observed spikes in the activity levels of N-acetyl-β-D-glucosaminidase and β-glucosidase in the bonnethead colon, and these chitin- and cellulose-degrading enzymes, respectively, are likely of microbial origin in this distal gut region. Taken in the context of intake and relatively long transit times of food through the gut, the colonic spikes in N-acetyl-β-D-glucosaminidase and β-glucosidase activities suggest that bonnetheads take a yield-maximizing strategy to the digestive process, with some reliance on microbial digestion in their hindguts. This is one of the first studies to examine digestive enzyme activities along the gut of any shark, and importantly, the data match with previous observations that sharks take an extended time to digest their meals (consistent with a yield-maximizing digestive strategy) and that the spiral intestine is the primary site of absorption in sharks. PMID:26239220

  14. Enzyme Specific Activity in Functionalized Nanoporous Supports

    SciTech Connect

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-26

    Enzyme specific activity can be increased or decreased to a large extent by changing protein loading density in functionalized nanoporous support, where organophosphorus hydrolase can display a constructive orientation and thus leave a completely open entrance for substrate even at higher protein loading density, but glucose oxidase can not.

  15. Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

    PubMed Central

    Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

    2015-01-01

    Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

  16. Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

    PubMed

    Su, S; Miska, K B; Fetterer, R H; Jenkins, M C; Wong, E A

    2015-03-01

    Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would

  17. ENZYME ADDITION TO THE ANAEROBIC DIGESTION OF MUNICIPAL WASTEWATER PRIMARY SLUDGE

    EPA Science Inventory

    The study evaluates the effects of enzyme augmentation on municipal wastewater (MWW) sludge anaerobic digestion. The primary objective was to examine the impact of using enzymes to enhance the degradation of the cellulosic and the oil- and grease-rich sludge fractions. The additi...

  18. Construction of a high-performance magnetic enzyme nanosystem for rapid tryptic digestion

    NASA Astrophysics Data System (ADS)

    Cheng, Gong; Zheng, Si-Yang

    2014-11-01

    A magnetic enzyme nanosystem have been designed and constructed by a polydopamine (PDA)-modification strategy. The magnetic enzyme nanosystem has well defined core-shell structure and a relatively high saturation magnetization (Ms) value of 48.3 emu g-1. The magnetic enzyme system can realize rapid, efficient and reusable tryptic digestion of proteins by taking advantage of its magnetic core and biofunctional shell. Various standard proteins (e.g. cytochrome C (Cyt-C), myoglobin (MYO) and bovine serum albumin (BSA)) have been used to evaluate the effectiveness of the magnetic enzyme nanosystem. The results show that the magnetic enzyme nanosystem can digest the proteins in 30 minutes, and the results are comparable to conventional 12 hours in-solution digestion. Furthermore, the magnetic enzyme nanosystem is also effective in the digestion of low-concentration proteins, even at as low as 5 ng μL-1 substrate concentration. Importantly, the system can be reused several times, and has excellent stability for storage. Therefore, this work will be highly beneficial for the rapid digestion and identification of proteins in future proteomics.

  19. Construction of a high-performance magnetic enzyme nanosystem for rapid tryptic digestion

    PubMed Central

    Cheng, Gong; Zheng, Si-Yang

    2014-01-01

    A magnetic enzyme nanosystem have been designed and constructed by a polydopamine (PDA)-modification strategy. The magnetic enzyme nanosystem has well defined core-shell structure and a relatively high saturation magnetization (Ms) value of 48.3 emu g−1. The magnetic enzyme system can realize rapid, efficient and reusable tryptic digestion of proteins by taking advantage of its magnetic core and biofunctional shell. Various standard proteins (e.g. cytochrome C (Cyt-C), myoglobin (MYO) and bovine serum albumin (BSA)) have been used to evaluate the effectiveness of the magnetic enzyme nanosystem. The results show that the magnetic enzyme nanosystem can digest the proteins in 30 minutes, and the results are comparable to conventional 12 hours in-solution digestion. Furthermore, the magnetic enzyme nanosystem is also effective in the digestion of low-concentration proteins, even at as low as 5 ng μL−1 substrate concentration. Importantly, the system can be reused several times, and has excellent stability for storage. Therefore, this work will be highly beneficial for the rapid digestion and identification of proteins in future proteomics. PMID:25374397

  20. Substitution of Wheat for Corn in Beef Cattle Diets: Digestibility, Digestive Enzyme Activities, Serum Metabolite Contents and Ruminal Fermentation.

    PubMed

    Liu, Y F; Zhao, H B; Liu, X M; You, W; Cheng, H J; Wan, F C; Liu, G F; Tan, X W; Song, E L; Zhang, X L

    2016-10-01

    The objective of this study was to evaluate the effect of diets containing different amounts of wheat, as a partial or whole substitute for corn, on digestibility, digestive enzyme activities, serum metabolite contents and ruminal fermentation in beef cattle. Four Limousin×LuXi crossbred cattle with a body weight (400±10 kg), fitted with permanent ruminal, proximal duodenal and terminal ileal cannulas, were used in a 4×4 Latin square design with four treatments: Control (100% corn), 33% wheat (33% substitution for corn), 67% wheat (67% substitution for corn), and 100% wheat (100% substitution for corn) on a dry matter basis. The results showed that replacing corn with increasing amounts of wheat increased the apparent digestibility values of dry matter, organic matter, and crude protein (p<0.05). While the apparent digestibility of acid detergent fiber and neutral detergent fiber were lower with increasing amounts of wheat. Digestive enzyme activities of lipase, protease and amylase in the duodenum were higher with increasing wheat amounts (p<0.05), and showed similar results to those for the enzymes in the ileum except for amylase. Increased substitution of wheat for corn increased the serum alanine aminotransferase concentration (p<0.05). Ruminal pH was not different between those given only corn and those given 33% wheat. Increasing the substitution of wheat for corn increased the molar proportion of acetate and tended to increase the acetate-to-propionate ratio. Cattle fed 100% wheat tended to have the lowest ruminal NH3-N concentration compared with control (p<0.05), whereas no differences were observed among the cattle fed 33% and 67% wheat. These findings indicate that wheat can be effectively used to replace corn in moderate amounts to meet the energy and fiber requirements of beef cattle. PMID:26954111

  1. Cellulose digestion in Monochamus marmorator Kby. (coleoptera: Cerambycidae): role of acquired fungal enzymes

    SciTech Connect

    Kukol, J.J.; Martin, M.M.

    1986-05-01

    Larvae of the balsam fir sawyer, Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus, Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungusfree diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enable M. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood.

  2. Influence of nutritional stress on digestive enzyme activities in juveniles of two marine clam species, Ruditapes decussatus and Venerupis pullastra

    NASA Astrophysics Data System (ADS)

    Albentosa, Marina; Moyano, Francisco J.

    2008-08-01

    The potential use of digestive activities as indicators of the nutritional status in bivalves is discussed in relation to the results obtained in two clam species exposed to starvation and refeeding. Activities of some digestive enzymes (amylase, laminarinase, cellulase, and protease) were measured in juveniles of two commercially interesting species of clams, Ruditapes decussatus and Venerupis pullastra. The specimens were fed normally, being after subjected to a 15-days starvation and a further refeeding period. Samples were obtained at different moments of such feeding schedule to evaluate enzymes as well as weight (live, dry and organic) and length, in order to calculate growth rates and feeding efficiencies. Starvation led to a major decrease in clam growth as measured by dry weight and a negative growth as measured by organic weight, this coinciding with a certain degree of growth of the shell and a consumption of soft tissue. This response occurred more rapidly in R. decussatus but was of a lower magnitude than in V. pullastra. Activity of carbohydrases decreased rapidly in both species with starvation, although protease activity was maintained in R. decussatus. Recovery after the end of starvation was not similar in both species; while R. decussatus attained similar growth rates and enzyme activities to those measured prior to nutritional stress, V. pullastra only recovered 50% of its initial values. For both species of bivalves it can be concluded that digestive enzymes, and more specifically amylase, could be used as indicative of their nutritional condition.

  3. Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea).

    PubMed

    Martínez-Lagos, R; Tovar-Ramírez, D; Gracia-López, V; Lazo, J P

    2014-06-01

    The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine-alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine-alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period. PMID:24189829

  4. Development of continuous microwave-assisted protein digestion with immobilized enzyme.

    PubMed

    Chen, Zhengyi; Li, Yongle; Lin, Shuhai; Wei, Meiping; Du, Fuyou; Ruan, Guihua

    2014-03-01

    In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research. PMID:24530398

  5. Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae.

    PubMed

    Caldeira, Waldir; Dias, Alcides B; Terra, Walter R; Ribeiro, Alberto F

    2007-01-01

    Bostrichiformia is the less known major series of Coleoptera regarding digestive physiology. The midgut of Dermestes maculatus has a cylindrical ventriculus with anterior caeca. There is no cell differentiation along the ventriculus, except for the predominance of cells undergoing apocrine secretion in the anterior region. Apocrine secretion affects a larger extension and a greater number of cells in caeca than in ventriculus. Ventricular cells putatively secrete digestive enzymes, whereas caecal cells are supposed to secrete peritrophic gel (PG) glycoproteins. Feeding larvae with dyes showed that caeca are water-absorbing, whereas the posterior ventriculus is water-secreting. Midgut dissection revealed a PG and a peritrophic membrane (PM) covering the contents in anterior and posterior ventriculus, respectively. This was confirmed by in situ chitin detection with FITC-WGA conjugates. Ion-exchange chromatography of midgut homogenates, associated with enzymatic assays with natural and synthetic substrates and specific inhibitors, showed that trypsin and chymotrypsin are the major proteinases, cysteine proteinase is absent, and aspartic proteinase probably is negligible. Amylase and trypsin occur in contents and decrease along the ventriculus; the contrary is true for cell-membrane-bound aminopeptidase. Maltase is cell-membrane-bound and predominates in anterior and middle midgut. Digestive enzyme activities in hindgut are negligible. This, together with dye data, indicates that enzymes are recovered from inside PM by a posterior-anterior flux of fluid outside PM before being excreted. The combined results suggest that protein digestion starts in anterior midgut and ends in the surface of posterior midgut cells. All glycogen digestion takes place in anterior midgut. PMID:17167750

  6. Activity, life time and effect of hydrolytic enzymes for enhanced biogas production from sludge anaerobic digestion.

    PubMed

    Odnell, Anna; Recktenwald, Michael; Stensén, Katarina; Jonsson, Bengt-Harald; Karlsson, Martin

    2016-10-15

    As an alternative to energy intensive physical methods, enzymatic treatment of sludge produced at wastewater treatment plants for increased hydrolysis and biogas production was investigated. Several hydrolytic enzymes were assessed with a focus on how enzyme activity and life time was influenced by sludge environments. It could be concluded that the activity life time of added enzymes was limited (<24 h) in both waste activated sludge and anaerobic digester sludge environments and that this was, for the majority of enzymes, due to endogenous protease activity. In biogas in situ experiments, subtilisin at a 1% mixture on basis of volatile solids, was the only enzyme providing a significantly increased biomethane production of 37%. However, even at this high concentration, subtilisin could not hydrolyze all available substrate within the life time of the enzyme. Thus, for large scale implementation, enzymes better suited to the sludge environments are needed. PMID:27498254

  7. Chemical characterization of hydrothermally pretreated and enzyme-digested wheat straw: An evaluation of recalcitrance.

    PubMed

    Merali, Zara; Marjamaa, Kaisa; Käsper, Andres; Kruus, Kristiina; Gunning, A Patrick; Morris, Vic J; Waldron, Keith W

    2016-05-01

    There is great interest in understanding changes that occur to cell wall constituents during saccharification of pretreated lignocellulose, particularly in relation to recalcitrance of the residues. This paper reports the effects of hydrothermal pretreatment followed by enzyme hydrolysis on the extractability and properties of recalcitrant wheat straw polymers. The results show that the undigested residue had lost much of its archestructure. Compositional analysis portrayed a considerable loss of cross-linking di-ferulic acid phenolics, hemicellulosic and cellulosic sugars. The remaining cellulosic and non-cellulosic polysaccharides were much more readily extractable in alkali and molecular profiling revealed the presence of low Mw oligomers in the fractions suggesting the partial enzyme hydrolysis of hemicelluloses and cellulose. Simultaneous saccharification and fermentation of the pretreated and enzyme-digested residues surprisingly resulted in ethanol yields of up to 99% of the theoretical. This is discussed in relation to the "recalcitrant" nature of the original pretreated and enzyme digested biomass. PMID:26769515

  8. Double clicking for site-specific coupling of multiple enzymes.

    PubMed

    Lim, Sung In; Cho, Jinhwan; Kwon, Inchan

    2015-09-14

    A method to site-specifically couple multiple enzymes is reported. The approach is based on the site-specific incorporation of a clickable non-natural amino acid into enzymes and two compatible click reactions. The multi-enzyme reaction system exhibited enhanced catalytic efficiency over the respective free enzymes. PMID:26191550

  9. Susceptibility of sweet potato (Ipomoea batatas) peel proteins to digestive enzymes.

    PubMed

    Maloney, Katherine P; Truong, Van-Den; Allen, Jonathan C

    2014-07-01

    Sweet potato proteins have been shown to possess antioxidant and antidiabetic properties in vivo. The ability of a protein to exhibit systemic effects is somewhat unusual as proteins are typically susceptible to digestive enzymes. This study was undertaken to better understand how digestive enzymes affect sweet potato proteins. Two fractions of industrially processed sweet potato peel, containing 6.8% and 8.5% protein and 80.5% and 83.3% carbohydrate, were used as a source of protein. Sweet potato proteins were incubated with pepsin, trypsin, and chymotrypsin and protein breakdown was visualized with SDS-PAGE. After pepsin digestion, samples were assayed for amylase inhibitory activity. Sporamin, the major storage protein in sweet potatoes, which functions as a trypsin inhibitor as well, exhibited resistance to pepsin, trypsin, and chymotrypsin. Sporamin from blanched peel of orange sweet potatoes was less resistant to pepsin digestion than sporamin from outer peel and from extract of the white-skinned Caiapo sweet potato. Trypsin inhibitory activity remained after simulated gastric digestion, with the Caiapo potato protein and peel samples exhibiting higher inhibitory activity compared to the blanched peel sample. Amylase and chymotrypsin inhibitory activity was not present in any of the samples after digestion. PMID:25473492

  10. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    PubMed Central

    Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

    2014-01-01

    The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

  11. Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

    PubMed

    Regnery, R L; Fu, Z Y; Spruill, C L

    1986-01-01

    The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts. PMID:3009528

  12. Inhibitory effect of Pistia tannin on digestive enzymes of Indian major carps: an in vitro study.

    PubMed

    Mandal, Sudipta; Ghosh, Koushik

    2010-12-01

    Aquatic weeds are one of the major unconventional feed ingredients tested for aquafeed formulation. Tannin content in the water lettuce, Pistia, has been quantified (26.67 mg g(-1); dry weight) and graded levels of which (12.5-200 μg) have been incorporated in the reaction mixtures to evaluate any change in the in vitro activity of the principal digestive enzymes from the three Indian major carps (IMC), namely rohu (Labeo rohita), catla (Catla catla) and mrigala (Cirrhinus mrigala). Result of the experiment revealed that the Pistia tannin (PT) significantly inhibit/lower the activities of the digestive enzymes from three IMCs in a dose-dependent manner, even at very low concentration. Significant variation in the reduction of the enzyme activities was noticed between the three fish species, as well as between the three enzymes studied. Among the three species studied, digestive enzymes from L. rohita were found to be the most sensitive to the PT, whereas enzymes from C. catla were found to be comparatively least affected. On the other hand, protease and lipase activities were comparatively more affected than the amylase activity. The results of the study suggest that more stress should be given on the elimination of tannin while incorporating feed ingredients of plant origin in fish diets. PMID:20369287

  13. The interplay of α-amylase and amyloglucosidase activities on the digestion of starch in in vitro enzymic systems.

    PubMed

    Warren, Frederick J; Zhang, Bin; Waltzer, Gina; Gidley, Michael J; Dhital, Sushil

    2015-03-01

    In vitro hydrolysis assays are a key tool in understanding differences in rate and extent of digestion of starchy foods. They offer a greater degree of simplicity and flexibility than dynamic in vitro models or in vivo experiments for quantifiable, mechanistic exploration of starch digestion. In the present work the influence of α-amylase and amyloglucosidase activities on the digestion of maize and potato starch granules was measured using both glucose and reducing sugar assays. Data were analysed through initial rates of digestion, and by 1st order kinetics, utilising logarithm of slope (LOS) plots. The rate and extent of starch digestion was dependent on the activities of both enzymes and the type of starch used. Potato required more enzyme than maize to achieve logarithmic reaction curves, and complete digestion. The results allow targeted design of starch digestion experiments through a thorough understanding of the contributions of α-amylase and amyloglucosidase to digestion rates. PMID:25498625

  14. Digestive Enzyme Supplementation for Autism Spectrum Disorders: A Double-Blind Randomized Controlled Trial

    ERIC Educational Resources Information Center

    Munasinghe, Sujeeva A.; Oliff, Carolyn; Finn, Judith; Wray, John A.

    2010-01-01

    To examine the effects of a digestive enzyme supplement in improving expressive language, behaviour and other symptoms in children with Autism Spectrum Disorder. Randomized, double-blind placebo-controlled trial using crossover design over 6 months for 43 children, aged 3-8 years. Outcome measurement tools included monthly Global Behaviour Rating…

  15. Influence of Molting and Starvation on Digestive Enzyme Activities and Energy Storage in Gammarus fossarum

    PubMed Central

    Charron, Laetitia; Geffard, Olivier; Chaumot, Arnaud; Coulaud, Romain; Jaffal, Ali; Gaillet, Véronique; Dedourge-Geffard, Odile; Geffard, Alain

    2014-01-01

    Among the many biological responses studied in ecotoxicology, energy-based biomarkers such as digestive enzyme activities and energy reserves appear to be useful predictive tools for detecting physiological disturbances in organisms. However, the use of these biological responses as biomarkers could be limited by the effects of confounding factors (biotic and abiotic) and physiological processes, such as the reproductive cycle. Thus, the optimal use of these biomarkers will be facilitated by understanding the effects of these factors on the energy metabolism of the sentinel species being studied. We considered abiotic factors (temperature and conductivity) in a previous study, whereas the present study investigated the effects of gender, the female reproductive stage, and food availability on the digestive enzyme activities and energy storage of Gammarus fossarum. The results indicated that, during the female reproductive cycle, the activities of digestive enzymes (amylase, cellulase, and trypsin) decreased significantly, whereas the levels of reserves (proteins, lipids, and sugar) increased until the last premolt stage. Restricted food diets only led to decreased amylase activities in both sexes. Food starvation also induced a decrease in the energy outcomes in females, whereas there were no effects in males. In general, the biochemical (digestive enzyme activities) and physiological (energy reserves) responses were more stable in males than in females. These results support the use of males fed ad libitum to limit the effects of confounding factors when using these energy biomarkers in Gammarus fossarum during biomonitoring programs. PMID:24788197

  16. Expression of digestive enzymes and nutrient transporters in Eimeria acervulina-challenged layers and broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. Eimeria-infected chickens develop lesions in the intestinal mucosa, which result in reduced feed efficiency and body weight gain. This growth reduction may be due to changes in expression of digestive enzymes and nutrient tran...

  17. Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored product pests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cereals have storage proteins with high amounts of the amino acids glutamine and proline. Therefore, storage pests need to have digestive enzymes that are efficient in hydrolyzing these types of proteins. Post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored product pe...

  18. Synergistic amylomaltase and branching enzyme catalysis to suppress cassava starch digestibility.

    PubMed

    Sorndech, Waraporn; Meier, Sebastian; Jansson, Anita M; Sagnelli, Domenico; Hindsgaul, Ole; Tongta, Sunanta; Blennow, Andreas

    2015-11-01

    Starch provides our main dietary caloric intake and over-consumption of starch-containing foods results in escalating life-style disease including diabetes. By increasing the content of α-1,6 branch points in starch, digestibility by human amylolytic enzymes is expected to be retarded. Aiming at generating a soluble and slowly digestible starch by increasing the content and changing the relative positioning of the branch points in the starch molecules, we treated cassava starch with amylomaltase (AM) and branching enzyme (BE). We performed a detailed molecular analysis of the products including amylopectin chain length distribution, content of α-1,6 glucosidic linkages, absolute molecular weight distribution and digestibility. Step-by-step enzyme catalysis was the most efficient treatment, and it generated branch structures even more extreme than those of glycogen. All AM- and BE-treated samples showed increased resistance to degradation by porcine pancreatic α-amylase and glucoamylase as compared to cassava starch. The amylolytic products showed chain lengths and branching patterns similar to the products obtained from glycogen. Our data demonstrate that combinatorial enzyme catalysis provides a strategy to generate potential novel soluble α-glucan ingredients with low dietary digestibility assets. PMID:26256365

  19. Kernel Composition, Starch Structure, and Enzyme Digestibility of Opaque-2 Maize and Quality Protein Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives of this study were to understand how opaque-2 (o2) mutation and quality protein maize (QPM) affect maize kernel composition and starch structure, property, and enzyme digestibility. Kernels of o2 maize contained less protein (9.6−12.5%) than those of the wild-type (WT) counterparts (12...

  20. Bovine lactoferrin digested with human gastrointestinal enzymes inhibits replication of human echovirus 5 in cell culture.

    PubMed

    Furlund, Camilla B; Kristoffersen, Anja B; Devold, Tove G; Vegarud, Gerd E; Jonassen, Christine M

    2012-07-01

    Many infant formulas are enriched with lactoferrin (Lf) because of its claimed beneficial effects on health. Native bovine Lf (bLf) is known to inhibit in vitro replication of human enteroviruses, a group of pathogenic viruses that replicate in the gut as their primary infection site. On the basis of a model digestion and human gastrointestinal enzymes, we hypothesized that bLf could retain its antiviral properties against enterovirus in the gastrointestinal tract, either as an intact protein or through bioactive peptide fragments released by digestive enzymes. To test our hypothesis, bLf was digested with human gastric juice and duodenal juice in a 2-step in vitro digestion model. Two gastric pH levels and reduction conditions were used to simulate physiological conditions in adults and infants. The antiviral activity of native bLf and of the digested fractions was studied on echovirus 5 in vitro, using various assay conditions, addressing several mechanisms for replication inhibition. Both native and digested bLf fractions revealed a significant inhibitory effect, when added before or simultaneously with the virus onto the cells. Furthermore, a significant stronger sustained antiviral effect was observed when bLf was fully digested in the gastric phase with fast pH reduction to 2.5, compared with native bLf, suggesting the release of antiviral peptides from bLf during the human digestion process. In conclusion, this study demonstrates that bLf may have a role in the prevention of human gastrointestinal virus infection under physiological conditions and that food containing bLf may protect against infection in vivo. PMID:22901558

  1. Fasting and refeeding cause rapid changes in intestinal tissue mass and digestive enzyme capacities of Atlantic salmon (Salmo salar L.).

    PubMed

    Krogdahl, Ashild; Bakke-McKellep, Anne Marie

    2005-08-01

    Fasting and refeeding effects on gastrointestinal morphology and digestive enzyme activities of Atlantic salmon, held in tanks of seawater at 9 degrees C and 31 per thousand salinity, were addressed in two trials. Trial 1: Fish (mean body mass 1190 g) were fasted for 40 days and intestines sampled at day 0, 2, 4, 11, 19 and 40. Trial 2: Fish (1334 g), fasted for 50 days, were refed and sampled at day 0, 3 and 7. Mass, length, protein, and maltase, lactase, and leucine aminopeptidase (LAP) activities were analyzed for stomach (ST), pyloric caeca (PC), proximal (PI), mid (MI), and distal intestine (DI). PC contributed 50% of gastrointestinal mass and 75% of enzyme capacity. Fasting decreased mass and enzyme capacities by 20-50% within two days, and 40-75% after 40 days. In PC, specific brush border membrane (BBM) maltase activity decreased whereas BBM LAP increased during fasting. Upon refeeding, enzyme capacities were mostly regenerated after one week. The results suggest that refeeding should start slowly with about 25% of estimated feed requirement during the first 3 days, but may then be stepped up rapidly. Investigations of digestive processes of fed fish should only be performed when intestines are feed-filled to avoid bias due to effects of fasting. PMID:16046160

  2. Digestive enzymes along digestive tract of a carnivorous fish Glyptosternum maculatum (Sisoridae, Siluriformes).

    PubMed

    Xiong, D M; Xie, C X; Zhang, H J; Liu, H P

    2011-02-01

    This study attempted to determine the presence, distribution and levels of digestive proteases, pepsin, amylase and lipase in the homogenates of digestive tract of a freshwater, cold-adapted, carnivorous teleost of Tibet, Glyptosternum maculatum (n= 10, mean weight: 129.6±28.9 g, mean length: 195.1±16.6 mm, approximately 6 years old), at different temperatures and pH levels. The descending order of protease activity was as follows: stomach (pepsin, 16.16±0.96 U/mg protein), anterior intestine (3.18±0.25 U/mg protein), posterior intestine (1.76±0.21 U/mg protein) and middle intestine (1.52±0.23 U/mg protein). Amylase activity levels in descending order were as follows: anterior intestine (0.0062±0.0007 U/mg protein), stomach (0.0032±0.0009 U/mg protein), middle intestine (0.0023±0.0005 U/mg protein) and posterior intestine (0.0023±0.0004 U/mg protein). The highest lipase activity was found in the anterior intestine (0.83±0.25 U/mg protein), followed by posterior intestine (0.51±0.19 U/mg protein), middle intestine (0.48±0.09 U/mg protein) and stomach (0.39±0.10 U/mg protein). The optimal temperature and pH level for protease were 50°C and 9.0-10.0, respectively, along the intestine. The pepsin optima in the stomach were 30°C and 2.0 respectively. The optimal temperature for amylase was found to be 30°C along the digestive tract and the optimal pH was 7.0 in the intestine and 6.0 in the stomach. The optimal temperature and pH levels of lipase were 30°C, pH 6.0 for the stomach and 40-50°C, pH 8.0 for the intestine, respectively. Hepato-somatic index was 1.35±0.2% and relative intestine length was 0.90±0.19. The weight ratio of the stomach and intestine to body weight was 1.63±0.43% and 1.57±0.24% respectively. PMID:20487102

  3. Effect of glucagon on digestive enzyme synthesis, transport and secretion in mouse pancreatic acinar cells.

    PubMed Central

    Singh, M

    1980-01-01

    ). 7. It is concluded that glucagon increased digestive enzyme secretion from pancreatic acinar cells by a direct action (in contrast to its reported indirect effect in vivo) and decreased digestive enzyme synthesis. The data on transport and secretion do not support the suggested role of glucagon as an inhibitor in the intact animal. PMID:6162027

  4. Norwalk Virus–specific Binding to Oyster Digestive Tissues

    PubMed Central

    Loisy, Fabienne; Atmar, Robert L.; Hutson, Anne M.; Estes, Mary K.; Ruvoën-Clouet, Nathalie; Pommepuy, Monique; Le Pendu, Jacques

    2006-01-01

    The primary pathogens related to shellfishborne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts (midgut, main and secondary ducts, and tubules) by carbohydrate structures with a terminal N-acetylgalactosamine residue in an α linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue. PMID:16707048

  5. Digestive enzyme activities in mudskipper Boleophthalmus pectinirostris and Chinese black sleeper Bostrichthys sinensis

    NASA Astrophysics Data System (ADS)

    Wu, Renxie; Hong, Wanshu; Zhang, Qiyong

    2010-07-01

    The mudskipper Boleophthalmus pectinirostris and Chinese black sleeper Bostrichthys sinensis occupy the intertidal zone. However, both species have their own unique diet. The former is an herbivore and the latter is a carnivore. In order to reveal the relationship between digestive enzyme activities and diets in the two species, the activities of protease (P), non-specific bile salt-activated lipase (BAL) and α-amylase (A) were determined in the stomach and intestine of adult mudskipper B. pectinirostris and Chinese black sleeper B. sinensis. The results showed that the activities of protease, BAL and α-amylase in the intestine of B. pectinirostris were significantly ( P<0.05) higher than those in the stomach. In B. sinensis, gastric protease activity was not different from the intestinal protease ( P>0.05), while BAL and α-amylase activities of the intestine were significantly ( P<0.05) higher than those of the stomach. The activity of gastric protease in B. sinensis was significantly ( P<0.05) higher than that in B. pectinirostris, while the activities of intestinal protease were not different between the two fish species ( P>0.05). BAL activities of the stomach and intestine in B. sinensis were significantly ( P<0.05) higher than those in B. pectinirostris, while α-amylase activities of the stomach and intestine in B. pectinirostris were significantly ( P<0.05) higher than those in B. sinensis. The ratios of P/BAL, A/P and A/BAL of the digestive tract in B. pectinirostris were 1.5, 107.3 and 158.6, respectively; and those in B. sinensis were 0.2, 1.6 and 0.2, respectively. It can be concluded that food digestion in the adult B. pectinirostris is mainly carried out in the intestine, whereas in the adult B. sinensis it is initiated in the stomach and finishes in the intestine. The activities of BAL and α-amylase in B. pectinirostris and B. sinensis are well correlated with their diets. However, a clear-cut correlation between protease activity and diets is

  6. Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes.

    PubMed

    Furlund, C B; Ulleberg, E K; Devold, T G; Flengsrud, R; Jacobsen, M; Sekse, C; Holm, H; Vegarud, G E

    2013-01-01

    Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is

  7. Effects of an enzyme complex on in vitro dry matter digestibility of feed ingredients for pigs.

    PubMed

    Kong, Changsu; Park, Chan Sol; Kim, Beob Gyun

    2015-01-01

    Feed ingredients of plant origin are commonly used in swine diets. However, the major components of plant cell walls, non-starch polysaccharides (NSPs), reduce nutrient digestibility. To improve the efficiency of feed utilization, exogenous enzyme products that degrade NSPs have been widely used in commercial animal feeds. Nonetheless, the effects of exogenous enzyme addition to swine diets on nutrient digestibility have not been determined. To this end, in vitro approaches may be used. The objective of this study was to determine the effects of an enzyme complex (EC) containing xylanase, protease, and phytase on the in vitro dry matter (DM) digestibility of nine feed ingredients including cereal grain energy sources (corn, wheat, and barley) and protein sources (soybean meal, rapeseed meal, palm kernel meal, cottonseed meal, copra meal, and distillers dried grains with solubles). Both in vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM were determined for the nine test ingredients, with or without EC addition. The EC addition increased the IVID of DM in copra meal (p = 0.047) and tended to increase the IVID of DM in corn, wheat, barley, palm kernel meal, cottonseed meal, and DDGS (p < 0.10). On the other hand, no significant effect was observed in soybean meal and rapeseed meal. The IVTTD of DM in the test ingredients was not affected by the addition of EC, except for cottonseed meal (52.1 vs. 50.6%, p = 0.053). In conclusion, the effects of EC addition on in vitro DM digestibility may vary, depending on the test ingredient and method used. PMID:26090308

  8. Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

    NASA Astrophysics Data System (ADS)

    Luo, Wen; Zhao, Yunlong; Zhou, Zhongliang; An, Chuanguang; Ma, Qiang

    2008-02-01

    The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR (FQ—PCR) methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

  9. Inside the trap: gland morphologies, digestive enzymes, and the evolution of plant carnivory in the Caryophyllales⋆

    PubMed Central

    Renner, Tanya; Specht, Chelsea D

    2013-01-01

    The digestion of prey by carnivorous plants is determined in part by suites of enzymes that are associated with morphologically and anatomically diverse trapping mechanisms. Chitinases represent a group of enzymes known to be integral to effective plant carnivory. In non-carnivorous plants, chitinases commonly act as pathogenesis-related proteins, which are either induced in response to insect herbivory and fungal elicitors, or constitutively expressed in tissues vulnerable to attack. In the Caryophyllales carnivorous plant lineage, multiple classes of chitinases are likely involved in both pathogenic response and digestion of prey items. We review what is currently known about trap morphologies, provide an examination of the diversity, roles, and evolution of chitinases, and examine how herbivore and pathogen defense mechanisms may have been coopted for plant carnivory in the Caryophyllales. PMID:23830995

  10. Influence of probiotics on the growth and digestive enzyme activity of white Pacific shrimp ( Litopenaeus vannamei)

    NASA Astrophysics Data System (ADS)

    Gómez, R. Geovanny D.; Shen, M. A.

    2008-05-01

    The influence of Bacillus probiotics on the digestive enzyme activity and the growth of Litopenaeus vannamei were determined in this study. The shrimp was treated with five percentages (1.5, 3.0, 4.5, 6.0 and 7.5) of probiotics ( Bacillus spp.) supplemented to the feed and cultured for 45d. The growth measured as the weight gain at the end of culturing was significantly ( P<0.05) higher in probiotic-treated shrimps than that of the control (without receiving probiotics). Activities of protease and amylase, two digestive enzymes of the midgut gland and the intestine were significantly ( P<0.05) higher in probiotic-treated shrimp than in the control.

  11. Digestive enzyme expression and epithelial structure of small intestine in neonatal rats after 16 days spaceflight

    NASA Astrophysics Data System (ADS)

    Miyake, M.; Yamasaki, M.; Hazama, A.; Ijiri, K.; Shimizu, T.

    It is important to assure whether digestive system can develop normally in neonates during spaceflight. Because the small intestine changes its function and structure drastically around weaning known as redifferentiation. Lactase expression declines and sucrase increases in small intestine for digestion of solid food before weaning. In this paper, we compared this enzyme transition and structural development of small intestine in neonatal rats after spaceflight. To find digestive genes differentially expressed in fight rats, DNA membrane macroarray was also used. Eight-day old rats were loaded to Space Shuttle Columbia, and housed in the animal facility for 16 days in space (STS-90, Neurolab mission). Two control groups (AGC; asynchronous ground control and VIV; vivarium) against flight group (FLT) were prepared. There was no difference in structure (crypt depth) and cell differentiation of epithelium between FLT and AGC by immunohistochemical analysis. We found that the amount of sucrase mRNA compared to lactase was decreased in FLT by RT-PCR. It reflected the enzyme transition was inhibited. Increase of 5 genes (APO A-I, APO A-IV, ACE, aFABP and aminopeptidase M) and decrease of carboxypeptidase-D were detected in FLT using macroarray. We think nutrition differences (less nourishment and late weaning) during spaceflight may cause inhibition of enzyme transition at least partly. The weightlessness might contribute to the inhibition through behavioral change.

  12. [Enzyme labeling methods and it's specificities].

    PubMed

    Kambegawa, A

    1995-09-01

    Enzyme labeled antigen for use in ELISA of Hapten (steroids, prostanoid, carbohydrate, nucleic acid, peptide, herbicide, insecticide and antibiotic) have usually been prepared by condensation of carboxy group of hapten with amino groups of lysine residue in enzyme. The horseradish peroxidase (HRP) is best suitable as labeling enzyme, therefore it is small molecular, and substrate turnover is much higher compared to the other enzymes. The mixed anhydride and carbodiimide methods have mainly been used the preparation of hapten conjugate BSA, but not satisfactory for enzyme labeling. The N-hydroxysuccinimide ester (NHS: active ester) method is satisfactory with respect to reproducibility and sensitivity. The sensitivity is related to the bridging phenomenon: One of the disadvantages of the homologous labels is that the antibody shows an affinity not only for the Hapten but also for the bridge which connects the Hapten to carrier protein. We have developed a sensitive bridge heterologous EIA for progesterone (P) using geometrical isomers of P-3 (E/Z) (O-carboxymethyl) oxime-N-hydroxysuccinimide esters [ef Ab of P-3 (E) CMO-BSA/P-3 (Z) CMO-HRP]3). The sensitivity of heterologous proved to be higher than a homologous EIA or a conventional RIA. It seem like that a 1:1 steroid-enzyme conjugate is suitable for obtaining a high sensitivity. The avidin-biotin (AB) system provides great versatility, since by conjugation with an appropriate label, the AB assay can be used with any chosen detector. Furthermore, IgG can be labels with biotin without significantly influencing their immunological activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7474375

  13. Effect of dietary genistein on growth performance, digestive enzyme activity, and body composition of Nile tilapia Oreochromis niloticus

    NASA Astrophysics Data System (ADS)

    Chen, Dong; Wang, Wei; Ru, Shaoguo

    2015-01-01

    An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance, body composition, and digestive enzymes activity of juvenile Nile tilapia ( Oreochromis niloticus). Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein: 0, 30, 300, and 3 000 μg/g. Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia (10.47±1.24 g). The results show that 30 and 300 μg/g dietary genistein had no significant effect on growth performance of Nile tilapia, but the higher level of genistein (3 000 μg/g) significantly depressed the final body weight and specific growth rate. There was no significant difference in survival rate, feed intake, feed efficiency ratio or whole body composition among all dietary treatments. An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity, and amylase activity in the liver and intestine, while a dietary level of 300 μg/g genistein depressed stomach protease and intestine amylase activities. However, no significant difference in stomach amylase activity was found among dietary treatments. Overall, the results of the present study indicate that a high level of dietary genistein (3 000 μg/g, or above) would significantly reduce the growth of Nile tilapia, partly because of its inhibitory effect on the activity of major digestive enzymes. Accordingly, the detrimental effects of genistein, as found in soybean products, should not be ignored when applied as an alternative ingredient source in aquaculture.

  14. Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin.

    PubMed Central

    Rose, Christiane; Voisin, Stéphanie; Gros, Claude; Schwartz, Jean-Charles; Ouimet, Tanja

    2002-01-01

    Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity. PMID:11964170

  15. Expression of hemocyanin and digestive enzyme messenger RNAs in the hepatopancreas of the Black Tiger Shrimp Penaeus monodon.

    PubMed

    Lehnert, Sigrid A; Johnson, Samuel E

    2002-10-01

    In order to define the cellular site of synthesis for hemocyanin and digestive enzymes in the decapod hepatopancreas, we studied the expression of messenger ribonucleic acids (RNAs) for these molecules in the epithelium lining hepatopancreas tubules. In situ hybridisation of gene probes for the digestive enzymes amylase, cathepsin-L, cellulase, chitinase-1 and trypsin to tissue sections of the shrimp hepatopancreas confirmed that the F-cells lining tertiary, secondary and primary ducts are the sites of synthesis for digestive enzyme messenger RNA (mRNA). The F-cells also contained mRNA for the hemocyanin gene. This finding raises important questions on the mechanism by which mature hemocyanin accumulates in the shrimp hemolymph. Our in situ hybridisation studies further showed that Penaeus monodon F-cells remain transcriptionally active for digestive enzyme mRNAs during periods of starvation. PMID:12381378

  16. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

    PubMed

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(P<0.01). Immunofluorescence staining showed that the SCs purity was (95.73±1.51)% in the improved method group,(84.66±2.68)% in the hemi-explants-culture method group,and (74.50±4.23)% in the explants-culture method group(P<0.01). Conclusion The improved enzyme digestion combined with explants-culture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration. PMID:27594149

  17. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    PubMed

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-01-01

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results. PMID:26345863

  18. Inhibitory activities of Ulva lactuca polysaccharides on digestive enzymes related to diabetes and obesity.

    PubMed

    BelHadj, Sahla; Hentati, Olfa; Elfeki, Abdelfattah; Hamden, Khaled

    2013-05-01

    The aim of this study was to evaluate the effect of alga Ulva lactuca polysaccharides (ULPS) on key enzymes related to diabetes and obesity. This marine natural product, ULPS, exerted potential inhibition on key enzymes related to starch digestion and absorption in both plasma and small intestine mainly α-amylase by 53% and 34% and maltase by 97 and 164% respectively, leading to a significant decrease in blood glucose rate by 297%. Moreover, ULPS potentially inhibited key enzymes of lipid metabolism and absorption as lipase activity in both plasma and small intestine by 235 and 287% respectively, which led to a notable decrease of blood LDL-cholesterol and triglycerides levels, and in the counterpart an increase in HDL-cholesterol level in surviving diabetic rats. Additively, ULPS significantly protected the liver-kidney functions, by decreasing of aspartate transaminase (AST), alanine transaminase (ALT) and gamma-glytamyl transpeptidase (GGT) activities and creatinine, urea and albumin rates in plasma. PMID:23638862

  19. Feeding habits and ontogenic changes in digestive enzyme patterns in five freshwater teleosts.

    PubMed

    Solovyev, M M; Kashinskaya, E N; Izvekova, G I; Gisbert, E; Glupov, V V

    2014-11-01

    Feeding habits and the activity of digestive enzymes (total alkaline proteases, α-amylase and lipase) from dace Leuciscus leuciscus, roach Rutilus rutilus, Prussian carp Carassius auratus gibelio, perch Perca fluviatilis and pikeperch Sander lucioperca fry were studied in the Malye Chany Lake-Kargat Estuary (western Siberia, Russia). The diet of fry from all studied species was mainly composed of chironomid larvae and zooplanktonic organisms (i.e. cladocera and copepoda), whereas carnivorous species such as P. fluviatilis and S. lucioperca also preyed on fry from other fishes while detritus and microalgae were also important in the diet of ommivorous species. When comparing diet similarity (Sørensen-Dice index, Q(S)) among fry at different stages of development, both omnivorous and carnivorous species showed a high level of similarity (0.67 < Q(S) < 0.89 and 0.73 < Q(S)< 0.89, respectively). Diet similarity values were in agreement with the overall digestive activity profile analysed by cluster analysis. Diet similarity suggested potential trophic competition when zooplanktonic and benthic prey began to decline towards autumn. The analysis of pancreatic digestive enzymes revealed a correlation among their activities and fry feeding habits with α-amylase:total proteases (A:P) values higher than 1 in omnivorous species and lower (A:P ≤ 1) in carnivorous species. PMID:25199648

  20. Daily rhythms of digestive enzyme activity and gene expression in gilthead seabream (Sparus aurata) during ontogeny.

    PubMed

    Mata-Sotres, José Antonio; Moyano, Francisco Javier; Martínez-Rodríguez, Gonzalo; Yúfera, Manuel

    2016-07-01

    In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24h cycle in larvae reared under a 12h light/12h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60days post-hatch. In each sampling day, larvae were sampled every 3h during a complete 24h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes. PMID:26987267

  1. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  2. Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

    PubMed

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

    2016-12-15

    Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. PMID:27451220

  3. Changes in diarrhea, nutrients apparent digestibility, digestive enzyme activities of weaned piglets in response to chitosan-zinc chelate.

    PubMed

    Qian, Lichun; Yue, Xiaojing; Hu, Luansha; Ma, Yuanfei; Han, Xinyan

    2016-04-01

    A total of 120 weanling barrows weighing 6.11 ± 0.20 kg were randomly allotted to four treatments with three replications (i.e. pen) of ten piglets per replicate. Pigs were received corn-soybean basal diet (control) or the same basal diet supplemented with the following sources of zinc: (i) 100 mg/kg of Zn as ZnSO4 ; (ii) 100 mg/kg of Zn as chitosan-Zn chelate (CS-Zn); and (iii) 100 mg/kg of Zn as ZnSO4 mixed with chitosan (CS + ZnSO4 ). The results showed that CS-Zn could highly improve average daily gain and average daily feed intake than those of ZnSO4 or CS+ ZnSO4 (P < 0.05). The pigs fed dietary CS-Zn had lower diarrhea incidence and higher apparent digestibility of crude protein than those of the pigs fed dietary ZnSO4 (P < 0.05). The protease activities in duodenal content of the pigs receiving CS-Zn diets was higher than that of the pigs fed dietary ZnSO4 or CS + ZnSO4 (P < 0.05). The amylase activity in duodenal content of the pigs fed dietary CS-Zn was higher than that of the pigs receiving ZnSO4 diets or basal diets (P < 0.05). These results indicated that dietary CS-Zn showed different bioactivities from ZnSO4 or CS + ZnSO4 in reducing the incidence of diarrhea, improving activities of digestive enzymes and growth performance of weaned pigs. PMID:26304729

  4. Effects of Hanseniaspora opuntiae C21 on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas

    NASA Astrophysics Data System (ADS)

    Ma, Yuexin; Liu, Zhiming; Yang, Zhiping; Bao, Pengyun; Zhang, Congyao; Ding, Jianfeng

    2014-07-01

    The effects of a diet containing Hanseniaspora opuntiae C21 on growth and digestive enzyme activity were estimated in juvenile Apostichopus japonicus. Groups of sea cucumbers were fed diets containing H. opuntiae C21 at 0 (control), 104, 105, and 106 CFU (colony-forming units)/g feed. Results showed that after 45 d the specific growth rate (SGR) of sea cucumbers fed a C21-supplemented diet at 10 4 CFU/g feed was significantly higher than that of the control ( P < 0.05). Intestinal trypsin and lipase activities were significantly enhanced by C21 administration at 104 and 105 CFU/g feed compared with the control ( P < 0.05). After feeding for 23-42 d, C21 was demonstrated by denaturing gradient gel electrophoresis to be present in the intestine of sea cucumbers. In addition, after feeding the C21-supplemented diets for 15 d, the sea cucumbers were switched to an unsupplemented diet and C21 was confirmed to be capable of colonizing the intestine for at least 31 d after cessation of feeding. In conclusion, C21 was shown to successfully colonize the intestine of juvenile A. japonicus via dietary supplementation, and improve growth and digestive enzyme activity.

  5. Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides.

    PubMed

    Yin, Junfa; Xu, Tian; Zhang, Ning; Wang, Hailin

    2016-08-01

    Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA. PMID:27416319

  6. Structural reorganisation of cellulose fibrils in hydrothermally deconstructed lignocellulosic biomass and relationships with enzyme digestibility

    PubMed Central

    2013-01-01

    Background The investigation of structural organisation in lignocellulose materials is important to understand changes in cellulase accessibility and reactivity resulting from hydrothermal deconstruction, to allow development of strategies to maximise bioethanol process efficiencies. To achieve progress, wheat straw lignocellulose and comparative model wood cellulose were characterised following increasing severity of hydrothermal treatment. Powder and fibre wide-angle X-ray diffraction techniques were employed (WAXD), complemented by enzyme kinetic measurements up to high conversion. Results Evidence from WAXD indicated that cellulose fibrils are not perfectly crystalline. A reduction in fibril crystallinity occurred due to hydrothermal treatment, although dimensional and orientational data showed that fibril coherency and alignment were largely retained. The hypothetical inter-fibril spacing created by hydrothermal deconstruction of straw was calculated to be insufficient for complete access by cellulases, although total digestion of cellulose in both treated straw and model pulp was observed. Both treated straw and model pulps were subjected to wet mechanical attrition, which caused separation of smaller fibril aggregates and fragments, significantly increasing enzyme hydrolysis rate. No evidence from WAXD measurements was found for preferential hydrolysis of non-crystalline cellulose at intermediate extent of digestion, for both wood pulp and hydrothermally treated straw. Conclusions The increased efficiency of enzyme digestion of cellulose in the lignocellulosic cell wall following hydrothermal treatment is a consequence of the improved fibril accessibility due to the loss of hemicellulose and disruption of lignin. However, incomplete accessibility of cellulase at the internal surfaces of fibrillar aggregates implies that etching type mechanisms will be important in achieving complete hydrolysis. The reduction in crystalline perfection following hydrothermal

  7. Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean.

    PubMed

    Pimentel, Marta S; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A; Pörtner, Hans O; Rosa, Rui

    2015-01-01

    Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems. PMID:26221723

  8. Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean

    PubMed Central

    Pimentel, Marta S.; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A.; Pörtner, Hans O.; Rosa, Rui

    2015-01-01

    Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems. PMID:26221723

  9. Tissue Specificity of Human Angiotensin I-Converting Enzyme

    PubMed Central

    Kryukova, Olga V.; Tikhomirova, Victoria E.; Golukhova, Elena Z.; Evdokimov, Valery V.; Kalantarov, Gavreel F.; Trakht, Ilya N.; Schwartz, David E.; Dull, Randal O.; Gusakov, Alexander V.; Uporov, Igor V.; Kost, Olga A.; Danilov, Sergei M.

    2015-01-01

    Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. PMID:26600189

  10. The effect of proanthocyanidin-rich hulls and proanthocyanidin extracts from bean (Vicia faba L.) hulls on nutrient digestibility and digestive enzyme activities in young chicks.

    PubMed

    Yuste, P; Longstaff, M; McCorquodale, C

    1992-01-01

    Proanthocyanidins were prepared from three bean (Vicia faba L.) varieties by extracting hulls in aqueous acetone. The amounts of freeze-dried extracts recovered were 74, 89 and 97 g/kg hull for the varieties Brunette, Statissa and Minica respectively. Chicks (3 weeks old) were fed on a maize-soya-bean control diet or the same control diet substituted with either 30 g proanthocyanidin extracts/kg or 300 g proanthocyanidin-rich hulls/kg. Chicks were tube-fed diets twice daily for 4 d. Nutrient digestibilities were calculated from amounts present in diets and freeze-dried excreta with the aid of titanium dioxide as a marker. Enzyme activities were measured in digesta removed from the jejunum. Extracts of proanthocyanidins depressed the digestibility of protein by 34%, starch by 3% and had no effect on the digestibility of lipid. Proanthocyanidin-rich hulls depressed the digestibility of protein by 62%, starch by 6% and lipid by 4%. Digestive enzyme activities were depressed to the same extent by extracts and hulls, trypsin (EC 3.4.21.4) by 55 and 62%, alpha-amylase (EC 3.2.1.1) by 75 and 78% and lipase (EC 3.1.1.3) by 31 and 32% for proanthocyanidin-extract and proanthocyanidin-rich-hull diets respectively. The susceptibility of substrates as well as enzymes to the effects of proanthocyanidins is discussed. PMID:1547203

  11. Specific Effects of Fiber Size and Fiber Swelling on Biomass Substrate Surface Area and Enzymatic Digestibility

    SciTech Connect

    Ju, Xiaohui; Grego, Courtnee; Zhang, Xiao

    2013-09-01

    To clarify the specific effect of biomass substrate surface area on its enzymatic digestibility, factors of fiber size reduction and swelling changes were investigated by using poplar substrates with controlled morphological and chemical properties after modified chemical pulping. Results showed that fiber size changes had insignificant influence on enzymatic hydrolysis, although the external surface area increased up to 41% with the reduction of fiber size. Swelling changes caused by increased biomass fiber porosities after PFI refining showed a significant influence on the efficiency of enzymatic hydrolysis. It is also found that chemical properties such as xylan and lignin content can influence the swelling effect. Xylan is confirmed to facilitate substrate hydrolysability by swelling, while lignin restricts swelling effect and thus minimizes the enzyme accessibility to substrates.

  12. Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets.

    PubMed

    Basmacioğlu Malayoğlu, H; Baysal, S; Misirlioğlu, Z; Polat, M; Yilmaz, H; Turan, N

    2010-02-01

    1. The study was conducted to determine the effects of dietary supplementation of enzyme and oregano essential oil at two levels, alone or together, on performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal based diets. 2. The following dietary treatments were used from d 0 to 21. Diet 1 (control, CONT): a commercial diet containing no enzyme or oregano essential oil, diet 2 (ENZY): supplemented with enzyme, diet 3 (EO250): supplemented with essential oil at 250 mg/kg feed, diet 4 (EO500): supplemented with essential oil at 500 mg/kg feed, diet 5 (ENZY + EO250): supplemented with enzyme and essential oil at 250 mg/kg, and diet 6 (ENZY + EO500): supplemented with enzyme and essential oil at 500 mg/kg. 3. Birds fed on diets containing ENZY, EO250 and ENZY + EO250 had significantly higher weight gain than those given CONT diet from d 0 to 7. No significant effects on feed intake, feed conversion ratio, mortality, organ weights except for jejunum weight and intestinal lengths was found with either enzyme or essential oil, alone or in combination, over the 21-d growth period. The supplementation of essential oil together with enzyme decreased jejunum weight compared with essential oil alone. 4. Supplementation with enzyme significantly decreased viscosity and increased dry matter of digesta, but did not alter pH of digesta. There was no effect of essential oil alone at either concentration on viscosity, dry matter or pH of digesta. A significant decrease in viscosity of digesta appeared when essential oil was used with together enzyme. 5. The supplementation of essential oil at both levels with or without enzyme significantly increased chymotrypsin activity in the digestive system, and improved crude protein digestibility. 6. The higher concentration of essential oil with and without enzyme significantly increased serum total cholesterol concentrations. No significant effect on immune response

  13. Digestible energy values of feed ingredients with or without addition of enzymes complex in growing pigs.

    PubMed

    Cozannet, P; Preynat, A; Noblet, J

    2012-12-01

    The DE values and digestible nutrients content of 6 diets were measured in 60-kg male growing pigs fed restricted amount of feed. Diets were prepared from 5 ingredients [wheat (Triticum aestivum), corn (Zea mays), barley (Hordeum vulgare), wheat bran, and soybean (Glycine max) meal; inclusion levels of ingredients were not correlated] with or without carbohydrose enzyme (Rovabio Excel AP; 3300 endo-β-1,4-xylanase visco units and 300 endo-1,3(4)-β-glucanase units/kg of feed; 150 g/t of feed) according to a 6 × 2 factorial arrangement; dietary NDF ranged from 10.6 to 20.1% of DM. Pigs (5 per treatment) were placed in metabolism cages that allowed total collections of feces and urine for 10 d after a 11-d adaptation. Samples of feed, urine, and feces were analyzed for GE, ash, and N. Digestibility of GE, N, and OM were calculated. The effects of diet and enzyme (Enz) were evaluated by ANOVA. In addition, the DE and digestible nutrient contents of ingredients were calculated by regression of nutritive values of diets on level of ingredient inclusions. Apparent total tract digestibility of OM, N, and GE of diets were associated with dietary NDF content (r = -0.97; P < 0.001) and were increased (P < 0.05) by Enz addition by 0.4, 1.6, and 0.5%-units (a difference between two percentage values) for OM, N, and GE digestibility, respectively. Improvement in DE value due to Enz averaged 0.09 MJ/kg DM (15.11 vs. 15.02 MJ/kg DM; P < 0.05). The ADG (891 vs. 850 g/d; P < 0.05) was also increased by Enz addition. The calculated DE content without Enz addition averaged 16.3, 16.4, 14.9, 10.5, and 17.2 MJ/kg DM for wheat, corn, barley, wheat bran, and soybean meal, respectively. The Enz addition increased the DE value of ingredients similarly, but the best response was observed for wheat (0.33 MJ/kg DM). PMID:23365332

  14. Effects of cadmium exposure on digestive enzymes, antioxidant enzymes, and lipid peroxidation in the freshwater crab Sinopotamon henanense.

    PubMed

    Wu, Hao; Xuan, Ruijing; Li, Yingjun; Zhang, Xiaomin; Wang, Qian; Wang, Lan

    2013-06-01

    In this study, the effects of cadmium (Cd) stress on the activities of disaccharidases (sucrase, lactase, and maltase), amylase, trypsin, pepsase, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) content in the alimentary system of freshwater crabs Sinopotamon henanense were studied. Results showed that the enzyme activities in the stomach, intestine, and hepatopancreas changed with Cd concentration. In terms of digestive enzymes, Cd exposure had an inhibitory effect on the activities of the disaccharidases, amylase, and pepsase (only in the stomach). Significant induction of trypsin activity by Cd at a lower concentration was observed, but as Cd concentration increased, trypsin activity decreased. Maltase activity showed a slight recovery after inhibition by Cd. The activities of SOD and CAT increased initially and decreased subsequently. Cd significantly inhibited the activity of GPx. MDA content increased with increasing concentration of Cd. These results showed that acute Cd exposure led to harmful effects on the alimentary system of crabs, which are likely linked to Cd induced oxidative stress. PMID:23224505

  15. Fibrolytic enzyme and ammonia application effects on the nutritive value, intake, and digestion kinetics of bermudagrass hay in beef cattle.

    PubMed

    Romero, J J; Zarate, M A; Queiroz, O C M; Han, J H; Shin, J H; Staples, C R; Brown, W F; Adesogan, A T

    2013-09-01

    The objectives were to compare the effect of exogenous fibrolytic enzyme (Biocellulase A20) or anhydrous ammonia (4% DM) treatment on the nutritive value, voluntary intake, and digestion kinetics of bermudagrass (Cynodon dactylon cultivar Coastal) hay harvested after 2 maturities (5- and 13-wk regrowths). Six individually housed, ruminally cannulated Brangus steers (BW 325 ± 10 kg) were used in an experiment with a 6 × 6 Latin square design with a 3 (additives) × 2 (maturities) factorial arrangement of treatments. Each period consisted of 14 d of adaptation and 7, 4, 1, 1, and 4 d for measuring in vivo digestibility, in situ degradability, no measurements, rumen liquid fermentation and passage indices, and rate of solid passage, respectively. Steers were fed hay for ad libitum intake and supplemented with sugarcane molasses and distillers grain (supplement total of 2.88 kg DM/d). Enzyme did not affect the nutritional composition of hay but ammonia treatment decreased hay NDF, hemicellulose, and ADL concentrations and increased the CP concentration particularly for the mature lignified 13-wk hay. The enzyme increased NDF and hemicellulose digestibility of the 5-wk hay but decreased those of the 13-wk hay. Ammoniation decreased intake of hay but increased digestibility of DM, OM, NDF, hemicellulose, ADF, and cellulose and increased the ruminal in situ soluble and potentially digestible fractions and the rate of DM degradation of the 13-wk hay. Also, ammoniation increased the concentrations of ruminal NH3, total VFA, acetate, and butyrate but enzyme treatment did not. Neither enzyme addition nor ammoniation affected rate of liquid and solid passage. In conclusion, ammoniation decreased the concentration of most fiber fractions, decreased the intake of hays, and increased their CP concentration, in vivo digestibility, and in situ degradability at both maturities whereas enzyme application increased fiber digestibility of the 5-wk hay but decreased it in the case of

  16. Effect of fibrolytic enzymes on the fermentation characteristics, aerobic stability, and digestibility of bermudagrass silage.

    PubMed

    Dean, D B; Adesogan, A T; Krueger, N; Littell, R C

    2005-03-01

    The aim of this study was to determine if the nutritive value and aerobic stability of bermudagrass (Cynodon dactylon) silage could be improved by addition of proprietary, exogenous cellulase/hemicellulase enzyme preparations at ensiling. A 5-wk regrowth of Tifton 85 bermudagrass was conserved without treatment (control) or after treatment with exogenous fibrolytic enzymes including Promote NET (Pr), Biocellulase X-20 (X20), Biocellulase A-20 (A20), and Enzyme CT. The respective enzymes were applied at half the recommended rate, the recommended rate, or twice the recommended rate corresponding to 0.65, 1.3, and 2.6 g/kg of DM, 7.3, 14.5, and 29 mg/kg of DM, at 7.3, 14.4, and 29 mg/kg of DM, and 89, 178, and 356 mg/kg of DM, for Pr, X20, A20, and CT, respectively. The enzymes were sprayed on the bermudagrass at ensiling (not added at feeding as suggested by the manufacturers) to test the objectives of the study. Six 1-kg replicates of chopped (5 cm) forage were ensiled for 145 d in 2.8-L mini silos. Three silos per treatment were used for chemical analysis and 3 for aerobic stability monitoring. The silage juice was analyzed for organic acids, pH, water-soluble carbohydrates (WSC), ammonia-N, and soluble N. Freeze-dried samples were analyzed for crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF). In vitro digestibility of DM (IVDMD), NDF (IVNDFD), and ADF (IVADFD) were determined after digesting the silages in buffered rumen fluid for 6 or 48 h in 2 ANKOM(II) Daisy Incubators. Compared with the other silages, those treated with Pr had lower DM losses, and lower pH and ammonia-N concentration than control silages. Residual WSC concentration was greater in Pr- and CT-treated silages than in control silages and greater in Pr-treated silages than CT-treated silages. Compared with control silages, NDF concentration was lower in silages treated with Pr, X20, and CT, and ADF concentration was lower in silages treated with Pr, X20, and A20

  17. Ruminant Nutrition Symposium: Improving cell wall digestion and animal performance with fibrolytic enzymes.

    PubMed

    Adesogan, A T; Ma, Z X; Romero, J J; Arriola, K G

    2014-04-01

    This paper aimed to summarize published responses to treatment of cattle diets with exogenous fibrolytic enzymes (EFE), to discuss reasons for variable EFE efficacy in animal trials, to recommend strategies for improving enzyme testing and EFE efficacy in ruminant diets, and to identify proteomic differences between effective and ineffective EFE. A meta-analysis of 20 dairy cow studies with 30 experiments revealed that only a few increased lactational performance and the response was inconsistent. This variability is attributable to several enzyme, feed, animal, and management factors that were discussed in this paper. The variability reflects our limited understanding of the synergistic and sequential interactions between exogenous glycosyl hydrolases, autochthonous ruminal microbes, and endogenous fibrolytic enzymes that are necessary to optimize ruminal fiber digestion. An added complication is that many of the standard methods of assaying EFE activities may over- or underestimate their potential effects because they are based on pure substrate saccharification and do not simulate ruminal conditions. Our recent evaluation of 18 commercial EFE showed that 78 and 83% of them exhibited optimal endoglucanase and xylanase activities, respectively, at 50 °C, and 77 and 61% had optimal activities at pH 4 to 5, respectively, indicating that most would likely act suboptimally in the rumen. Of the many fibrolytic activities that act synergistically to degrade forage fiber, the few usually assayed, typically endoglucanase and xylanase, cannot hydrolyze the recalcitrant phenolic acid-lignin linkages that are the main constraints to ruminal fiber degradation. These factors highlight the futility of random addition of EFE to diets. This paper discusses reasons for the variable animal responses to dietary addition of fibrolytic enzymes, advances explanations for the inconsistency, suggests a strategy to improve enzyme efficacy in ruminant diets, and describes differences

  18. Effects of exogenous enzymes and dietary energy on performance and digestive physiology of broilers

    PubMed Central

    2013-01-01

    The study was conducted to compare the effects of XG with AG and BM at different metabolizable energy diets on growth performance, digestive physiology and energy utilization of broilers fed with corn-SBM diet. A 2 × 4 factorial design was used with two basal diets (the positive control group, PC; negative control with ME reduction 100 kcal/kg, NC) and with or without the addition of three exogenous enzymes (0.02% BM; 0.01% AG; 0.05% XG) respectively. 1,200 one-day-old broilers were randomly allocated to 8 treatments with 10 pens of 15 broilers. There was no significant difference on BW, BWG, and FI at 0-21d, 21-42d or 0-42d for diet, enzymes or their interactions, but FI at 22-42d and 0-42d were tend to be decreased with the addition of enzymes. The F/G was significantly improved by the addition of enzymes especially in NC diet. The dietary AME and TME in PC or NC diet were significantly increased by XG or AG in NC diet. The villus length and V/C of ileum were significantly increased by the addition of BM or XG. XG improved the activities of trypsin, chymotrypsin and amylase, BM improved the activity of trypsin at 21d, and AG improved the activity of chymotrypsin at 21d. Comparing to PC diet, the addition of enzymes in PC or NC diet decreased feed cost per kg body weight gain especially in NC diet (except AG in PC diet) with the highest profits for XG in NC diet. In conclusion, supplementation of 0.02% BM or 0.01% AG or 0.05% XG could improve feed conversion of broilers in corn-soybean meal diet by improving energy utilization and digestive physiology, and also supplementation of 0.05% XG had a preferable efficacy in low energy diet. PMID:23556436

  19. Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley

    PubMed Central

    Li, Wei-Fen; Feng, Jie; Xu, Zi-Rong; Yang, Cai-Mei

    2004-01-01

    AIM: To investigate effects of non-starch polysaccharides(NSP) enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. METHODS: Sixty crossbred piglets averaging 13.5 kg were randomly assigned to two treatment groups with three replications (pens) based on sex and mass. Each group was fed on the diet based on barley with or without added NSP enzymes (0.15%) for a 40-d period. At the end of the experiment the pigs were weighed. Three piglets of each group were chosen and slaughtered. Pancreas, digesta from the distal end of the duodenum and jejunal mucosa were collected for determination. Activities of the digestive enzymes trypsin, chymotrypsin, amylase and lipase were determined in the small intestinal sections as well as in homogenates of pancreatic tissue. Maltase, sucrase, lactase and γ-glutamyl transpeptidase (γ-GT) activities were analyzed in jejunal mucosa. RESULTS: Supplementation with NSP enzymes improved growth performance of piglets. It showed that NSP enzymes had no effect on digestive enzyme activities in pancreas, but decreased the activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents by 57.56%, 76.08%, 69.03% and 40.22%(P < 0.05) compared with control, and increased γ-GT activities in jejunal mucosa by 118.75%(P < 0.05). CONCLUSION: Supplementation with NSP enzymes in barley based diets could improve piglets’ growth performance, decrease activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents and increase γ-GT activities in jejunal mucosa. PMID:15040032

  20. Effects of dietary stachyose on growth performance, digestive enzyme activities and intestinal morphology of juvenile turbot ( Scophthalmus maximus L)

    NASA Astrophysics Data System (ADS)

    Hu, Haibin; Zhang, Yanjiao; Mai, Kangsen; Ai, Qinghui; Xu, Wei; Zhang, Wenbing; Li, Yanxian; Liu, Jintao

    2015-10-01

    A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot ( Scophthalmus maximus L). Five isonitrogenous (49.58% crude protein) and isolipidic (10.50% crude lipid) diets were formulated to contain 0 (Control), 0.625% (S-0.625), 1.25% (S-1.25), 2.5% (S-2.5) and 5% (S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly ( P< 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher ( P< 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose ( P<0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased ( P< 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose (1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.

  1. Effects of exogenous enzymes and application method on nutrient intake, digestibility and growth performance of Pelibuey lambs.

    PubMed

    López-Aguirre, Daniel; Hernández-Meléndez, Javier; Rojo, Rolando; Sánchez-Dávila, Fernando; López-Villalobos, Nicolás; Salem, Abdel-Fattah Z M; Martínez-González, Juan Carlos; Vázquez-Armijo, José Fernando; Ruíz, Salomón

    2016-01-01

    Pelibuey sheep is the main breed in the tropical and subtropical regions of Mexico, and high demand of sheep meat has favored the finishing of lambs in feedlots with diets containing high levels of grains. The objective of this study was to evaluate the effects of exogenous enzymes (EE) and application method on nutrient intake and digestibility and performance of growing Pelibuey lambs. Treatments were based on comparison of two different methods of adding an enzyme product (sprayed on the total mixed ration or applied orally to the lambs) versus control treatment (no added enzyme). Twenty-one Pelibuey lambs, weighing 15.7 kg (SD = 1.8 kg) initial body weight, were individually housed in shaded pens and assigned randomly to one of the three enzyme treatments. At the end of study (lasting for 45 days), three lambs from each treatment were randomly selected and adapted to a pants and harness designed for fecal collection to measure nutrient digestibilities. Total body gain and average daily gain were affected (P < 0.05) by supplemental EE. The application method of EE had significant (P < 0.05) effect on FCE and FCR, but no effects were observed on nutrient intake. Supplemental EE did improve (P < 0.05) the digestibilities of dry matter, organic matter, neutral and acid detergent fiber, but no differences were observed in crude protein digestibility. The application method of EE had significant (P < 0.05) effect on the digestibility of acid detergent fiber. Supplemental EE can improve body weight gain and nutrient digestibilities without affecting nutrient intake in Pelibuey lambs, but the results of feed conversion efficiency and acid detergent fiber digestibility depend on the application method used of the EE. PMID:27610318

  2. Preparation of slowly digestible sweet potato Daeyumi starch by dual enzyme modification.

    PubMed

    Jo, A Ra; Kim, Ha Ram; Choi, Seung Jun; Lee, Joon Seol; Chung, Mi Nam; Han, Seon Kyeong; Park, Cheon-Seok; Moon, Tae Wha

    2016-06-01

    Sweet potato Daeyumi starch was dually modified using glycogen branching enzyme (BE) from Streptococcus mutans and amylosucrase (AS) from Neisseria polysaccharea to prepare slowly digestible starch (SDS). Dually modified starches had higher SDS and resistant starch (RS) contents than control starch. The branched chain length distributions of the BE-modified starches indicated an increase in short side-chains [degree of polymerization (DP)≤12] compared with native starch. AS treatment of the BE-modified starches decreased the proportion of short side-chains and increased the proportion of long side-chains (DP≥25) and molecular mass. It also resulted in a B-type X-ray diffraction pattern and an increased relative crystallinity. Regarding thermal properties, the BE-modified starches showed no endothermic peak, whereas the BEAS-modified starches had a broader melting temperature range and lower melting enthalpy compared to native starch. The combined enzymatic treatment resulted in novel glucan polymers with slow digestion properties. PMID:27083356

  3. The effect of mixed-enzyme addition in anaerobic digestion on methane yield of dairy cattle manure.

    PubMed

    Sutaryo, Sutaryo; Ward, Alastair James; Møller, Henrik Bjarne

    2014-01-01

    This study investigates the effect of applying a mixture of enzymes (ME) to dairy cattle manure (DCM) as substrate in anaerobic digestion (AD). The aims of this study were to evaluate different methods of ME application to DCM at different temperatures and to investigate the effect of adding ME during the pre-treatment of the solid fractions of dairy cattle manure (SFDCM). The results showed that there was no positive effect of direct ME addition to substrate at either mesophilic (35 degrees C) or thermophilic (50 degrees C) process temperatures, but there was a significant 4.44% increase in methane yield when DCM, which had been incubated with ME addition at 50 degrees C for three days, was fed to a digester when compared to a control digester operating at the same retention time. Methane production was detected during the pre-treatment incubation, and the total sum methane yield during pre-treatment and digestion was found to be 8.33% higher than in the control. The addition of ME to the SFDCM in a pre-incubation stage of 20 h at 35 degrees C gave a significant increase in methane yield by 4.15% in a digester treating a mixed substrate (30% liquid fractions DCM and 70% enzyme-treated SFDCM) when compared with the control digester treating a similar mixed substrate with inactivated enzyme addition. The results indicate that direct physical contact of enzyme molecules and organic material in DCM prior to AD, without the intervention of extracellular enzymes from the indigenous microorganism population, was needed in order to increase methane yields. PMID:25145202

  4. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    NASA Astrophysics Data System (ADS)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-01-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  5. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    NASA Astrophysics Data System (ADS)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-09-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  6. Sensitivity of blood-clotting factors and digestive enzymes to inhibition by organophosphorus pesticides.

    PubMed

    Quistad, G B; Casida, J E

    2000-01-01

    Organophosphorus pesticide toxicology is normally evaluated in relation to inhibition of cholinesterases (acetyl and butyryl), neuropathy target esterase, and carboxylesterases, with less attention given to other physiologically important hydrolases. This study considers the relative organophosphate sensitivities of the aforementioned serine hydrolases compared with purified blood-clotting factors (thrombin, plasmin, and kallikrein) and digestive enzymes (alpha-chymotrypsin, trypsin, and elastase), assayed under similar conditions. Inhibitors that we examined are organophosphorus insecticides or their activated metabolites (paraoxon, chlorpyrifos oxon, and profenofos) and other toxicants (phenyl saligenin cyclic phosphonate and tribufos) for comparison with values that are found in the literature for the fluorophosphonates (isoflurophate and sarin). Thrombin is the most sensitive blood-clotting factor with IC-50 values of 19 to 160 microM for tribufos, the cyclic phosphonate, isoflurophate, and profenofos; plasmin and kallikrein are less affected (IC-50 >100 microM). Alpha-Chymotrypsin, trypsin, and elastase are most sensitive to the cyclic phosphonate (IC-50 1.3-15 microM) and less so to isoflurophate, sarin, and profenofos (IC-50 values from 3.6 to greater than 100 microM). The cholinesterases, carboxylesterase, and neuropathy target esterase are the most sensitive to inhibition with IC-50 values for the insecticides of less than 0.001 to 0.6, 0.002 to 0.009, and 0.15 to 100 microM, respectively. The generally low potency of these organophosphates for blood-clotting factors and digestive enzymes suggests that associated toxic effects are unlikely at sublethal doses. PMID:10561082

  7. Consequences of Lower Food Intake on the Digestive Enzymes Activities, the Energy Reserves and the Reproductive Outcome in Gammarus fossarum

    PubMed Central

    Charron, Laetitia; Geffard, Olivier; Chaumot, Arnaud; Coulaud, Romain; Jaffal, Ali; Gaillet, Véronique; Dedourge-Geffard, Odile; Geffard, Alain

    2015-01-01

    Digestive enzyme activity is often used as a sensitive response to environmental pollution. However, only little is known about the negative effects of stress on digestive capacities and their consequences on energy reserves and reproduction, although these parameters are important for the maintenance of populations. To highlight if changes in biochemical responses (digestive enzymes and reserves) led to impairments at an individual level (fertility), Gammarus fossarum were submitted to a lower food intake throughout a complete female reproductive cycle (i.e. from ovogenesis to offspring production). For both males and females, amylase activity was inhibited by the diet stress, whereas trypsin activity was not influenced. These results underline similar sensitivity of males and females concerning their digestive capacity. Energy reserves decreased with food starvation in females, and remained stable in males. The number of embryos per female decreased with food starvation. Lower digestive activity in males and females therefore appears as an early response. These results underline the ecological relevance of digestive markers, as they make it possible to anticipate upcoming consequences on reproduction in females, a key biological variable for population dynamics. PMID:25880985

  8. Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid.

    PubMed

    Stygar, Dominika; Michalczyk, Katarzyna; Dolezych, Bogdan; Nakonieczny, Miroslaw; Migula, Pawel; Zaak, Maria; Sawczyn, Tomasz; Karcz-Socha, Iwona; Kukla, Michal; Zwirska-Korczala, Krystyna; Buldak, Rafal

    2013-01-01

    In the present study we describe the effect of chloronicotinoid pesticide (imidacloprid) on the digestive enzymes activity of the Cameraria ohridella larvae after lasting 1 year sublethal exposure to imidacloprid pesticide. Caterpillars - L4 stage (fourth instar, hyperphagic tissue-feeding phase) - were collected from chemically protected white horse chestnut trees 1 year after imidacloprid treatment, and compared with caterpillars collected from non-treated trees in a previous study. Enzymes activity of α-amylase, disaccharidases, glycosidases and proteases was assayed. The presence of pesticide in ingested food changed the digestive enzymes profile of caterpillars. The analysis of correlations between different digestive enzymes showed many significant correlations (P<0.05) among glycolytic activities like β-glucosidase and α-galactosidase activities. Statistically significant correlations for proteolytic activity were found between trypsin and chymotrypsin activity and aminopeptidase activity that occurred only in the 1st generation. PCA distinguished five primary components with eigenvalues higher than 1, from which the first two explain almost 59% of analyzed results. Surprisingly, in the pesticide treated groups significantly higher activities of sucrase and lactase in relation to control were found. In general, glycosidase (α-glucosidase, β-glucosidase and β-galactosidase) activities showed a similar pattern of activity in different generations. These results contrast with those obtained with control larvae, where significant differences in activities of α-glucosidase, β-glucosidase and β-galactosidase may result from the different quantity and quality food intake by subsequent generations of larvae. No inter-generation differences in total proteolytic activity were observed in treated larvae. The absolute value of total proteolytic activity was higher than that in the control group. The pesticide present in the vascular system of the horse chestnut

  9. Expression of an antimicrobial peptide, digestive enzymes and nutrient transporters in the intestine of E. praecox-infected chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters and an antimicrobial peptide following an Eimeria praecox challenge of chickens at d...

  10. Rapid preparation of functional polysaccharides from Pyropia yezoensis by microwave-assistant rapid enzyme digest system.

    PubMed

    Lee, Ji-Hyeok; Kim, Hyung-Ho; Ko, Ju-Young; Jang, Jun-Ho; Kim, Gwang-Hoon; Lee, Jung-Suck; Nah, Jae-Woon; Jeon, You-Jin

    2016-11-20

    This study describes a simple preparation of functional polysaccharides from Pyropia yezoensis using a microwave-assistant rapid enzyme digest system (MAREDS) with various carbohydrases, and evaluates their antioxidative effects. Polysaccharide hydrolysates were prepared using MAREDS under different hydrolytic conditions of the carbohydrases and microwave powers. Polysaccharides less than 10kDa (Low molecular weight polysaccharides, LMWP, ≤10kDa) were efficiently obtained using an ultrafiltration (molecular weight cut-off of 10kDa). MAREDS increases AMG activation via an increased degree of hydrolysis; the best AMG hydrolysate was prepared using a 10:1 ratio of substrate to enzyme for 2h in MAREDS with 400W. LMWP consisted of galactose (27.3%), glucose (64.5%), and mannose (8.3%) from the AMG hydrolysate had stronger antioxidant effects than the high molecular weight polysaccharides (>10kDa). We rapidly prepared functional LMWPs by using MAREDS with carbohydrases, and suggest that LMWP might be potentially a valuable algal polysaccharide antioxidant. PMID:27561523

  11. Digestive enzyme activities are higher in the shortfin mako shark, Isurus oxyrinchus, than in ectothermic sharks as a result of visceral endothermy.

    PubMed

    Newton, Kyle C; Wraith, James; Dickson, Kathryn A

    2015-08-01

    Lamnid sharks are regionally endothermic fishes that maintain visceral temperatures elevated above the ambient water temperature. Visceral endothermy is thought to increase rates of digestion and food processing and allow thermal niche expansion. We tested the hypothesis that, at in vivo temperatures, the endothermic shortfin mako shark, Isurus oxyrinchus, has higher specific activities of three digestive enzymes-gastric pepsin and pancreatic trypsin and lipase-than the thresher shark, Alopias vulpinus, and the blue shark, Prionace glauca, neither of which can maintain elevated visceral temperatures. Homogenized stomach or pancreas tissue obtained from sharks collected by pelagic longline was incubated at both 15 and 25 °C, at saturating substrate concentrations, to quantify tissue enzymatic activity. The mako had significantly higher enzyme activities at 25 °C than did the thresher and blue sharks at 15 °C. This difference was not a simple temperature effect, because at 25 °C the mako had higher trypsin activity than the blue shark and higher activities for all enzymes than the thresher shark. We also hypothesized that the thermal coefficient, or Q 10 value, would be higher for the mako shark than for the thresher and blue sharks because of its more stable visceral temperature. However, the mako and thresher sharks had similar Q 10 values for all enzymes, perhaps because of their closer phylogenetic relationship. The higher in vivo digestive enzyme activities in the mako shark should result in higher rates of food processing and may represent a selective advantage of regional visceral endothermy. PMID:25893905

  12. Influence of dietary zinc and copper on digestive enzyme activity and intestinal morphology in weaned pigs.

    PubMed

    Hedemann, M S; Jensen, B B; Poulsen, H D

    2006-12-01

    The current study was conducted to investigate the effects of high dietary concentrations of Zn as zinc oxide and Cu as copper sulfate on the activity of digestive enzymes in the pancreas and the intestinal mucosa, intestinal morphology, and mucin histochemistry in pigs after weaning. Thirty-two pigs were weaned at 4 wk of age. The pigs were fed standard weaning diets supplemented with Zn (100 or 2,500 ppm) and Cu (0 or 175 ppm) in a 2 x 2 factorial arrangement of treatments for a 14-d period. In pancreatic tissue, the activity of amylase, carboxypeptidase A, chymotrypsin, trypsin, and lipase increased (P < 0.01) in pigs fed 2,500 ppm of Zn, whereas the activity of carboxypeptidase B and carboxylester hydrolase was unaffected. Copper had no effect on the activity of pancreatic enzymes. In small intestinal contents, the total activity of amylase and carboxypeptidase A was greater in pigs fed 100 ppm of Zn (P < 0.05), whereas feeding 2,500 ppm of Zn increased the chymotrypsin activity (P < 0.001). The remaining enzymes were unaffected by dietary Zn concentration. The villi were longer in the cranial small intestine (P < 0.001) in pigs fed 100 ppm of Zn than in pigs fed 2,500 ppm of Zn, but otherwise there were no clear effects of Zn and Cu supplementation on intestinal morphology. In the cranial small intestine, the activity of maltase (P < 0.001), sucrase (P < 0.001), and lactase was greater in pigs fed 100 ppm of Zn, even though there was a Zn x Cu interaction (P < 0.05) in lactase activity. In the middle and caudal small intestine, no clear differences between dietary treatments were observed. The activity of gamma-glutamyl transpeptidase in the intestinal mucosa was not affected by dietary Zn or Cu. In pigs fed 100 ppm of Zn, the activity of aminopeptidase N was greater in the caudal small intestine, but dietary Zn or Cu had no effect on aminopeptidase N in the cranial and middle small intestine. No effect of dietary Zn or Cu supplementation was found on

  13. Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

    PubMed

    Nguyen, Elizabeth V; Imanishi, Susumu Y; Haapaniemi, Pekka; Yadav, Avinash; Saloheimo, Markku; Corthals, Garry L; Pakula, Tiina M

    2016-02-01

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed. PMID:26689635

  14. Effects of Dietary Supplementation with the Combination of Zeolite and Attapulgite on Growth Performance, Nutrient Digestibility, Secretion of Digestive Enzymes and Intestinal Health in Broiler Chickens

    PubMed Central

    Zhou, P.; Tan, Y. Q.; Zhang, L.; Zhou, Y. M.; Gao, F.; Zhou, G. H.

    2014-01-01

    This study was designed to investigate the effects of basal diets supplemented with a clay product consisting of zeolite and attapulgite (ZA) at 1:1 ratio on growth performance, digestibility of feed nutrients, activities of digestive enzymes in small intestine and intestinal health in broiler chickens. In experiment 1, 112 one-day-old male chickens were randomly divided into 2 groups with 8 replicates of 7 chickens each. In experiment 2, 84 one-day-old male chickens were randomly allocated into 2 groups consisting 6 replicates of 7 chickens each. The experimental diets both consisted of a maize-soybean basal control diet supplemented with 0% or 2% ZA. The diets were fed from 1 to 42 days of age. The results showed that ZA supplementation could increase body weight gain (BWG) and feed intake (FI), but had no significant effect on feed conversion ratio. The apparent digestibility values of crude protein and gross energy were significantly increased (p<0.05) by ZA from 14 to 16 d and 35 to 37 d. Dietary ZA treatment significantly increased (p<0.05) the activities of amylase, lipase and trypsin in jejunal digesta and the activities of maltase and sucrase in jejunal mucosa on days 21 and 42. The ZA supplementation also significantly increased (p<0.05) the catalase activity, reduced (p<0.05) the malondialdehyde concentration in the jejunal mucosa. In addition, a decrease of serum diamine oxidase activity and an increase (p<0.05) in concentration of secretory immunoglobulin A in jejunal mucosa were observed in birds treated with ZA on 21 and 42 days. It is concluded that ZA supplementation (2%) could partially improve the growth performance by increasing BWG and FI. This improvement was achieved through increasing the secretion of digestive enzymes, enhancing the digestibilites of nutrients, promoting intestinal health of broiler chickens. PMID:25178375

  15. Inhibition of Key Digestive Enzymes by Cocoa Extracts 1 and Procyanidins

    PubMed Central

    Gu, Yeyi; Hurst, William J.; Stuart, David A.; Lambert, Joshua D.

    2011-01-01

    We determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL) and secreted phospholipase A2 (PLA2), and characterized the kinetics of such inhibition. Lavado, regular and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2 to 10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL and PLA2. Lavado cocoa extract was the most potent inhibitor (IC50 = 8.5 – 47 μg/mL). An inverse correlation between Log IC50 and DP (R2 > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer and decamer inhibited PL activity in a mixed mode. The pentamer and decamer non-competitively inhibited PLA2 activity, whereas regular cocoa extract inhibited PLA2 competitively. Our study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro, and may, in conjunction with a low calorie diet, play a role in body weight management. PMID:21495725

  16. A sweet new wave: structures and mechanisms of enzymes that digest polysaccharides from marine algae.

    PubMed

    Hehemann, Jan-Hendrik; Boraston, Alisdair B; Czjzek, Mirjam

    2014-10-01

    Marine algae contribute approximately half of the global primary production. The large amounts of polysaccharides synthesized by these algae are degraded and consumed by microbes that utilize carbohydrate-active enzymes (CAZymes), thus creating one of the largest and most dynamic components of the Earth's carbon cycle. Over the last decade, structural and functional characterizations of marine CAZymes have revealed a diverse set of scaffolds and mechanisms that are used to degrade agars, carrageenan, alginate and ulvan-polysaccharides from red, brown and green seaweeds, respectively. The analysis of these CAZymes is not only expanding our understanding of their functions but is enabling the enhanced annotation of (meta)-genomic data sets, thus promoting an improved understanding of microbes that drive this marine component of the carbon cycle. Furthermore, this information is setting a foundation that will enable marine algae to be harnessed as a novel resource for biorefineries. In this review, we cover the most recent structural and functional analyses of marine CAZymes that are specialized in the digestion of macro-algal polysaccharides. PMID:25136767

  17. Pectinases from Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): putative accessory digestive enzymes.

    PubMed

    Evangelista, Danilo Elton; de Paula, Fernando Fonseca Pereira; Rodrigues, André; Henrique-Silva, Flávio

    2015-01-01

    The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthesized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)-denominated Sl-PME and Sl-endoPG, respectively-were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These

  18. Integrated approach using multistep enzyme digestion, TiO2 enrichment, and database search for in-depth phosphoproteomic profiling.

    PubMed

    Han, Dohyun; Jin, Jonghwa; Yu, Jiyoung; Kim, Kyunggon; Kim, Youngsoo

    2015-01-01

    Protein phosphorylation is a major PTM that regulates important cell signaling mechanisms. In-depth phosphoproteomic analysis provides a method of examining this complex interplay, yielding a mechanistic understanding of the cellular processes and pathogenesis of various diseases. However, the analysis of protein phosphorylation is challenging, due to the low concentration of phosphoproteins in highly complex mixtures and the high variability of phosphorylation sites. Thus, typical phosphoproteome studies that are based on MS require large amounts of starting material and extensive fractionation steps to reduce the sample complexity. To this end, we present a simple strategy (integrated multistep enzyme digestion, enrichment, database search-iMEED) to improve coverage of the phosphoproteome from lower sample amounts which is faster than other commonly used approaches. It is inexpensive and adaptable to low sample amounts and saves time and effort with regard to sample preparation and mass spectrometric analysis, allowing samples to be prepared without prefractionation or specific instruments, such as HPLC. All MS data have been deposited in the ProteomeXchange with identifier PXD001033 (http://proteomecentral.proteomexchange.org/dataset/PXD001033). PMID:25159016

  19. [Effects of sub-lethal dosages abamectin on food intake and digestive enzyme activities of silkworm Bombyx mori L].

    PubMed

    Zhu, Jiu-sheng; Wang, Jing; Gao, Hai-yan; Qin, Shu; Qiao, Xiong-wu; Han, Ju-cai

    2008-11-01

    Mulberry leaves treated with sub-lethal dosages (LC5, LC10 and LC20) abameetin were fed to the 5th instar larvae of silkworm (Bombyx mori L.), and the food intake and digestive enzyme activities of the larvae were studied by using gravimetric method and measuring enzyme activities. The results showed that sub-lethal dosages abameetin significantly inhibited the growth and food intake of the larvae, with their body mass and its increase rate as well as their relative growth rate being significantly lower than the control, and accompanied with the decreases of food intake, its relative consumption rate, and feces amount. The efficiency of the conversion of ingested food (ECI) and that of the conversion of digested food (EDI) also reduced, but the approximate digestibility (AD) increased significantly. The amylase and sucrase activities in the midgut of the larvae treated with abameetin decreased significantly for a longer time at the beginning, and then recovered to the same as or a higher level than the control, whereas the trehalase activity decreased significantly for a shorter time at the beginning, then increased significantly, and finally recovered to the normal. It was suggested that sub-lethal dosages abameetin had definite toxicity to the silkworm, and the toxic effect was increased with increasing dosage, which could result in the turbulence of silkworm's digestive system, and further, affect its food intake and its growth and development. PMID:19238858

  20. The effect of chaya (Cnidoscolus aconitifolius) leaf meal and of exogenous enzymes on amino acid digestibility in broilers.

    PubMed

    Sarmiento-Franco, L; McNab, J M; Pearson, A; Belmar-Casso, R

    2003-07-01

    1. The apparent ileal nitrogen (N) and amino acid digestibilities in chaya leaf meal (CLM) (Cnidoscolus aconitifolius) with added enzymes, and the same variables in diets containing different amounts of CLM were studied in chickens. 2. In the first experiment pectinase, beta-glucanase, and pectinase + beta-glucanase were added to CLM. In the second experiment, there were three diets based on maize and soybean: 0, 150 and 250 g/kg CLM. 3. Pectinase significantly increased both lysine and overall amino acid digestibilities in CLM. 4. In experiment 2, the amino acid digestibility in birds fed on CLM250 was lower than that from birds fed on either control or CLM150. Only the digestibilities of alanine, arginine and proline were lower in birds fed on CLM150 than in those fed on the control diet. Nitrogen digestibility was lower in birds fed on the CLM250 diet than on either control or CLM150 diets. These findings were attributed to the increasing concentration of fibre with increasing dietary CLM. PMID:12964630

  1. Lignocellulolytic enzyme activity, substrate utilization, and mushroom yield by Pleurotus ostreatus cultivated on substrate containing anaerobic digester solids.

    PubMed

    Isikhuemhen, Omoanghe S; Mikiashvilli, Nona A

    2009-11-01

    Solid waste from anaerobic digestion of litter from the commercial production of broiler chickens has limited use as fertilizer. Its disposal is a major problem for digester operators who are seeking alternative use for anaerobic digester solids, also referred to as solid waste (SW). The use of SW as substrates for the cultivation of Pleurotus ostreatus strain MBFBL400 was investigated. Lignocellulolytic enzymes activity, substrate utilization, and mushroom yield were evaluated in ten different substrate combinations (SCs) containing varying amounts of solid waste, wheat straw, and millet. Nutritional content of mushrooms produced on the different substrates was also determined. Substrates containing 70-80% wheat straw, 10-20% SW, and 10-20% millet were found to produce the highest mushroom yield (874.8-958.3 g/kg). Loss of organic matter in all SCs tested varied from 45.8% to 56.2%, which had positive correlation with the biological efficiency. Laccase, peroxidase, and carboxymethylcellulase (CMCase) activities were higher before fruiting, whereas xylanase showed higher activities after mushroom fruiting. SW increased the nutritional content in mushrooms harvested, and the combination of wheat straw and SW with millet significantly improved mushroom yield. Our findings demonstrated the possibility of utilizing anaerobic digester solids in mushroom cultivation. The application of SW as such could improve the financial gains in the overall economy of anaerobic digester plants. PMID:19618225

  2. Digestive enzymes of two freshwater fishes (Limia vittata and Gambusia punctata) with different dietary preferences at three developmental stages.

    PubMed

    Falcón-Hidalgo, Banessa; Forrellat-Barrios, Alina; Carrillo Farnés, Olimpia; Ubieta Hernández, Kenia

    2011-02-01

    The variation of activity of some digestive enzymes was studied in three age groups of two freshwater endemic fishes from Cuba: Limia vittata and Gambusia punctata. Trypsin, chymotrypsin and amylase activities showed a different pattern between both species. Trypsin and chymotrypsin activity increased with the age of fishes, while amylase activity decreased. The highest activity of trypsin and chymotrypsin was registered in G. punctata while the highest amylase activity was detected in L. vittata. Zymograms revealed proteases with molecular masses similar to trypsin and chymotrypsin reported for other fish species. Amylase electrophoresis showed the presence of this enzyme; in L. vittata amylase zymograms showed two bands with molecular masses of 175 and 100 kDa and in G. punctata four bands of 175, 100, 46 and 30 kDa respectively were found. The activity of the digestive enzymes can be used as an effective indicator of the feeding habits and the development of the digestive tracts in L. vittata and G. punctata. PMID:21044696

  3. In vitro study on digestion of peptides in Emmental cheese: analytical evaluation and influence on angiotensin I converting enzyme inhibitory peptides.

    PubMed

    Parrot, Sandrine; Degraeve, Pascal; Curia, Céline; Martial-Gros, Adèle

    2003-04-01

    A simple in vitro protocol simulating gastrointestinal digestion of proteins and peptides to investigate the effect of digestive enzymes on the biological activity of peptides present in dairy products was developed. This protocol consisted in a 30 min incubation with pepsin followed by a 4 h incubation with trypsin or pancreatin. It was applied to an Emmental cheese water-soluble extract (WSE) and to a casein solution (as a control). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) allowed to monitor the digestion of proteins. Reversed-phase high-performance liquid chromatography (RP-HPLC) allowed to monitor the conversion of proteins and peptides into peptides and amino acids: it is proposed to use the mean retention time corresponding to the overall retention time distribution of molecules to assess the effect of digestive enzymes. The biological activity focused in this study was the angiotensin I converting enzyme (ACE) inhibitory activity. Digestion of Emmental WSE induced an increase of the ACE inhibition as compared to undigested WSE while a 10 kDa ultrafiltered WSE lost a part of its ACE inhibitory activity after digestion process. These results strongly suggest that digestive enzymes diminished the ACE inhibition by the peptides present in Emmental cheese WSE, while the digestion of peptides of high molecular weight would generate new ACE inhibitory peptides. PMID:12744284

  4. Combined techniques for characterising pasta structure reveals how the gluten network slows enzymic digestion rate.

    PubMed

    Zou, Wei; Sissons, Mike; Gidley, Michael J; Gilbert, Robert G; Warren, Frederick J

    2015-12-01

    The aim of the present study is to characterise the influence of gluten structure on the kinetics of starch hydrolysis in pasta. Spaghetti and powdered pasta were prepared from three different cultivars of durum semolina, and starch was also purified from each cultivar. Digestion kinetic parameters were obtained through logarithm-of-slope analysis, allowing identification of sequential digestion steps. Purified starch and semolina were digested following a single first-order rate constant, while pasta and powdered pasta followed two sequential first-order rate constants. Rate coefficients were altered by pepsin hydrolysis. Confocal microscopy revealed that, following cooking, starch granules were completely swollen for starch, semolina and pasta powder samples. In pasta, they were completely swollen in the external regions, partially swollen in the intermediate region and almost intact in the pasta strand centre. Gluten entrapment accounts for sequential kinetic steps in starch digestion of pasta; the compact microstructure of pasta also reduces digestion rates. PMID:26041231

  5. The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

    PubMed Central

    Qi, Meirigeng; Valiente, Luis; McFadden, Brian; Omori, Keiko; Bilbao, Shiela; Juan, Jemily; Rawson, Jeffrey; Scott, Stephen; Ferreri, Kevin; Mullen, Yoko; El-Shahawy, Mohamed; Dafoe, Donald; Kandeel, Fouad; Al-Abdullah, Ismail H.

    2015-01-01

    Background We evaluated 3 commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality after isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using 3 different enzyme groups: liberase HI (n = 63), collagenase NB1/neutral protease (NP) (n = 43), and liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) (n = 115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate, and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the 3 enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index, hemoglobin A1c, cold ischemia time, and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the liberase MTF C/T group (73.5 ± 1.5 %) when compared to the liberase HI group (63.6 ± 2.3 %) (P < 0.001) and the collagenase NB1/NP group (61.7 ± 2.9%) (P < 0.001). The stimulation index for GSIS was significantly higher in the liberase MTF C/T group (5.3 ± 0.5) as compared to the liberase HI (2.9 ± 0.2) (P < 0.0001) and the collagenase NB1/NP (3.6 ± 2.9) (P = 0.012) groups. Furthermore, the liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic non-obese diabetic severe combined immunodeficiency mice (65%), which was significantly higher than the liberase HI (42%, P = 0.001) and the collagenase NB1/NP enzymes (41%, P < 0.001). Conclusions Liberase MTF C/T is superior to liberase HI and collagenase NB1/NP in terms of digestion efficacy and GSIS in vitro. Moreover, liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to liberase HI and

  6. Soybean hull and enzyme inclusion effects on diet digestibility and growth performance in beef steers consuming corn-based diets.

    PubMed

    Russell, J R; Sexten, W J; Kerley, M S

    2016-06-01

    A beef feedlot study was conducted to determine the effects of increasing soybean hull (SH) inclusion and enzyme addition on diet digestibility and animal performance. The hypothesis was SH inclusion and enzyme addition would increase fiber digestibility with no negative effect on animal performance. Eight treatments (TRT) were arranged in a 4 × 2 factorial using four diets and two enzyme (ENZ) inclusion rates. The diets were composed primarily of whole shell corn (WSC) with 0%, 7%, 14%, or 28% SH replacing corn. The ENZ was a commercial proprietary mix of , and (Cattlemace, R&D Life Sciences, Menomonie, WI) included in the diets at 0% (S0, S7, S14, S28) or 0.045% DM basis (S0e, S7e, S14e, S28e). Eighty steers (287 ± 31 kg, SD) were stratified by weight and blocked into pens with 1 heavy and 1 light pen per TRT (2 pen/TRT, 5 steers/pen). Steers were fed for 70 d with titanium dioxide included in the diets for the final 15 d. Fecal samples were collected on d 70 to determine diet digestibility. Diets were balanced for AA and RDP requirement based on available ME. Individual DMI was measured using a GrowSafe system. Diet, ENZ, and diet × ENZ effects were analyzed using the MIXED procedure of SAS. Initial BW was applied as a covariate for final BW (FBW), and DMI was included as a covariate for all digestibility measures. The diet × ENZ interaction had no effect on FBW, ADG, DMI, or any digestibility measure ( ≥ 0.11). Steers fed ENZ tended to have greater FBW ( = 0.09) and had numerically greater ADG than steers not fed ENZ. Diet influenced DMI ( < 0.01), as steers fed S7 diets had the greatest DMI ( ≤ 0.3), steers fed S0 diets had the least DMI ( ≤ 0.002), and DMI of steers fed S14 and S28 diets did not differ ( = 0.5). There was a diet × ENZ interaction for G:F ( = 0.02) in which S0, S0e, S14e, and S28e did not differ ( ≥ 0.3) and were greatest ( ≤ 0.05). There was no effect of diet or ENZ on DM, OM, or CP digestibility ( ≥ 0.2). Diet had an effect

  7. Performance Responses, Nutrient Digestibility, Blood Characteristics, and Measures of Gastrointestinal Health in Weanling Pigs Fed Protease Enzyme.

    PubMed

    Tactacan, Glenmer B; Cho, Seung-Yeol; Cho, Jin H; Kim, In H

    2016-07-01

    Although exogenous protease enzymes have been used in poultry diets quite extensively, this has not been the case for pig diets. In general, due to their better gut fermentative capacity and longer transit time, pigs have greater capacity to digest dietary proteins than poultry. However, in early-weaned piglets, the stress brought about by weaning adversely affects the digestion of dietary proteins. Therefore, a study was conducted to determine the effects of a commercial protease enzyme in weanling pigs. Indices of growth, nutrient digestibility, blood profiles, fecal microflora, fecal gas emission and fecal scores were measured during the study. A total of 50 weanling pigs (6.42±0.12 kg) at 28 d of age were randomly assigned to receive 1 of 2 dietary treatments: i) control diet (corn-soy based) with no supplemental protease (CON), and ii) control diet+200 g/ton protease (PROT) for 42 d. A completely randomized design consisting of 2 treatments, 5 replicates, and 5 pigs in each replicate was used. Growth performance in terms of body weight (27.04±0.38 kg vs 25.75±0.39 kg; p<0.05) and average daily gain (491±7.40 g vs 460±7.46 g; p<0.05) in PROT fed pigs were increased significantly, but gain per feed (0.700±0.01 vs 0.678±0.01; p>0.05) was similar between treatments at d 42. Relative to CON pigs, PROT fed pigs had increased (p<0.05) apparent total tract digestibility (84.66%±0.65% vs 81.21%±1.13% dry matter and 84.02%±0.52% vs 80.47%±1.22% nitrogen) and decreased (p<0.05) NH3 emission (2.0±0.16 ppm vs 1.2±0.12 ppm) in the feces at d 42. Except for a decreased (p<0.05) in blood creatinine level, no differences were observed in red blood cell, white blood cell, lymphocyte, urea nitrogen, and IgG concentrations between treatments. Fecal score and fecal microflora (Lactobacillus and E. coli) were also similar between CON and PROT groups. Overall, the supplementation of protease enzyme in weanling pigs resulted in improved growth rate and nutrient

  8. Performance Responses, Nutrient Digestibility, Blood Characteristics, and Measures of Gastrointestinal Health in Weanling Pigs Fed Protease Enzyme

    PubMed Central

    Tactacan, Glenmer B.; Cho, Seung-Yeol; Cho, Jin H.; Kim, In H.

    2016-01-01

    Although exogenous protease enzymes have been used in poultry diets quite extensively, this has not been the case for pig diets. In general, due to their better gut fermentative capacity and longer transit time, pigs have greater capacity to digest dietary proteins than poultry. However, in early-weaned piglets, the stress brought about by weaning adversely affects the digestion of dietary proteins. Therefore, a study was conducted to determine the effects of a commercial protease enzyme in weanling pigs. Indices of growth, nutrient digestibility, blood profiles, fecal microflora, fecal gas emission and fecal scores were measured during the study. A total of 50 weanling pigs (6.42±0.12 kg) at 28 d of age were randomly assigned to receive 1 of 2 dietary treatments: i) control diet (corn-soy based) with no supplemental protease (CON), and ii) control diet+200 g/ton protease (PROT) for 42 d. A completely randomized design consisting of 2 treatments, 5 replicates, and 5 pigs in each replicate was used. Growth performance in terms of body weight (27.04±0.38 kg vs 25.75±0.39 kg; p<0.05) and average daily gain (491±7.40 g vs 460±7.46 g; p<0.05) in PROT fed pigs were increased significantly, but gain per feed (0.700±0.01 vs 0.678±0.01; p>0.05) was similar between treatments at d 42. Relative to CON pigs, PROT fed pigs had increased (p<0.05) apparent total tract digestibility (84.66%±0.65% vs 81.21%±1.13% dry matter and 84.02%±0.52% vs 80.47%±1.22% nitrogen) and decreased (p<0.05) NH3 emission (2.0±0.16 ppm vs 1.2±0.12 ppm) in the feces at d 42. Except for a decreased (p<0.05) in blood creatinine level, no differences were observed in red blood cell, white blood cell, lymphocyte, urea nitrogen, and IgG concentrations between treatments. Fecal score and fecal microflora (Lactobacillus and E. coli) were also similar between CON and PROT groups. Overall, the supplementation of protease enzyme in weanling pigs resulted in improved growth rate and nutrient

  9. Loss of Native Flavanols during Fermentation and Roasting Does Not Necessarily Reduce Digestive Enzyme-Inhibiting Bioactivities of Cocoa.

    PubMed

    Ryan, Caroline M; Khoo, Weslie; Ye, Liyun; Lambert, Joshua D; O'Keefe, Sean F; Neilson, Andrew P

    2016-05-11

    Polyphenol profiles and in vitro digestive enzyme inhibitory activities were compared between cocoa extracts from unfermented beans (UB), fermented beans (FB), unfermented liquor (UL), and fermented liquor (FL). Total polyphenols, total flavanols, and individual flavanols were significantly different between UB/FB and UL/FL. All extracts effectively inhibited α-glucosidase (lowest IC50 = 90.0 μg/mL, UL) and moderately inhibited α-amylase (lowest IC50 = 183 μg/mL, FL) and lipase (lowest IC25 = 65.5 μg/mL, FB). Our data suggest that fermentation does not reduce α-glucosidase inhibition, while roasting may enhance inhibition. For α-amylase, both fermentation and roasting improved inhibition. Finally, for lipase, both fermentation and roasting attenuated inhibition. Conclusive correlations between inhibition and mDP, total polyphenol, and flavanol contents were not found. Our data suggest that enzyme inhibition activities of cocoa are not uniformly reduced by polyphenol/flavanol losses during fermentation and roasting. This paradigm-challenging finding suggests other cocoa constituents, potentially formed during processing, contribute to digestive enzyme inhibition. PMID:27094258

  10. BSSL and PLRP2: key enzymes for lipid digestion in the newborn examined using the Caco-2 cell line.

    PubMed

    Andersson, Eva-Lotta; Hernell, Olle; Bläckberg, Lars; Fält, Helen; Lindquist, Susanne

    2011-11-01

    In rodents, bile salt-stimulated lipase (BSSL) and pancreatic lipase-related protein 2 (PLRP2) are the dominant lipases expressed in the exocrine pancreas in early life when milk is the main food. The aim of the present study was to evaluate whether BSSL and PLRP2 are also key enzymes in neonatal intestinal fat digestion. Using Caco-2 cells as a model for the small intestinal epithelium, purified human enzymes were incubated in the apical compartment with substrates, bile salt composition and concentrations physiologic to newborn infants. Both BSSL and PLRP2 hydrolyzed triglycerides (TG) to free FA and glycerol. Released FA were absorbed by the cells and reesterfied to TG. Together, BSSL and PLRP2 had a synergistic effect, increasing cellular uptake and reesterification 4-fold compared with the sum of each lipase alone. A synergistic effect was also observed with retinyl ester as a substrate. PLRP2 hydrolyzed cholesteryl ester but not as efficiently as BSSL, and the two had an additive rather than synergistic effect. We conclude the key enzymes in intestinal fat digestion are different in newborns than later in life. Further studies are needed to fully understand this difference and its implication for designing optimal neonatal nutrition. PMID:21865348

  11. Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies.

    PubMed

    Secundino, Nagila; Kimblin, Nicola; Peters, Nathan C; Lawyer, Phillip; Capul, Althea A; Beverley, Stephen M; Turco, Salvatore J; Sacks, David

    2010-07-01

    Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO(4)] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2(-) or lpg5A(-)/5B(-)) or LPG alone (lpg1(-)) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36-72 h after the infective feed, all parasites developed well except the lpg2(-) and lpg5A(-)/5B(-) mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2(-) in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2(-) promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage. PMID:20088949

  12. Alterations of digestive enzyme activities, intestinal morphology and microbiota in juvenile paddlefish, Polyodon spathula, fed dietary probiotics.

    PubMed

    Fang, Cheng; Ma, Mingyang; Ji, Hong; Ren, Tongjun; Mims, Steven D

    2015-02-01

    The effects of dietary supplementation of probiotics on digestive enzymes activities, intestinal morphology and microbiota in juvenile paddlefish (Polyodon spathula) were studied. A total of 400 fish were reared in two cages and fed with a basal diet (control group, CG) or diet supplemented with commercial probiotics (treatment group, TG) for 80 days. Enzymes activities analysis indicated that protease and α-amylase activities increased (P < 0.01 or P < 0.05) in TG. Light microscopy observation demonstrated the decrease of wall thickness and muscularis thickness in foregut (P < 0.01), the increase of those in hindgut (P < 0.05), the increase of folds height in foregut (P < 0.01) and midgut in TG (P < 0.05). DGGE results of PCR-amplified 16S rRNA confirmed that the richness and diversity of intestinal microbial species increased in TG. The similarity between the commercial bacteria product and intestinal microbiota of TG were higher than the microbiota from CG. The quantities of bacterium, Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria, present an increasing trend from foregut to hindgut both in two groups. To our knowledge, this is the first in vivo study to reveal the effect of dietary probiotics on intestinal digestive enzymes activities, morphology and microbiota in paddlefish. PMID:25403154

  13. Digestive development of the early-weaned pig. 2. Effect of level of food intake on digestive enzyme activity during the immediate post-weaning period.

    PubMed

    Kelly, D; Smyth, J A; McCracken, K J

    1991-03-01

    Gastric intubation was adopted as a means of comparing the effect of two feeding levels, continuous nutrient supply (C) and restricted nutrient supply (R), on the digestive development of pigs weaned at 14 d of age, during the first 5 d post-weaning. The absolute weights of the stomach and the pancreas were significantly greater (P less than 0.001) in C compared with R pigs. The effect was not significant for pancreas weight when expressed per kg body-weight but was significant (P less than 0.05) for stomach weight. The weights of the small intestine (SI), SI mucosa and total mucosal protein were significantly higher (P less than 0.001) in C pigs but protein content per g mucosa was similar in the C and R groups. There was no significant effect of treatment on the activity of lactase (beta-glucosidase; EC 3.2.1.23) or sucrase (sucrose-alpha-glucosidase; EC 3.2.1.48) irrespective of the basis of comparison used. The specific activity (mumol/min per g protein) of maltase (alpha-glucosidase; EC 3.2.1.20) and of glucoamylase (glucan-1,4-alpha-glucosidase; EC 3.2.1.3) were similar in C and R groups but activities of maltase (mumol/g mucosa) (P less than 0.05), and maltase and glucoamylase (mol/d) (P less than 0.01) were significantly higher in C pigs. Villous height and crypt depth were significantly greater in C pigs (P less than 0.001 and P less than 0.05 respectively). Enteroglucagon was significantly (P less than 0.05) higher in C compared with R pigs. Xylose absorption and the digestibility of energy were not affected by treatment. Digestibility of dry matter, organic matter, crude protein (nitrogen x 6.25) and carbohydrate were significantly higher (P less than 0.001, P less than 0.01, P less than 0.05 and P less than 0.001 respectively) in R pigs compared with C pigs but the differences were small, ranging from 1.3 to 2.5%. These results demonstrate that (1) nutrient intake in the weaned pig affects the anatomy, morphology and function of the gut, (2) there is

  14. Histochemical distribution of digestive enzymes in the intestine of the common two-banded seabream, Diplodus vulgaris, Geoffroy St-Hilaire 1817.

    PubMed

    Tlak Gajger, I; Nejedli, S; Kozarić, Z

    2013-06-01

    The histochemical localization of non-specific esterase, alkaline and acid phosphatase as well as aminopeptidase in the intestine of the free-living common two-banded sea bream (Diplodus vulgaris) was investigated. Fish were caught near the town of Zadar (Adriatic Sea, Croatia). Samples of pyloric caeca and three parts of the intestine proper (anterior, middle and posterior) were used for the description of non-specific esterase, alkaline and acid phosphatase as well as aminopeptidase. Non-specific esterase activity was found in the cytoplasm of enterocytes in pyloric caeca and in all investigated intestinal segments. The activity was stronger in the anterior and posterior part of the intestine than in the pyloric caeca and middle segment of the intestine. Intestinal alkaline phosphatase was detected in brush border of enterocytes of all investigated intestinal segments. Enzymatic activity gradually decreased in a posterior direction. Acid phosphatase activity was observed as a fine granular reaction product in the supranuclear region of enterocytes. This activity was almost equal in pyloric caeca as well as in the anterior intestinal segment, while it was stronger in the middle and posterior intestinal segment. Aminopeptidase was present along the intestinal epithelium brush border in all investigated parts of the digestive tube. The intensity of aminopeptidase increased posteriorly. The possible role of investigated enzymes in intracellular digestion and transport is discussed. PMID:22812413

  15. Changes in small intestinal morphology and digestive enzyme activity with oral administration of copper-loaded chitosan nanoparticles in rats.

    PubMed

    Han, Xin-Yan; Du, Wen-Li; Huang, Qi-Chun; Xu, Zi-Rong; Wang, Yi-Zheng

    2012-03-01

    The experiment was conducted to evaluate the effect of copper-loaded chitosan nanoparticles on the small intestinal morphology and activities of digestive enzyme and mucosal disaccharase in rats. Forty male Sprague-Dawley rats, with average body weight of 82 g, were randomly allotted to five groups (n = 8). All rats were received a basal diet (control) or the same basal diet added with 80 mg/kg BW CuSO(4), 80 mg/kg BW chitosan (CS-I), 80 mg/kg BW copper-loaded chitosan nanoparticles (CSN-I), 160 mg/kg BW copper-loaded chitosan nanoparticles (CSN-II), respectively. The experiment lasted 21 days. The results showed that the villus heights of the small intestinal mucosa in groups CSN-I and CSN-II were higher than those of the control, group CuSO(4) or CS-I. The crypt depth of duodenum and ileum mucosa in group CSN-I or CSN-II was depressed. Compared with the control, there were no significant effects of CuSO(4) or CS-I on the villus height and crypt depth of small intestinal mucosa. Supplementation with CSN improved the activities of trypsin, amylase and lipase in the small intestinal contents and maltase, sucrase and lactase of duodenum, jejunum, and ileum mucosa while there were no significant effects of CuSO(4) on the digestive enzyme activities of the small content compared with the control. The results indicated that intestinal morphology, activities of digestive enzyme in digesta and mucosal disaccharase were beneficially changed by treatment of copper-loaded chitosan nanoparticles. PMID:21882065

  16. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    NASA Astrophysics Data System (ADS)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-02-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  17. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    PubMed Central

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  18. Effect of anthocyanin-rich corn silage on digestibility, milk production and plasma enzyme activities in lactating dairy cows.

    PubMed

    Hosoda, Kenji; Eruden, Bayaru; Matsuyama, Hiroki; Shioya, Shigeru

    2012-06-01

    Anthocyanin in purple corn (Zea mays L.) has been reported to show several functional and biological attributes, displaying antioxidant, antiobesity and antidiabetic effects in monogastric animals. The objective of this study was to investigate the effect of feeding anthocyanin-rich corn (Zea mays L., Choko C922) silage on digestibility, milk production and plasma enzyme activities in lactating dairy cows. The cows were fed diets based on the control corn or the anthocyanin-rich corn silage (AR treatment) in a crossover design. The anthocyanin-rich corn silage-based diet had a lower starch content, nutrient digestibility and total digestible nutrients content when compared to the control diet. The milk yield, lactose and solids-not-fat contents in the AR-treatment cows were lower than in the control cows. The feeding of the anthocyanin-rich corn silage led to a reduction in aspartate aminotransferase (AST) activity and an increase in superoxide dismutase (SOD) activity in the plasma. These data suggest that the anthocyanin-rich corn has a lowering effect on AST activity with concomitant enhancement of SOD activity in lactating dairy cows. However, a new variety of anthocyanin-rich corn with good nutritional value is needed for practical use as a ruminant feed. PMID:22694328

  19. Effects of dietary essential oil components on growth performance, digestive enzymes and lipid metabolism in female broiler chickens.

    PubMed

    Lee, K W; Everts, H; Kappert, H J; Frehner, M; Losa, R; Beynen, A C

    2003-07-01

    1. The present experiment was conducted to describe the effects of thymol, cinnamaldehyde and a commercial preparation of essential oil components (CRINA Poultry), in female broilers. Feed and water were provided for ad libitum consumption. 2. Feed intake, weight gain and feed:gain ratio were not different among the treatments. Water intake was significantly lowered by cinnamaldehyde. Relative liver weight (g/100 g of body weight) was highest in birds given thymol, but this was seen only at the age of 21 d and not at 40 d. Patterns of digestive enzymes in pancreatic tissue were similar for the 4 treatments. 3. Amylase activity in intestinal digesta was highest in chickens given CRINA Poultry for 21 d, but the effect had disappeared after 40 d. Ileal digestibility coefficients for starch and protein were high and identical for all treatments. 4. Fatty acid composition of diet was reflected in that of adipose tissue. Plasma lipid concentrations were not changed by any dietary treatment. 5. Thus, the present results show no effect of essential oil constituents on growth performance in female broiler chickens, but it cannot be excluded that positive effects would have been observed under less hygienic environmental conditions or when using a less digestible diet. PMID:12964629

  20. Site-Specific Epithelial-Mesenchymal Interactions in Digestive Neuroendocrine Tumors

    PubMed Central

    Dumortier, Jérôme; Ratineau, Christelle; Scoazec, Jean-Yves; Pourreyron, Céline; Anderson, Wena; Jacquier, Marie-France; Blanc, Martine; Bernard, Christine; Bellaton, Claire; Remy, Lionel; Chayvialle, Jean-Alain; Roche, Colette

    2000-01-01

    Little is known about the functional interactions between digestive neuroendocrine tumor cells and their stromal microenvironment. The focus of our study is whether mesenchymal cells modulate peptide expression, cell proliferation, and invasiveness in digestive neuroendocrine tumor cells. We designed an experimental in vivo and in vitro study using the mouse enteroendocrine cell line STC-1. In vivo, STC-1 cells were injected subcutaneously in 18 immunosuppressed newborn rats. At day 21, all animals presented poorly differentiated neuroendocrine tumors with lung metastases. Subcutaneous tumors were usually limited by a capsule containing basement membrane components and myofibroblasts that presented a low mitotic index. Lung tumors were devoid of capsule and poor in myofibroblasts, and their mitotic index was high. The profile of peptide expression in STC-1 tumors was different from that of cultured STC-1 cells. In vitro, STC-1 cells were cultured with fibroblasts of different origins, including dermis, lung, digestive tract, and liver. Based on their origin, myofibroblasts differentially modulated hormone synthesis, proliferation, spreading, and adhesion of STC-1 cells. In conclusion, our results show that site-specific functional interactions between mesenchymal and neuroendocrine cells may contribute to modulating the behavior of digestive neuroendocrine tumors, depending on their growth site. PMID:10666396

  1. Inquiry-Based Experiments for Large-Scale Introduction to PCR and Restriction Enzyme Digests

    ERIC Educational Resources Information Center

    Johanson, Kelly E.; Watt, Terry J.

    2015-01-01

    Polymerase chain reaction and restriction endonuclease digest are important techniques that should be included in all Biochemistry and Molecular Biology laboratory curriculums. These techniques are frequently taught at an advanced level, requiring many hours of student and faculty time. Here we present two inquiry-based experiments that are…

  2. Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber ( Apostichopus japonicus)

    NASA Astrophysics Data System (ADS)

    Ma, Yuexin; Sun, Feixue; Zhang, Congyao; Bao, Pengyun; Cao, Shuqing; Zhang, Meiyan

    2014-12-01

    A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0 (control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control ( P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet ( P < 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets ( P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.

  3. One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha).

    PubMed

    Palais, F; Dedourge-Geffard, O; Beaudon, A; Pain-Devin, S; Trapp, J; Geffard, O; Noury, P; Gourlay-Francé, C; Uher, E; Mouneyrac, C; Biagianti-Risbourg, S; Geffard, A

    2012-04-01

    A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised. PMID:22252290

  4. Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

    PubMed Central

    Hou, Yanan; Ernst, Stephen A.; Stuenkel, Edward L.; Lentz, Stephen I.; Williams, John A.

    2015-01-01

    The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway. PMID:25951179

  5. Development of digestive tract and enzyme activities during the early ontogeny of the tropical gar Atractosteus tropicus.

    PubMed

    Frías-Quintana, C A; Márquez-Couturier, G; Alvarez-González, C A; Tovar-Ramírez, D; Nolasco-Soria, H; Galaviz-Espinosa, M A; Martínez-García, R; Camarillo-Coop, S; Martínez-Yañez, R; Gisbert, E

    2015-10-01

    Changes in digestive enzyme activity and histology were studied in Atractosteus tropicus embryos, larvae and juvenile periods. Alkaline protease, chymotrypsin, carboxypeptidase A, lipase and α-amylase were detected in all periods and gradually increased until reaching the maximum peak in juveniles; meanwhile, acid protease was first detected at 5 days after hatching (dah) when first feeding started and trypsin and leucine aminopeptidase activities were detected from 19 dah, their values being increased gradually until reaching a maximum value at 31 dah. Acid and alkaline phosphatase activities increased from yolk-sac absorption (3 dah) until day 31 after hatching. Zymogram for acid protease showed two bands in active forms (0.4 and 0.5 Rfs) from day 5 after hatching and a third protease form (0.3 Rf) that appears at 31 dah. Two active forms (26.3 and 24.9 kDa) were detected using SDS-PAGE alkaline proteases zymogram at 5 dah, and an additional active form (44.1 kDa) was detected at 7 dah. Regarding the histological development of the digestive system, the exocrine pancreas containing zymogen granules was already visible at 3 dah, whereas at 5 dah first gastric glands were already detected in the stomach. Between 7 and 9 dah, the digestive tract of A. tropicus resembled that of a juvenile specimen with a well-developed and short oesophagus, stomach divided into a glandular and non-glandular (pyloric) stomach, folded intestine with pyloric caeca and a well-developed spiral valve (posterior intestine). Considering this, larvae of A. tropicus are capable of digesting several foods from yolk absorption (3 dah), maximizing its activities at 15 dah, age at which the organisms maximize its capability to absorb nutrients from diets provided. PMID:25987007

  6. Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase.

    PubMed

    Finer-Moore, J S; Montfort, W R; Stroud, R M

    1990-07-31

    The structures of thymidylate synthase (TS) from Escherichia coli, in ternary complexes with substrate and an analogue of the cofactor, are the basis of a stereochemical model for a key reaction intermediate in the catalyzed reaction. This model is used to compare the reaction chemistry and chirality of the transferred methyl group with structures of the components, to identify those residues that participate, and to propose a stereochemical mechanism for catalysis by TS. Effects of chemical modification of specific amino acid residues and site-directed mutations of residues are correlated with structure and effects on enzyme mechanism. The ordered binding sequence of substrate deoxyuridine monophosphate and methylenetetrahydrofolate can be understood from the structure, where each forms a large part of the binding site for the other. The catalytic site serves to orient the reactants, which are sequestered along with many water molecules within a cavernous active center. Conformational changes during the reaction could involve nearby residues in ways that are not obvious in this complex. PMID:2223755

  7. Inquiry-based experiments for large-scale introduction to PCR and restriction enzyme digests.

    PubMed

    Johanson, Kelly E; Watt, Terry J

    2015-01-01

    Polymerase chain reaction and restriction endonuclease digest are important techniques that should be included in all Biochemistry and Molecular Biology laboratory curriculums. These techniques are frequently taught at an advanced level, requiring many hours of student and faculty time. Here we present two inquiry-based experiments that are designed for introductory laboratory courses and combine both techniques. In both approaches, students must determine the identity of an unknown DNA sequence, either a gene sequence or a primer sequence, based on a combination of PCR product size and restriction digest pattern. The experimental design is flexible, and can be adapted based on available instructor preparation time and resources, and both approaches can accommodate large numbers of students. We implemented these experiments in our courses with a combined total of 584 students and have an 85% success rate. Overall, students demonstrated an increase in their understanding of the experimental topics, ability to interpret the resulting data, and proficiency in general laboratory skills. PMID:26503481

  8. Nutrient value of spray field forages fed to pigs and the use of feed enzymes to enhance nutrient digestibility.

    PubMed

    Passos, A A; Andrade, C; Phillips, C E; Coffey, M T; Kim, S W

    2015-04-01

    This study determined the DE, ME, apparent total tract digestibility (ATTD) of N, and N retention of spray field forages (Bermuda grass, forage sorghum, and sweet sorghum) fed to pigs and the effects of the supplemental feed enzymes on energy and N utilization. A basal diet was formulated with 96% corn and 4% amino acids, minerals, and vitamins. Test diets contained 85% basal diet + 15% Bermuda grass, forage sorghum, or sweet sorghum. Allzyme SSF (Alltech, Nicholasville, KY) was used as a feed enzyme, which was composed of cellulase, glucanase, xylanase, phytase, and protease. The basal diet and test diets were evaluated by using 4 sets of 2 × 2 Latin square designs consisting of 2 pigs and 2 periods with a total of 32 barrows (38.7 ± 7.9 kg). Each period (10-d adjustment and 4-d collection) had 2 Latin squares. The 2 treatments were levels of enzyme supplementation (0 or 200 mg/kg). Pigs received experimental diets twice daily (0700 and 1700 h) at a fixed amount based on BW of pigs (0.09 × BW0.75 kg). On d 10, chromic oxide (0.5%) was added to the diets at 1700 h as an external marker. Fecal and urine samples were collected during 4 consecutive days. The basal diet contained 3,850 kcal DE/kg, 3,769 kcal ME/kg, 86.06% ATTD of N, and 71.10% N retention and was not affected by enzyme supplementation. Bermuda grass contained 893 kcal DE/kg, 845 kcal ME/kg, -16.50% ATTD of N, and -37.49% N retention and tended to be improved by enzyme supplementation to 1,211 kcal DE/kg (P = 0.098), 1,185 kcal ME/kg (P = 0.081), and -10.54% N retention (P = 0.076). The ATTD of N of Bermuda grass increased (P < 0.05) to 0.08% by enzyme supplementation. The forage sorghum contained 1,520 kcal DE/kg, 1,511 kcal ME/kg, -0.72% ATTD of N, and -16.99% N retention. The sweet sorghum contained 1,086 kcal DE/kg, 1,061 kcal ME/kg, -75.47% ATTD of N, and -49.22% N retention. Enzyme supplementation did not improve energy digestibility of forage sorghum and sweet sorghum. Nitrogen in these

  9. Effect of insoluble fiber supplementation applied at different ages on digestive organ weight and digestive enzymes of layer-strain poultry.

    PubMed

    Yokhana, J S; Parkinson, G; Frankel, T L

    2016-03-01

    Two experiments were conducted to study effects of dietary insoluble fiber (IF) on digestive enzyme function in layer poultry. In Experiment 1, 8 wk old pullets were fed a control diet (Group C) or a diet (Group IF) supplemented with 1% IF (Arbocel RC). After 5 wk, 6 pullets per group were killed and organ samples collected. The remaining pullets in Group C were divided into two groups: half were fed the control diet (Group C) and half were given the IF diet (Group C-IF). Similarly, half the pullets in Group IF continued on the IF diet (Group IF) and half on the control diet (Group IF-C). At 10 wk, organ samples were collected. BW at wk 5 (IF, 1364.8 g; C, 1342.9 g) and 10 wk (IF, 1678.1 g; IF-C, 1630.5 g; C-IF, 1617.1 g; C, 1580.4 g) were not different. At wk 5, the relative proventricular weight (0.41 g/100 g BW) and activities of pepsin (75.3 pepsin units/g proventriculus/min) and pancreatic general proteolytic activity (GP) (122.9 μmol tyrosine produced/g tissue) were greater (P < 0.05) than those of Group C (proventricular relative weight, 0.36; pepsin activity, 70.6; GP activity, 94.3). At wk 10, relative weights of liver and gizzard of Group IF were heavier (P < 0.05) than other treatments; activities of pepsin, GP, trypsin and chymotrypsin of IF pullets were significantly greater than other treatments as was mRNA expression for pepsinogens A (25.9 vs. 22.9) and C (13.1 vs. 10.8). In Experiment 2, 19 wk old hens were fed a control diet or a diet containing 0.8% IF (Arbocel RC) for 12 wk. Final BW after 12 wk was not different (IF, 1919.4 g; C, 1902.1 g). Pancreatic GP activity was greater (P < 0.05) in Group IF hens than Group C at wk 12 (122.2 vs. 97.0 μmol tyrosine released/min/g tissue)) as was relative gizzard weight (1.32 vs 1.10 g/100 g BW). The significantly improved digestive organ weights and enzyme activities in IF pullets may contribute to an improvement in feed utilization. PMID:26574026

  10. Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies

    PubMed Central

    Secundino, Nagila; Kimblin, Nicola; Peters, Nathan C.; Lawyer, Phillip; Capul, Althea A.; Beverley, Stephen M.; Turco, Salvatore J.; Sacks, David

    2010-01-01

    Summary Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO4] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2− or lpg5A−/5B−) or LPG alone (lpg1−) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36–72 h after the infective feed, all parasites developed well except the lpg2− and lpg5A−/5B− mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2− in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2− promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage. PMID:20088949

  11. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes.

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the "hot-spot" within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  12. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    PubMed Central

    Chu, Wen-Ting; Wang, Jin

    2016-01-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design. PMID:27298067

  13. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    NASA Astrophysics Data System (ADS)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  14. Effect of Exogenous Fibrolytic Enzyme Application on the Microbial Attachment and Digestion of Barley Straw In vitro

    PubMed Central

    Wang, Y.; Ramirez-Bribiesca, J. E.; Yanke, L. J.; Tsang, A.; McAllister, T. A.

    2012-01-01

    The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and β-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 μg/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 μg/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 μg/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of 15N into particle-associated microbial N (15N-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) 15N-PAMN at 4 h only, but EFE on AS increased (p<0.001) 15N-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) 15N-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it. PMID:25049480

  15. Microbial diversity and digestive enzyme activities in the gut of earthworms found in sawmill industries in Abeokuta, Nigeria.

    PubMed

    Bamidele, Julius A; Idowu, Adewunmi B; Ademolu, Kehinde O; Atayese, Adijat O

    2014-09-01

    The growing demand for wood has resulted in large volumes of wood wastes that are daily released to the soil from the activities of sawmills in South-Western Nigeria. In an attempt to setup a bioremediation model for sawdust, this study therefore aimed at evaluating microbial diversity, and the level of digestive enzymes in the gut of earthworms (Eudrilus eugeniae, Libyodrilus violaceous and Hyperiodrilus africanus) of sawmill origin. Four major sawmills located in Abeokuta (7°9'12" N- 3°19'35" E), namely Lafenwa, Sapon, Isale-Ake and Kotopo sawmills were used for this study. The arboretum of the Federal University of Agriculture, Abeokuta was used as control. Gut microbial analysis was carried out using the pour-plate method while digestive enzyme activities in the earthworm guts were done by the spectrophotometric method. Higher microbial counts (28.5 ± 0.1 x 10(3)-97.0 ± 0.1 x 10(3) cfu for bacteria and 7.0 ± 0.1x 10(3)-96.0 ± 0.1 x 10(3) cfu for fungi) and microbial diversity were recorded in the gut of earthworms of the sawmill locations than those of the control site (17.5 ± 0.1 x10(3) cfu for bacteria and 4.5 ± 0.1 x 10(3) cfu for fungi). Streptococcus mutans and Proteus spp. were common in the gut of E. eugeniae, and L. violaceous from the study sawmills, while Streptococcus mutans were also identified in H. africanus, but absent in the gut of E. eugeniae from the control site. Cellulase (48.67 ± 0.02 mg/g) and lipase (1.81 ± 0.01 mg/g) activities were significantly higher (p < 0.05) in the gut of earthworms from the control site than those of the study sawmills. Furthermore, amylase (α and β) activity was highest in the gut of earthworms from the sawmills. Variations observed in the gut microbial and digestive enzyme activities of earthworms from the study sawmills as compared to the control site suggests that earthworms, especially E. eugeniae, could be a better organism for use as bioremediator of wood wastes. PMID:25412548

  16. Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

    PubMed

    Funeriu, Daniel P; Eppinger, Jörg; Denizot, Lucile; Miyake, Masato; Miyake, Jun

    2005-05-01

    The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined. PMID:15821728

  17. Strategies in protein sequencing and characterization: multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry.

    PubMed

    Nardiello, Donatella; Palermo, Carmen; Natale, Anna; Quinto, Maurizio; Centonze, Diego

    2015-01-01

    A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis. PMID:25479873

  18. Effects of xylitol on carbohydrate digesting enzymes activity, intestinal glucose absorption and muscle glucose uptake: a multi-mode study.

    PubMed

    Chukwuma, Chika Ifeanyi; Islam, Md Shahidul

    2015-03-01

    The present study investigated the possible mechanism(s) behind the effects of xylitol on carbohydrate digesting enzymes activity, muscle glucose uptake and intestinal glucose absorption using in vitro, ex vivo and in vivo experimental models. The effects of increasing concentrations of xylitol (2.5%-40% or 164.31 mM-2628.99 mM) on alpha amylase and alpha glucosidase activity in vitro and intestinal glucose absorption and muscle glucose uptake were investigated under ex vivo conditions. Additionally, the effects of an oral bolus dose of xylitol (1 g per kg BW) on gastric emptying and intestinal glucose absorption and digesta transit in the different segments of the intestinal tract were investigated in normal and type 2 diabetic rats at 1 hour after dose administration, when phenol red was used as a recovery marker. Xylitol exhibited concentration-dependent inhibition of alpha amylase (IC₅₀ = 1364.04 mM) and alpha glucosidase (IC₅₀ = 1127.52 mM) activity in vitro and small intestinal glucose absorption under ex vivo condition. Xylitol also increased dose dependent muscle glucose uptake with and without insulin, although the uptake was not significantly affected by the addition of insulin. Oral single bolus dose of xylitol significantly delayed gastric emptying, inhibited intestinal glucose absorption but increased the intestinal digesta transit rate in both normal and diabetic rats compared to their respective controls. The data of this study suggest that xylitol reduces intestinal glucose absorption via inhibiting major carbohydrate digesting enzymes, slowing gastric emptying and fastening the intestinal transit rate, but increases muscle glucose uptake in normal and type 2 diabetic rats. PMID:25656339

  19. Plant pathogens as a source of diverse enzymes for lignocellulose digestion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant cell wall is a major barrier that many plant pathogens must surmount for successful invasion of their plant hosts. Full genome sequencing of a number of plant pathogens has revealed often large, complex, and redundant enzyme systems for degradation of plant cell walls. Recent surveys have ...

  20. Effects of Enzyme Complex Supplementation to a Paddy-based Diet on Performance and Nutrient Digestibility of Meat-type Ducks

    PubMed Central

    Kang, P.; Hou, Y. Q.; Toms, Derek; Yan, N. D.; Ding, B. Y.; Gong, Joshua

    2013-01-01

    Paddy rice is rarely used as a feed because of its high fiber content. In this study, two experiments were conducted to study the effects of supplementing an enzyme complex consisting of xylanase, beta-glucanase and cellulase, to paddy-based diets on the performance and nutrient digestibility in meat-type ducks. In the both experiments, meat-type ducks (Cherry Valley) were randomly assigned to four treatments. Treatment 1 was a basal diet of corn-soybean; treatment 2 was a basal diet of corn-paddy-soybean; treatment 3, had enzyme complex added to the corn-paddy-soybean basal diet at levels of 0.5 g/kg diet; and treatment 4, had enzyme complex added to the corn-paddy-soybean diet at levels of 1.0 g/kg diet. The results showed that the enzyme complex increased the ADG, and decreased the ADFI and F/G significantly (p<0.05) in the ducks, and the ADFI for the ducks fed the corn-paddy-soybean diet showed no difference compared to the ducks fed corn-soybean diets at all stages of the experiment (p<0.05). When corn was partially replaced by paddy, the digestibility of CP and NDF was decreased and increased, respectively (p<0.05), and the level of enzyme complex had a significant effect on both CP and NDF digestibility (p<0.05). As for the AME, addition of enzyme complex increased it significantly (p<0.05), but both diet types and levels of enzyme complex had no effect (p>0.05). The outcome of this research indicates that the application of enzyme complex made up of xylanase, beta-glucanase, and cellulase, in the corn-paddy-soybean diet, can improve performance and nutrition digestibility in meat-type ducks. PMID:25049784

  1. Efficient and Specific Trypsin Digestion of Microgram to Nanogram Quantities of Proteins in Organic-Aqueous Solvent Systems

    SciTech Connect

    Strader, Michael B; Tabb, Dave L; Hervey, IV, William Judson; Pan, Chongle; Hurst, Gregory {Greg} B

    2006-01-01

    Mass spectrometry-based identification of the components of multiprotein complexes often involves solution-phase proteolytic digestion of the complex. The affinity purification of individual protein complexes often yields nanogram to low-microgram amounts of protein, which poses several challenges for enzymatic digestion and protein identification. We tested different solvent systems to optimize trypsin digestions of samples containing limited amounts of protein for subsequent analysis by LC-MS-MS. Data collected from digestion of 10-, 2-, 1-, and 0.2- g portions of a protein standard mixture indicated that an organicaqueous solvent system containing 80% acetonitrile consistently provided the most complete digestion, producing more peptide identifications than the other solvent systems tested. For example, a 1-h digestion in 80% acetonitrile yielded over 52% more peptides than the overnight digestion of 1 g of a protein mixture in purely aqueous buffer. This trend was also observed for peptides from digested ribosomal proteins isolated from Rhodopseudomonas palustris. In addition to improved digestion efficiency, the shorter digestion times possible with the organic solvent also improved trypsin specificity, resulting in smaller numbers of semitryptic peptides than an overnight digestion protocol using an aqueous solvent. The technique was also demonstrated for an affinityisolated protein complex, GroEL. To our knowledge, this report is the first using mass spectrometry data to show a linkage between digestion solvent and trypsin specificity. Mass spectrometry (MS) has become a widely used method for studying proteins, protein complexes, and whole proteomes because of innovations in soft ionization techniques, bioinformatics, and chromatographic separation techniques.1-7 An example of a high-throughput mass spectrometry strategy commonly used for this purpose is a variation of the "shotgun" approach, involving in-solution digestion of a protein complex followed by

  2. The in vivo infusion of hydrogen peroxide induces oxidative stress and differentially affects the activities of small intestinal carbohydrate digestive enzymes in the neonatal pig.

    PubMed

    Lackeyram, D; Mine, Y; Widowski, T; Archbold, T; Fan, M Z

    2012-12-01

    Chronic fatigue syndrome (CFS) is characterized by persistent and relapsing fatigue that involves oxidative stress in its pathogenesis. We tested the hypothesis that a decrease in key carbohydrate-digesting enzyme activity in the gut is one of the major biological mechanisms of developing CFS in liquid formula-fed neonatal pigs with in vivo infusion of H(2)O(2). Piglets at 7 to 10 d of age were fitted with an intraperitoneal catheter, allowed a 3-d post surgical recovery, and infused with either H(2)O(2) at 5 mmol/kg BW (PER; n = 8) or the same volume of saline (CON; n = 8) in six 20-ml doses daily for a period of 10 d. During this period, animal behavior was monitored, blood samples collected, and jejunal enzyme activity kinetic experiments for lactase, sucrase, maltase, and maltase-glucoamylase were conducted. Plasma concentration of reduced glutathione remained similar (P > 0.05) to the pre-infusion level over the study duration in the CON group whereas this was 65% lower (P < 0.05) than the pre-infusion level in the PER group. Piglets experiencing oxidative stress had an overall lower (P < 0.05) physical mobility and the maximal jejunal specific activities [μmol/(mg protein · min)] for lactase (PER, 6.54 ± 0.68 vs. CON, 12.65 ± 0.69) and maltase (PER, 57.39 ± 1.02 vs. CON, 75.60 ± 1.04), respectively. However, differences were not observed (P > 0.05) in the maximal specific activities [μmol/(mg protein · min)] of sucrase (PER, 10.50 ± 1.37 vs. CON, 12.40 ± 1.55) and maltase-glucoamylase (PER, 0.71 ± 0.08 vs. CON, 0.70 ± 0.07) between the 2 groups. In conclusion, infusion of a suitable dose of H(2)O(2) induced CFS in the neonatal pigs. Oxidative stress in vivo differentially affected the maximal activities of important small intestinal carbohydrate-digesting enzymes in neonatal pigs fed a dairy milk-based liquid formula. PMID:23365398

  3. [Studies on digestive enzyme activity of Whitmania pigra in different months old].

    PubMed

    Shi, Hong-zhuan; Liu, Hong; Guo, Qiao-sheng; Wang, Jia; Liu, Fei; Li, Meng-meng

    2015-07-01

    Studies on the variation of amylase, lipase and lrotease activity of Whitmania pigra in 0-6 months old using 3, 5-dinitro- salicylic acid colorimetry, right-nitrophenyl palmitate ester (ρ-NPP) colorimetry and folin-phenol method. The results showed that pro- tease activity remained low before 1.5 months old and with the highest activity in 2 months old, but after showing a small peak in 4 months, alkaline protease rapid declined. Amylase was low at born, then gradually increased the activity of the highest in 2.5 months old. Lipase with a strong vitality at birth, then 1 month with minimum and 2 months peaked, but appeared a small peak in 4 months old. In summary, only lipase exhibits strong activity at birth, lipase with the strongest activity in the digestive tract during develop- ment. Protease, lipase and amylase with the strongest activity at 2-3 months old, but were decreased after 4 months old. PMID:26666029

  4. Debranching and Crystallization of Waxy Maize Starch in Relation to Enzyme Digestibility

    SciTech Connect

    Cai, L.; Shi, Y; Rong, L; Hsiao, B

    2010-01-01

    Molecular and crystal structures as well as morphology during debranching and crystallization of waxy maize starch at a high solid content (25%, w/w) were investigated, and the results were related to the digestibility of debranched products. The starch was cooked at 115-120 C for 10 min, cooled to 50 C and debranched by isoamylase. After 1 h of debranching, wormlike objects with 5-10 nm width and ca. 30 nm length were observed by transmission electron microscopy. Further release of linear chains and crystallization led to assembly of semi-crystalline structures in the form of nano-particles and subsequent growth of nano-particles into large aggregates. After 24 h at 50 C, a debranched starch product with an A-type X-ray diffraction pattern, a high melting temperature (90-140 C), and high resistant starch content (71.4%) was obtained. Small-angle X-ray scattering results indicated that all debranched products were surface fractal in a dry state (4% moisture) but had a mass fractal structure when hydrated (e.g. 45% moisture).

  5. Validation of a specific quality of life questionnaire for functional digestive disorders

    PubMed Central

    Chassany, O; Marquis, P; Scherrer, B; Read, N; Finger, T; Bergmann, J; Fraitag, B; Geneve, J; Caulin, C

    1999-01-01

    BACKGROUND—Dyspepsia and irritable bowel syndrome are suitable conditions for assessment of quality of life. Their similarities justify the elaboration of a single specific questionnaire for the two conditions. 
AIMS—To examine the process leading to the validation of the psychometric properties of the functional digestive disorders quality of life questionnaire (FDDQL). 
METHODS—Initially, the questionnaire was given to 154 patients, to assess its acceptability and reproducibility, analyse its content, and reduce the number of items. Its responsiveness was tested during two therapeutic trials which included 428 patients. The questionnaire has been translated into French, English, and German. The psychometric validation study was conducted in France, United Kingdom, and Germany by 187 practitioners. A total of 401patients with dyspepsia or irritable bowel syndrome, defined by the Rome criteria, filled in the FDDQL and generic SF-36 questionnaires. 
RESULTS—The structure of the FDDQL scales was checked by factorial analysis. Its reliability was expressed by a Cronbach's α coefficient of 0.94. Assessment of its discriminant validity showed that the more severe the functional digestive disorders, the more impaired the quality of life (p<0.05). Concurrent validity was supported by the correlation found between the FDDQL and SF-36 questionnaire scales. The final version of the questionnaire contains 43 items belonging to eight domains. 
CONCLUSIONS—The properties of the FDDQL questionnaire, available in French, English, and German, make it appropriate for use in clinical trials designed to evaluate its responsiveness to treatment among patients with dyspepsia and irritable bowel syndrome. 

 Keywords: digestive disorders; irritable bowel syndrome; dyspepsia; quality of life; clinical trial; validation PMID:10075960

  6. Effect of orally administered betel leaf (Piper betle Linn.) on digestive enzymes of pancreas and intestinal mucosa and on bile production in rats.

    PubMed

    Prabhu, M S; Platel, K; Saraswathi, G; Srinivasan, K

    1995-10-01

    The influence of two varieties of betel leaf (Piper betle Linn.) namely, the pungent Mysore and non-pungent Ambadi, was examined on digestive enzymes of pancreas and intestinal mucosa and on bile secretion in experimental rats. The betel leaves were administered orally at two doses which were either comparable to human consumption level or 5 times this. The results indicated that while these betel leaves do not influence bile secretion and composition, they have a significant stimulatory influence on pancreatic lipase activity. Besides, the Ambadi variety of betel leaf has a positive stimulatory influence on intestinal digestive enzymes, especially lipase, amylase and disaccharidases. A slight lowering in the activity of these intestinal enzymes was seen when Mysore variety of betel leaf was administered, and this variety also had a negative effect on pancreatic amylase. Further, both the betel leaf varieties have shown decreasing influence on pancreatic trypsin and chymotrypsin activities. PMID:8575807

  7. Zinc oxide-montmorillonite hybrid influences diarrhea, intestinal mucosal integrity, and digestive enzyme activity in weaned pigs.

    PubMed

    Hu, Caihong; Song, Juan; You, Zhaotong; Luan, Zhaoshuang; Li, Weifen

    2012-11-01

    One hundred-eighty piglets (Duroc × Landrace × Yorkshire), with an average initial weight of 7.4 kg weaned at 27 ± 1 days of age, were used to evaluate the effects of dietary zinc oxide-montmorillonite hybrid (ZnO-MMT) on growth performance, diarrhea, intestinal mucosal integrity, and digestive enzyme activity. All pigs were allotted to five treatments and fed with the basal diets supplemented with 0, 250, 500, and 750 mg/kg of Zn as ZnO-MMT or 2,000 mg/kg of Zn as ZnO. The results showed that supplementation with 500 or 750 mg/kg of Zn from ZnO-MMT and 2,000 mg/kg of Zn from ZnO improved average daily gain, enhanced average daily feed intake, decreased fecal scores at 4, 8, and 14 days postweaning, reduced intestinal permeability which was evident from the reduced lactulose recovery and urinary lactulose/mannitol ratio, and improved the activities of protease, amylase, lipase, trypsin, and chymotrypsin both in pancreas and small intestinal contents of pigs as compared with the control. Supplemental 250 mg/kg of Zn from ZnO-MMT also decreased fecal scores at 8 and 14 days postweaning, decreased urinary lactulose/mannitol ratio, and improved chymotrypsin activity in pancreas and small intestinal contents as well as protease activity in small intestinal contents compared with control. Moreover, the above indexes of weanling pigs fed with 500 or 750 mg/kg of Zn as ZnO-MMT did not differ from those fed with 2,000 mg/kg of Zn as ZnO. The results demonstrated that supplementation with 500 or 750 mg/kg of Zn from ZnO-MMT was as efficacious as 2,000 mg/kg of Zn from ZnO in improving growth performance, alleviating postweaning diarrhea, and enhancing intestinal mucosal integrity and the digestive enzyme activities in pancreas and small intestinal contents of pigs. The results that feeding lower concentrations of ZnO-MMT to weanling pigs maintained performance will be beneficial for the environment and for sustaining swine production. PMID:22539019

  8. Specificity of bacteriolytic enzyme II from a soil amoeba, Hartmannella glebae.

    PubMed Central

    Hemelt, D M; Mares, B; Upadhyay, J M

    1979-01-01

    Two bacteriolytic enzymes were produced when Hartmanella glebae was grown in the presence of both Enterobacter aerogenes and Alcaligenes faecalis. The identification of enzyme I as N-acetylmuramidase was reported earlier. Enzyme II was purified by gel filtration on a Bio-Gel A column. A recovery of 68.76% with 72.3-fold purification was obtained. It was found that 5 and 10 mM MgCl2 significantly increased the bacteriolytic activity. It is a basic protein. The cell walls of Micrococcus lysodeikticus were lysed by the enzyme, and the products of digestion were purified by Amberlite CG-120 and Sephadex G-15 chromatography to facilitate the detection of amino sugars. After reduction of the oligosaccharides with sodium borohydride and acid hydrolysis, the amino sugars were identified by paper chromatography. It was found that enzyme II cleaved the glycosidic bond between N-acetylmuramic and and N-acetylglucosamine of the peptidoglycan moiety of the cell walls. Thus, the enzyme was identified as endo-beta-N-acetylmuramidase. PMID:533270

  9. Effect of source and amount of fiber on kinetics of digestion and specific gravity of forage particles in the rumen.

    PubMed

    Wattiaux, M A; Mertens, D R; Satter, L D

    1991-11-01

    This experiment investigated the relationship between kinetics of digestion and change in specific gravity during in situ incubation. Nine cows were fed three sources of fiber (corn silage, alfalfa silage, or alfalfa hay) in diets formulated to contain 25, 30, or 35% NDF in three simultaneous 3 x 3 Latin squares. Method of alfalfa preservation did not influence rate of digestion or rate of increase in specific gravity of forage particles measured by a flotation technique. Prior to incubation, specific gravity of forage particles was in increasing order: alfalfa hay, alfalfa silage, and then corn silage. Essentially, all particles with a specific gravity less than 1.0 shifted to a higher specific gravity fraction by hydration within the first 4 h of incubation. From 4 to 56 h of incubation, percentage of residual DM that settled in solution having specific gravity of 1.3 increased linearly from 21 to 27% for corn silage but exponentially from 3 to 20% for alfalfa forages. Fractional rates of DM and NDF digestion and increase in percentage of residual DM having a specific gravity greater than 1.3 increased with the amount of fiber in the alfalfa diets and were correlated positively, suggesting that rate of increase in specific gravity, which affects rate of passage from the rumen, is influenced by rate of digestion of forage particles. PMID:1661750

  10. Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems.

    PubMed

    McBain, A J; MacFarlane, G T

    2001-09-01

    Supplementation of the human diet with prebiotic substances such as inulin and non-digestible oligosaccharides (NDO), e.g., galacto-oligosaccharides (GOS), has been associated with various health benefits. However, little information is available regarding the spatial location of their metabolism in human gut bacterial ecosystems. Therefore, the present study investigated the metabolism of inulin and GOS with respect to bacterial growth, bifidobacterial stimulatory properties and anti-mutagenicity potential, in a three-stage continuous culture model of the colon which reproduces the physicochemical characteristics of the proximal (V1) and distal (V2, V3) colons. Fermentation of both carbohydrates was rapid, and occurred primarily in V1, as evidenced by acid formation. Inulin metabolism was associated with 10-fold stimulation of lactobacillus populations, together with smaller increases in bifidobacterial cell counts in V1. However, peptostreptococci, enterococci and Clostridium perfringens also increased in this fermentation vessel. In contrast, GOS was only weakly bifidogenic in V1, although these bacteria did proliferate in V2. GOS also increased lactobacilli by an order of magnitude in V1. However, overall changes in microbial populations resulting from inulin or GOS addition were minimal in V2 and V3. Potential beneficial effects of inulin metabolism included minor reductions in beta-glucosidase and beta-glucuronidase, whereas GOS strongly suppressed these enzymes, together with arylsulphatase (AS). Growth of putatively health promoting micro-organisms was not only associated with reductions in enzymes linked to genotoxicity. For example, both carbohydrates stimulated synthesis of nitroreductase and azoreductase, throughout the fermentation system, while inulin increased AS. Colonic transit time is an important factor in bacterial metabolism in the large bowel, and these data suggest that, in some circumstances, NDO fermentation will occurprincipally in the

  11. Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis.

    PubMed

    Yu, Ching-Ching; Kuo, Yu-Ying; Liang, Chien-Fu; Chien, Wei-Ting; Wu, Huan-Ting; Chang, Tsung-Che; Jan, Fan-Dan; Lin, Chun-Cheng

    2012-04-18

    Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and

  12. Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed effic...

  13. Metabolite profile, antioxidant capacity, and inhibition of digestive enzymes in infusions of peppermint (Mentha piperita) grown under drought stress.

    PubMed

    Figueroa-Pérez, Marely G; Rocha-Guzmán, Nuria Elizabeth; Pérez-Ramírez, Iza F; Mercado-Silva, Edmundo; Reynoso-Camacho, Rosalía

    2014-12-10

    Peppermint (Mentha piperita) infusions represent an important source of antioxidants, which can be enhanced by inducing abiotic stress in plants. The aim of this study was to evaluate the effect of drought stress on peppermint cultivation as well as the metabolite profile, antioxidant capacity, and inhibition of digestive enzymes of resulting infusions. At 45 days after planting, irrigation was suppressed until 85 (control), 65, 35, 24, and 12% soil moisture (SM) was reached. The results showed that 35, 24, and 12% SM decreased fresh (20%) and dry (5%) weight. The 35 and 24% SM treatments significantly increased total phenolic and flavonoid contents as well as antioxidant capacity. Coumaric acid, quercetin, luteolin, and naringenin were detected only in some drought treatments; however, in these infusions, fewer amino acids and unsaturated fatty acids were identified. The 24 and 12% SM treatments slightly improved inhibition of pancreatic lipase and α-amylase activity. Therefore, induction of moderate water stress in peppermint is recommended to enhance its biological properties. PMID:25439559

  14. Elastase Digests

    PubMed Central

    Rietschel, Benjamin; Arrey, Tabiwang N.; Meyer, Bjoern; Bornemann, Sandra; Schuerken, Malte; Karas, Michael; Poetsch, Ansgar

    2009-01-01

    Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases. PMID:19116210

  15. Comparison of Ultrasonic and CO2 Laser Pretreatment Methods on Enzyme Digestibility of Corn Stover

    PubMed Central

    Tian, Shuang-Qi; Wang, Zhen-Yu; Fan, Zi-Luan; Zuo, Li-Li

    2012-01-01

    To decrease the cost of bioethanol production, biomass recalcitrance needs to be overcome so that the conversion of biomass to bioethanol becomes more efficient. CO2 laser irradiation can disrupt the lignocellulosic physical structure and reduce the average size of fiber. Analyses with Fourier transform infrared spectroscopy, specific surface area, and the microstructure of corn stover were used to elucidate the enhancement mechanism of the pretreatment process by CO2 laser irradiation. The present work demonstrated that the CO2 laser had potential to enhance the bioconversion efficiency of lignocellulosic waste to renewable bioethanol. The saccharification rate of the CO2 laser pretreatment was significantly higher than ultrasonic pretreatment, and reached 27.75% which was 1.34-fold of that of ultrasonic pretreatment. The results showed the impact of CO2 laser pretreatment on corn stover to be more effective than ultrasonic pretreatment. PMID:22605970

  16. Comparison of ultrasonic and CO₂laser pretreatment methods on enzyme digestibility of corn stover.

    PubMed

    Tian, Shuang-Qi; Wang, Zhen-Yu; Fan, Zi-Luan; Zuo, Li-Li

    2012-01-01

    To decrease the cost of bioethanol production, biomass recalcitrance needs to be overcome so that the conversion of biomass to bioethanol becomes more efficient. CO(2) laser irradiation can disrupt the lignocellulosic physical structure and reduce the average size of fiber. Analyses with Fourier transform infrared spectroscopy, specific surface area, and the microstructure of corn stover were used to elucidate the enhancement mechanism of the pretreatment process by CO(2) laser irradiation. The present work demonstrated that the CO(2) laser had potential to enhance the bioconversion efficiency of lignocellulosic waste to renewable bioethanol. The saccharification rate of the CO(2) laser pretreatment was significantly higher than ultrasonic pretreatment, and reached 27.75% which was 1.34-fold of that of ultrasonic pretreatment. The results showed the impact of CO(2) laser pretreatment on corn stover to be more effective than ultrasonic pretreatment. PMID:22605970

  17. Influence of feeding alternative fiber sources on the gastrointestinal fermentation, digestive enzyme activities and mucosa morphology of growing Greylag geese.

    PubMed

    He, L W; Meng, Q X; Li, D Y; Zhang, Y W; Ren, L P

    2015-10-01

    The objective of this trial was to study the influence of dietary fiber sources on the gastrointestinal fermentation, digestive enzyme activity, and mucosa morphology of growing Greylag geese. In total, 240 Greylag geese (28-day-old) were allocated to 4 treatments (15 pens/treatment) differing in dietary fiber source: corn straw silage (CSS group), steam-exploded corn straw (SECS group), steam-exploded wheat straw (SEWS group), or steam-exploded rice straw (SERS group). At 112 days of age, 15 birds per group were euthanized to collect samples. No difference (P > 0.05) was found on all the gastrointestinal pH values and ammonia-nitrogen concentrations between the groups. The CSS and SERS groups had a lower (P < 0.05) proportion of acetic acid in the gizzard than the SECS and SEWS groups. The CSS group had a higher VFA concentration in the jejunum (P < 0.05) and acetic acid proportion (P < 0.01) in the ceca, and a lower (P < 0.01) butyric acid proportion than the other groups except for the SECS group. The SECS group had a higher (P < 0.01) acetic acid proportion and lower (P < 0.05) proportions of propionic acid and valeric acid in the ceca than the SEWS and SERS groups. Different fiber sources resulted in different VFA profiles, especially in the gizzard and ceca. Almost all gastrointestinal protease activities of the CSS group were higher (P < 0.05) than the other groups, along with lower (P < 0.01) amylase activities in the duodenum, jejunum, ileum, and ceca. Lipase activity in proventriculus was highest (P < 0.01) in the SEWS group and its cecal activity was lower (P < 0.01) in the SECS and SEWS groups than the CSS and SERS groups with a higher (P < 0.01) lipase activity in the CSS group than the SERS group. The SECS and SERS groups had a higher cellulase activity in the ceca than the CSS and SEWS groups, with a higher (P < 0.01) rectal cellulase activity in the SERS group than the other groups. There was no

  18. Impact of transgenic oilseed rape expressing oryzacystatin-1 (OC-1) and of insecticidal proteins on longevity and digestive enzymes of the solitary bee Osmia bicornis.

    PubMed

    Konrad, Roger; Connor, Melanie; Ferry, Natalie; Gatehouse, Angharad M R; Babendreier, Dirk

    2009-04-01

    The risk that insect-resistant transgenic plants may pose for solitary bees was assessed by determining longevity of adult Osmia bicornis (O. rufa) chronically exposed to transgenic oilseed rape expressing oryzacystatin-1 (OC-1) or to the purified insecticidal proteins recombinant rOC-1, Kunitz soybean trypsin inhibitor (SBTI), Galanthus nivalis agglutinin (GNA), or Bacillus thuringiensis toxin Cry1Ab dissolved in sugar solution (at 0.01 and 0.1%, w:v, Cry1Ab only at 0.01%). Compared to control bees, longevity was significantly reduced by SBTI and GNA at both concentrations and by rOC-1 at 0.1%, but not by Cry1Ab or rOC-1 at 0.01%. Longevity on the OC-1 oilseed rape was not significantly different from the control plants. The effects of SBTI and rOC-1 on longevity were investigated through characterization of the digestive proteinases of O. bicornis and analysis of the response in proteinase profiles to ingestion of these proteinase inhibitors. A relatively complex profile of at least four types of soluble proteolytic enzymes was identified. Serine proteinases were found to be predominant, with metallo and especially cysteine proteinases making a smaller albeit significant contribution. The compensatory response to in vivo enzyme inhibition was similar for SBTI and rOC-1 although less pronounced for rOC-1. It consisted of a non-specific overproduction of native proteinases, both sensitive and insensitive, and the induction of a novel aspartic proteinase. PMID:19135058

  19. Characterization of digestive enzymes from de-oiled mackerel (Scomber japonicus) muscle obtained by supercritical carbon dioxide and n-hexane extraction as a comparative study.

    PubMed

    Asaduzzaman, A K M; Chun, Byung-Soo

    2015-06-01

    The oil in mackerel muscle was extracted using an environmental friendly solvent, supercritical carbon dioxide (SC-CO2) at a semi-batch flow extraction process and an n-hexane. The SC-CO2 was carried out at temperature 45 °C and pressures ranging from 15 to 25 MPa. The flow rate of CO2 (27 g/min) was constant at the entire extraction period of 2 h. The highest oil extracted residues after SC-CO2 extraction was used for activity measurement of digestive enzymes. Four digestive enzymes were found in water soluble extracts after n-hexane and SC-CO2 treated samples. Amylase, lipase and trypsin activities were higher in water soluble extracts after SC-CO2 treated samples except protease. Among the four digestive enzymes, the activity of amylase was highest and the value was 44.57 uM/min/mg of protein. The water soluble extracts of SC-CO2 and n-hexane treated mackerel samples showed same alkaline optimum pH and pH stability for each of the digestive enzymes. Optimum temperature of amylase, lipase, protease and trypsin was 40, 50, 60 and 30 °C, respectively of both extracts. More than 80 % temperature stability of amylase, lipase, protease and trypsin were retained at mentioned optimum temperature in water soluble extracts of both treated samples. Based on protein patterns, prominent protein band showed in water soluble extracts after SC-CO2 treated samples indicates no denaturation of protein than untreated and n-hexane. PMID:26028731

  20. Effect of Mediterranean saltbush (Atriplex halimus) ensilaging with two developed enzyme cocktails on feed intake, nutrient digestibility and ruminal fermentation in sheep.

    PubMed

    Alsersy, Haidy; Salem, Abdelfattah Z M; Borhami, Borhami E; Olivares, Jaime; Gado, Hany M; Mariezcurrena, Maria D; Yacuot, Mohamed H; Kholif, Ahmed E; El-Adawy, Mounir; Hernandez, Saul R

    2015-01-01

    The aim of this study was to assess the effects of feeding Atriplex halimus (AH) silage treated with two developed enzyme cocktails to sheep on feed intake, nutrient digestibility and ruminal fermentation. The AH silage was treated without or with 2 L of ZAD1(®) or ZAD2(®) /1000 kg with 5% molasses and ensiled for 30 days. Barley grain (300 g/head/day) was fed as an energy supplement once daily at 10.00 hours and AH silage with or without enzyme treatment was offered ad libitum to animals twice daily at 09.00 and 16.00 hours. Sheep were fed on four experimental forage diets comprised of AH silage and barley (D1), AH silage treated with ZAD1(®) and barley (D2), AH silage treated with ZAD2(®) and barley (D3) and AH silage treated with a combination of ZAD1(®) and ZAD2(®) (1:1) and barley (D4). Ensiling AH with enzymes reduced its contents of neutral detergent fiber and acid detergent fiber. The dry matter intake of AH of D2, D3 and D4 decreased (P < 0.001) as compared to D1. However, enzyme-treated diets had greater total digestible nutrients intake (P < 0.001) as compared to D1. The nutrients digestibility for D2, D3 and D4 were higher than those for D1 (P < 0.001), and were higher for D3 as compared to both D2 and D4. Sheep fed on D3 had highest (P < 0.001) ruminal total volatile fatty acids concentration, ammonia nitrogen concentration and microbial protein yield. It could be concluded that AH silage treated with ZAD1(®) or ZAD2(®) improved digestibility and rumen fermentation in sheep. PMID:25228428

  1. Influence of Palm Kernel Meal Inclusion and Exogenous Enzyme Supplementation on Growth Performance, Energy Utilization, and Nutrient Digestibility in Young Broilers

    PubMed Central

    Abdollahi, M. R.; Hosking, B. J.; Ning, D.; Ravindran, V.

    2016-01-01

    The objective of the present study was to investigate the influence of palm kernel meal (PKM) inclusion and exogenous enzyme supplementation on growth performance, nitrogen-corrected apparent metabolizable energy (AMEn), coefficient of apparent ileal digestibility (CAID) and total tract retention of nutrients in young broilers fed corn-based diets. Four inclusion levels of PKM (no PKM [PKM0], 8% [PKM8], 16% [PKM16], and 24% [PKM24]) and two enzyme additions were evaluated in a 4×2 factorial arrangement of treatments. A total of 384, one-d-old male broilers (Ross 308) were individually weighed and allocated to 48 cages (eight broilers/cage), and cages were randomly assigned to eight dietary treatments. Results indicated that the inclusion of 8% and 16% PKM increased (p<0.05) the weight gain compared to the PKM0 diet. Birds fed the PKM8 diets had the highest (p<0.05) feed intake. Weight gain and feed intake were severely reduced (p<0.05) by feeding the PKM24 diet. Enzyme supplementation increased weight gain (p<0.05), independent of PKM inclusion level. In PKM0 and PKM8 diets, enzyme addition significantly (p<0.05) lowered feed conversion ratio (FCR); whereas enzyme addition had no effect on FCR of birds fed PKM16 and PKM24 diets. In PKM0 and PKM16 diets, enzyme addition significantly (p<0.05) increased CAID of nitrogen and energy but had no effect in the PKM8 and PKM24 diets. Inclusion of PKM into the basal diet, irrespective of inclusion level, enhanced (p<0.05) starch and fat digestibility. Inclusion of PKM at 16% and 24% resulted in similar CAID of neutral detergent fiber (NDF) but higher (p<0.05) than that of the PKM0 and PKM8 diets. Enzyme addition, regardless of the level of PKM inclusion, significantly (p<0.05) increased CAID of NDF. There was a significant (p<0.05) decrease in AMEn with PKM inclusion of 24%. The present data suggest that inclusion of PKM in broiler diets could be optimized if PKM-containing diets are formulated based on digestible amino

  2. Affinity chromatography of trypsin and related enzymes. III. Purification of Streptomyces griseus trypsin using an affinity adsorbent containing a tryptic digest of protamine as a ligand.

    PubMed

    Yokosawa, H; Hanba, T; Ishii, S

    1976-04-01

    A new, simple method has been developed for the purification of Streptomyces griseus trypsin [EC 3.4.21.4] from Pronase. Only a single operation of affinity chromatography on an agarose derivative, which was easily prepared by coupling a tryptic digest of salmine to cyanogen bromide-activated Sepharose 4B, was required. A high degree of homogeneity was demonstrated for the purified enzyme by disc electrophoresis, SDS-polyacrylamide gel electrophoresis and gel filtration, as well as by active-site titration. The behavior of a carboxypeptides B [EC 3.4.12.3]-like enzyme present in Pronase is also discussed. PMID:819428

  3. Influence of Palm Kernel Meal Inclusion and Exogenous Enzyme Supplementation on Growth Performance, Energy Utilization, and Nutrient Digestibility in Young Broilers.

    PubMed

    Abdollahi, M R; Hosking, B J; Ning, D; Ravindran, V

    2016-04-01

    The objective of the present study was to investigate the influence of palm kernel meal (PKM) inclusion and exogenous enzyme supplementation on growth performance, nitrogen-corrected apparent metabolizable energy (AMEn), coefficient of apparent ileal digestibility (CAID) and total tract retention of nutrients in young broilers fed corn-based diets. Four inclusion levels of PKM (no PKM [PKM0], 8% [PKM8], 16% [PKM16], and 24% [PKM24]) and two enzyme additions were evaluated in a 4×2 factorial arrangement of treatments. A total of 384, one-d-old male broilers (Ross 308) were individually weighed and allocated to 48 cages (eight broilers/cage), and cages were randomly assigned to eight dietary treatments. Results indicated that the inclusion of 8% and 16% PKM increased (p<0.05) the weight gain compared to the PKM0 diet. Birds fed the PKM8 diets had the highest (p<0.05) feed intake. Weight gain and feed intake were severely reduced (p<0.05) by feeding the PKM24 diet. Enzyme supplementation increased weight gain (p<0.05), independent of PKM inclusion level. In PKM0 and PKM8 diets, enzyme addition significantly (p<0.05) lowered feed conversion ratio (FCR); whereas enzyme addition had no effect on FCR of birds fed PKM16 and PKM24 diets. In PKM0 and PKM16 diets, enzyme addition significantly (p<0.05) increased CAID of nitrogen and energy but had no effect in the PKM8 and PKM24 diets. Inclusion of PKM into the basal diet, irrespective of inclusion level, enhanced (p<0.05) starch and fat digestibility. Inclusion of PKM at 16% and 24% resulted in similar CAID of neutral detergent fiber (NDF) but higher (p<0.05) than that of the PKM0 and PKM8 diets. Enzyme addition, regardless of the level of PKM inclusion, significantly (p<0.05) increased CAID of NDF. There was a significant (p<0.05) decrease in AMEn with PKM inclusion of 24%. The present data suggest that inclusion of PKM in broiler diets could be optimized if PKM-containing diets are formulated based on digestible amino

  4. Precocious alteration of digestive enzyme activities in small intestine and pancreas by chronic oral administration of protease inhibitor in suckling rats.

    PubMed

    Harada, E; Syuto, B

    1991-01-01

    1. The role of endogenous CCK in the development of digestive enzyme activities in small intestine and pancreas was investigated in suckling rats. Synthetic protease inhibitor (camostat 100 micrograms/g bwt) was orally administered twice daily for 5 days from 11 days of age. 2. Pancreatic hypertrophy and hyperplasia, and alteration of pancreatic enzyme composition, especially decreases in amylase activity and increases in trypsin and chymotrypsin activities were produced by camostat treatment. These changes were completely suppressed by simultaneous administration of the potent CCK receptor antagonist L-364,718 (1 microgram/g bwt). 3. With camostat treatment, intestinal lactase activity decreased to 41%, while maltase and sucrase activities increased 3 and 2.5 times respectively. These changes in enzyme activities were not affected by the application of L-364,718. 4. The mucosal disaccharidase and pancreatic enzyme activities could not be modified by chronic subcutaneous injection of camostat. The precocious induction of maltase and sucrase activities by camostat treatment was also observed in the adrenalectomized pups. 5. These results indicate that pancreatic growth accompanied by alteration of digestive enzyme composition in the suckling rats is regulated by endogenous CCK, but the precocious induction of disaccharidase activities is not mediated by endogenous CCK released by camostat treatment. PMID:1685962

  5. An inhibitory C-terminal region dictates the specificity of A-adding enzymes

    PubMed Central

    Tretbar, Sandy; Neuenfeldt, Anne; Betat, Heike; Mörl, Mario

    2011-01-01

    For efficient aminoacylation, tRNAs carry the conserved 3′-terminal sequence C-C-A, which is synthesized by highly specific tRNA nucleotidyltransferases (CCA-adding enzymes). In several prokaryotes, this function is accomplished by separate enzymes for CC- and A-addition. As A-adding enzymes carry an N-terminal catalytic core identical to that of CCA-adding enzymes, it is unclear why their activity is restricted. Here, it is shown that C-terminal deletion variants of A-adding enzymes acquire full and precise CCA-incorporating activity. The deleted region seems to be responsible for tRNA primer selection, restricting the enzyme’s specificity to tRNAs ending with CC. The data suggest that A-adding enzymes carry an intrinsic CCA-adding activity that can be reactivated by the introduction of deletions in the C-terminal domain. Furthermore, a unique subtype of CCA-adding enzymes could be identified that evolved out of A-adding enzymes, suggesting that mutations and deletions in nucleotidyltransferases can lead to altered and even more complex activities, as a simple A-incorporation is converted into sequence-specific addition of C and A residues. Such activity-modifying events may have had an important role in the evolution of tRNA nucleotidyltransferases. PMID:22167803

  6. Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples.

    PubMed Central

    Rodtong, S; Dobbinson, S; Thode-Andersen, S; McConnell, M A; Tannock, G W

    1993-01-01

    Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples. Images PMID:8285690

  7. Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

    PubMed

    Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

    2015-08-01

    Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation. PMID:26080680

  8. Cow-specific diet digestibility predictions based on near-infrared reflectance spectroscopy scans of faecal samples.

    PubMed

    Mehtiö, T; Rinne, M; Nyholm, L; Mäntysaari, P; Sairanen, A; Mäntysaari, E A; Pitkänen, T; Lidauer, M H

    2016-04-01

    This study was designed to obtain information on prediction of diet digestibility from near-infrared reflectance spectroscopy (NIRS) scans of faecal spot samples from dairy cows at different stages of lactation and to develop a faecal sampling protocol. NIRS was used to predict diet organic matter digestibility (OMD) and indigestible neutral detergent fibre content (iNDF) from faecal samples, and dry matter digestibility (DMD) using iNDF in feed and faecal samples as an internal marker. Acid-insoluble ash (AIA) as an internal digestibility marker was used as a reference method to evaluate the reliability of NIRS predictions. Feed and composite faecal samples were collected from 44 cows at approximately 50, 150 and 250 days in milk (DIM). The estimated standard deviation for cow-specific organic matter digestibility analysed by AIA was 12.3 g/kg, which is small considering that the average was 724 g/kg. The phenotypic correlation between direct faecal OMD prediction by NIRS and OMD by AIA over the lactation was 0.51. The low repeatability and small variability estimates for direct OMD predictions by NIRS were not accurate enough to quantify small differences in OMD between cows. In contrast to OMD, the repeatability estimates for DMD by iNDF and especially for direct faecal iNDF predictions were 0.32 and 0.46, respectively, indicating that developing of NIRS predictions for cow-specific digestibility is possible. A data subset of 20 cows with daily individual faecal samples was used to develop an on-farm sampling protocol. Based on the assessment of correlations between individual sample combinations and composite samples as well as repeatability estimates for individual sample combinations, we found that collecting up to three individual samples yields a representative composite sample. Collection of samples from all the cows of a herd every third month might be a good choice, because it would yield a better accuracy. PMID:26412206

  9. Effects of extrusion and supplementation of exogenous enzymes to diets containing Chinese storage brown rice on the carbohydrase activity in the digestive tract of piglets.

    PubMed

    He, J; Liu, C; Fu, C; Li, J

    2010-04-01

    Two experiments were conducted to study the effects of extrusion of Chinese storage brown rice and of exogenous enzymes supplementation to diets containing Chinese storage brown rice on the carbohydrase activity in digestive tract of piglets. In Experiment 1, 96 weaned piglets [initially 6.95 +/- 0.05 kg body weight (BW)] were used in a 2 x 2 factorial design, the animals were fed the diets containing extruded Chinese storage brown rice or non-treated Chinese storage brown rice and supplemented with or without exogenous enzymes. Each treatment had six replicate pens and four piglets in each pen. The results demonstrated that extrusion significantly increased (p < 0.05) the activity of duodenal maltase after 14 days of treatment and glucoamylase after 28 days of treatment, jejunal lactase, maltase, isomaltase, sucrase and alpha-amylase after 28 days of treatment, and jejunal alpha-amylase after 14 days of treatment; enzyme supplementation positively influenced (p < 0.05) the activity of pancreatic alpha-amylase after 14 and 28 days of treatment, pancreatic glucoamylase after 28 days of treatment and ileal trehalase after 14 days of treatment. Similarly, interaction between extrusion and enzyme addition existed after 14 days of treatment on the activity of pancreatic alpha-amylase and duodenal maltase and on the activity of duodenal glucoamylase and isomaltase, jejunal alpha-amylase, lactase, maltase, isomaltase and jejunal alpha-amylase after 28 days of treatment. In Experiment 2, six piglets (initially 21 +/- 1.85 kg BW) fitted with ileal 'T'-cannulas in a 6 x 6 Latin Square Design were used to study the effects of extrusion and addition of exogenous enzymes on ileal carbohydrase activity and nutrients digestibility. The results showed that exogenous enzymes significantly (p < 0.05) increased ileal alpha-amylase, glucoamylase and trehalase activity. The interaction between extrusion and enzyme supplementation had positive effect (p < 0.05) on the ileal lactase

  10. Potential anti-obesogenic properties of non-digestible carbohydrates: specific focus on resistant dextrin.

    PubMed

    Hobden, Mark R; Guérin-Deremaux, Laetitia; Rowland, Ian; Gibson, Glenn R; Kennedy, Orla B

    2015-08-01

    Alterations in the composition and metabolic activity of the gut microbiota appear to contribute to the development of obesity and associated metabolic diseases. However, the extent of this relationship remains unknown. Modulating the gut microbiota with non-digestible carbohydrates (NDC) may exert anti-obesogenic effects through various metabolic pathways including changes to appetite regulation, glucose and lipid metabolism and inflammation. The NDC vary in physicochemical structure and this may govern their physical properties and fermentation by specific gut bacterial populations. Much research in this area has focused on established prebiotics, especially fructans (i.e. inulin and fructo-oligosaccharides); however, there is increasing interest in the metabolic effects of other NDC, such as resistant dextrin. Data presented in this review provide evidence from mechanistic and intervention studies that certain fermentable NDC, including resistant dextrin, are able to modulate the gut microbiota and may alter metabolic process associated with obesity, including appetite regulation, energy and lipid metabolism and inflammation. To confirm these effects and elucidate the responsible mechanisms, further well-controlled human intervention studies are required to investigate the impact of NDC on the composition and function of the gut microbiota and at the same time determine concomitant effects on host metabolism and physiology. PMID:25721052

  11. Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

    SciTech Connect

    Tomasic, Ivan B.; Metcalf, Matthew C.; Guce, Abigail I.; Clark, Nathaniel E.; Garman, Scott C.

    2010-09-03

    The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired the enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

  12. Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

    PubMed

    Tomasic, Ivan B; Metcalf, Matthew C; Guce, Abigail I; Clark, Nathaniel E; Garman, Scott C

    2010-07-01

    The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease. PMID:20444686

  13. DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

    PubMed

    Sukovich, David J; Modavi, Cyrus; de Raad, Markus; Prince, Robin N; Anderson, J Christopher

    2015-07-17

    Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. We demonstrate the DLEnCA methodology using glucosyltransferases as an illustration. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected postreaction using qPCR or a similar means of DNA analysis. OleD and two glucosyltransferases from Arabidopsis were used to verify the assay's generality toward glucosyltransferases. We further show DLEnCA's utility by mapping out the substrate specificity for these enzymes. PMID:25621860

  14. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme

    PubMed Central

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P.-Y.; Wang, Steven S.-S.

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer’s disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12–16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12–16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12–16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1–7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1–7)C and qf-Aβ(12–16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells. PMID:27096746

  15. Prediction of Detailed Enzyme Functions and Identification of Specificity Determining Residues by Random Forests

    PubMed Central

    Nagao, Chioko; Nagano, Nozomi; Mizuguchi, Kenji

    2014-01-01

    Determining enzyme functions is essential for a thorough understanding of cellular processes. Although many prediction methods have been developed, it remains a significant challenge to predict enzyme functions at the fourth-digit level of the Enzyme Commission numbers. Functional specificity of enzymes often changes drastically by mutations of a small number of residues and therefore, information about these critical residues can potentially help discriminate detailed functions. However, because these residues must be identified by mutagenesis experiments, the available information is limited, and the lack of experimentally verified specificity determining residues (SDRs) has hindered the development of detailed function prediction methods and computational identification of SDRs. Here we present a novel method for predicting enzyme functions by random forests, EFPrf, along with a set of putative SDRs, the random forests derived SDRs (rf-SDRs). EFPrf consists of a set of binary predictors for enzymes in each CATH superfamily and the rf-SDRs are the residue positions corresponding to the most highly contributing attributes obtained from each predictor. EFPrf showed a precision of 0.98 and a recall of 0.89 in a cross-validated benchmark assessment. The rf-SDRs included many residues, whose importance for specificity had been validated experimentally. The analysis of the rf-SDRs revealed both a general tendency that functionally diverged superfamilies tend to include more active site residues in their rf-SDRs than in less diverged superfamilies, and superfamily-specific conservation patterns of each functional residue. EFPrf and the rf-SDRs will be an effective tool for annotating enzyme functions and for understanding how enzyme functions have diverged within each superfamily. PMID:24416252

  16. Effect of full-fat rice bran on palatability and digestibility of diets supplemented with enzymes in adult dogs.

    PubMed

    Pacheco, G F E; Marcolla, C S; Machado, G S; Kessler, A M; Trevizan, L

    2014-10-01

    Two experiments were conducted to evaluate the effect of full-fat rice bran (FFRB) inclusion in dry diets with and without enzyme blend (EB) supplementation for adult dogs. The diets contained 0, 20, or 40% of FFRB, replacing the equivalent amount of wheat flour (WF). Experiment 1 evaluated the consumption and preference of diets using a simple choice method with 3 comparisons (0 vs. 20, 0 vs. 40, and 20 vs. 40% FFRB). Experiment 2 investigated the effect of EB supplementation on the apparent digestibility coefficients (ADC) of nutrients and GE, fecal characteristics, urinary pH, Ca and P balance, and ME of the diets. In Exp. 1, the results indicated that FFRB included in diets up to 40% did not affect the preference or consumption of food by dogs (P < 0.05). In Exp. 2, increasing levels of FFRB in the diet linearly reduced the ADC of nutrients, GE, and ME (P < 0.05). The addition of EB had no effect on any of the variables examined. Regression analysis enabled estimation of the ADC in FFRB; and ADC of DM, CP, ether extract, GE; and the apparent coefficient of ME were 60.5, 74.8, 88.4, 70.8, and 66.4%, respectively. The inclusion of 20 or 40% FFRB in the diets did not affect urinary pH but caused an imbalance in the Ca and P metabolism when included at 40% (P < 0.05), which could be one of the limitations for greater inclusion of FFRB. The ME of FFRB was estimated to be 3,443 kcal/kg DM. The FFRB appears to be palatable for adult dogs, and although ADC was reduced by 40% FFRB in the diet, this ingredient has the potential for inclusion at 20% of diets for dogs, depending on the other ingredients used to achieve adequate Ca and P balance. The inclusion greater than 20% tends to increase P in the diet and reverse the relationship between Ca and P. PMID:25184849

  17. Combining Structure and Sequence Information Allows Automated Prediction of Substrate Specificities within Enzyme Families

    PubMed Central

    Röttig, Marc; Rausch, Christian; Kohlbacher, Oliver

    2010-01-01

    An important aspect of the functional annotation of enzymes is not only the type of reaction catalysed by an enzyme, but also the substrate specificity, which can vary widely within the same family. In many cases, prediction of family membership and even substrate specificity is possible from enzyme sequence alone, using a nearest neighbour classification rule. However, the combination of structural information and sequence information can improve the interpretability and accuracy of predictive models. The method presented here, Active Site Classification (ASC), automatically extracts the residues lining the active site from one representative three-dimensional structure and the corresponding residues from sequences of other members of the family. From a set of representatives with known substrate specificity, a Support Vector Machine (SVM) can then learn a model of substrate specificity. Applied to a sequence of unknown specificity, the SVM can then predict the most likely substrate. The models can also be analysed to reveal the underlying structural reasons determining substrate specificities and thus yield valuable insights into mechanisms of enzyme specificity. We illustrate the high prediction accuracy achieved on two benchmark data sets and the structural insights gained from ASC by a detailed analysis of the family of decarboxylating dehydrogenases. The ASC web service is available at http://asc.informatik.uni-tuebingen.de/. PMID:20072606

  18. In vitro evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides.

    PubMed

    Mattison, Christopher P; Dinter, Jens; Berberich, Matthew J; Chung, Si-Yin; Reed, Shawndrika S; Le Gall, Sylvie; Grimm, Casey C

    2015-07-01

    Ara h 1 is a major peanut allergen. Processing-induced modifications may modulate the allergenic potency of Ara h 1. Carboxymethyl lysine (CML) modifications are a commonly described nonenzymatic modification on food proteins. In the current study, we tested the ability of digestive and endolysosomal proteases to cleave CML-modified and unmodified Ara h 1 peptides. Mass spectrometric analyses of the digested peptides demonstrate that carboxymethylation of lysine residues renders these peptides refractory to trypsin digestion. We did not detect observable differences in the simulated gastric fluid or endolysosomal digestion between the parental and CML-modified peptides. One of the tested peptides contains a lysine residue previously shown to be CML modified laying in a previously mapped linear IgE epitope, but we did not observe a difference in IgE binding between the modified and parental peptides. Our findings suggest a molecular mechanism for the increased resistance of peanut allergens modified by thermal processing, such as Ara h 1, to digestion in intestinal fluid after heating and could help explain how food processing-induced modifications may lead to more potent food allergens by acting to protect intact IgE epitopes from digestion by proteases targeting lysine residues. PMID:26288719

  19. In vitro evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides

    PubMed Central

    Mattison, Christopher P; Dinter, Jens; Berberich, Matthew J; Chung, Si-Yin; Reed, Shawndrika S; Le Gall, Sylvie; Grimm, Casey C

    2015-01-01

    Ara h 1 is a major peanut allergen. Processing-induced modifications may modulate the allergenic potency of Ara h 1. Carboxymethyl lysine (CML) modifications are a commonly described nonenzymatic modification on food proteins. In the current study, we tested the ability of digestive and endolysosomal proteases to cleave CML-modified and unmodified Ara h 1 peptides. Mass spectrometric analyses of the digested peptides demonstrate that carboxymethylation of lysine residues renders these peptides refractory to trypsin digestion. We did not detect observable differences in the simulated gastric fluid or endolysosomal digestion between the parental and CML-modified peptides. One of the tested peptides contains a lysine residue previously shown to be CML modified laying in a previously mapped linear IgE epitope, but we did not observe a difference in IgE binding between the modified and parental peptides. Our findings suggest a molecular mechanism for the increased resistance of peanut allergens modified by thermal processing, such as Ara h 1, to digestion in intestinal fluid after heating and could help explain how food processing-induced modifications may lead to more potent food allergens by acting to protect intact IgE epitopes from digestion by proteases targeting lysine residues. PMID:26288719

  20. Short communication: Effect of casein haplotype on angiotensin-converting enzyme inhibitory and antioxidant capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes.

    PubMed

    Perna, Annamaria; Simonetti, Amalia; Gambacorta, Emilio

    2016-09-01

    The aim of this work was to investigate the effect of casein haplotype (αS1, β, and κ) on antioxidative and angiotensin-converting enzyme (ACE) inhibitory capacities of milk casein from Italian Holstein cows before and following in vitro digestion with gastrointestinal enzymes. The antioxidant capacity was measured using 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power assays, whereas ACE inhibition was determined by ACE-inhibitory assay. The ACE-inhibitory and antioxidant capacities of milk casein increased during in vitro gastrointestinal digestion. Casein haplotype significantly influenced the antioxidative and ACE-inhibitory capacities of digested casein. In particular, BB-A(2)A(1)-AA casein and BB-A(1)A(1)-AA casein showed the highest ACE-inhibitory capacity, BB-A(2)A(2)-AA casein showed the highest antioxidant capacity, whereas BB-A(2)A(2)-BB casein showed the lowest biological capacity. To date, few studies have been done on the effect of casein haplotype on biological capacity of milk casein, thus the present study sets the basis for a new knowledge that could lead to the production of milk with better nutraceutical properties. PMID:27289148

  1. Attribute based specification, comparison and selection of feed stock for anaerobic digestion using MADM approach.

    PubMed

    Rao, P Venkateswara; Baral, Saroj S

    2011-02-28

    Organic wastes are common in nature and generated at different sources which need to be treated before disposing into the environment. Anaerobic digestion (AD) process is a primary technique used for digestion and reduction of the ill effects of disposing the organic waste. Selection of appropriate feed stock for anaerobic digestion among the available options is a primary concern and the process efficiency and stability largely depend on this. The present paper describes a methodology for evaluation, comparison, ranking and optimum selection of a feed stock for anaerobic digestion. A 30 attribute coding scheme is proposed to evaluate the existing alternatives for feed stock of anaerobic digester. A three stage procedure which includes the elimination search is proposed to evaluate the available alternatives with the help of attributes quickly. Technique for order preference by similarity to ideal solution (TOPSIS) is a Multiple Attribute Decision Making (MADM) approach and graphical methods namely line graph and spider diagrams are used for optimum selection of feed stock among the available options. MATLAB code is written to execute the three stage procedure. The proposed methodology is explained through an illustrated example. PMID:21247688

  2. Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio.

    PubMed

    Kralj, Slavko; van Leeuwen, Sander S; Valk, Vincent; Eeuwema, Wieger; Kamerling, Johannis P; Dijkhuizen, Lubbert

    2008-12-01

    The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into alpha-d-glucans (labelled reuterans) with mainly alpha-(1-->4) glucosidic linkages (50% and 70%, respectively), plus alpha-(1-->6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA/O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1-3; O1-3), and used to construct six additional GTFA/O hybrids. All hybrid enzymes were able to synthesize alpha-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2/O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1/O1 regions make a strong contribution. Some GTFO derived hybrid/mutant enzymes displayed strongly increased transglucosylation/hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligo- and polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation/hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in alpha-(1-->4) and alpha-(1-->6) linkage ratios. PMID:19016850

  3. Oral administration recombinant porcine epidermal growth factor enhances the jejunal digestive enzyme genes expression and activity of early-weaned piglets.

    PubMed

    Lee, D N; Chuang, Y S; Chiou, H Y; Wu, F Y; Yen, H T; Weng, C F

    2008-08-01

    This study attempted to determine ingested porcine epidermal growth factor (pEGF) on the gastrointestinal tract development of early-weaned piglets. Thirty-two piglets (14-day weaned) were randomly allotted to supplemented with 0 (control), 0.5, 1.0, or 1.5 mg pEGF/kg diet. Each treatment consisted of four replicates with two pigs per pen for a 14 days experimental period. Piglets were sacrificed and gastrointestinal tract samples were collected to measure mucosa morphology, mRNA expression and activities of digestive enzymes in the gastrointestinal tract of piglets at the end of the experiment. Diets supplemented with pEGF failed to influence growth performance but tended to increase jejunal mucosa weight (p < 0.09) and protein content (p < 0.07). Piglets supplemental pEGF induced incrementally the gastric pepsin activity (p < 0.05) and stimulated jejunal alkaline phosphatase (ALP) and lactase activities accompanied with the increase of jejunal ALP and maltase mRNA expression. No effect of pEGF on the activities of all enzymes in ileum except the stimulation of ileal aminopeptide N mRNA expression. These results reveal that dietary pEGF supplementation might enhance gene expression and activities of digestive enzymes in the stomach and jejunum of piglets. PMID:18662356

  4. Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF.

    PubMed Central

    Tolson, D A; Nicholson, N H

    1998-01-01

    The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between the nucleotides U and C, which makes unambiguous assignment difficult using a MALDI-TOF instrument. Here we report our experiences with sequence specific endonucleases and chemical methods followed by MALDI-TOF to resolve these sequence ambiguities. We have found chemical methods superior to endonucleases both in terms of correct specificity and extent of sequence coverage. This methodology can be used in combination with exonuclease digestion to rapidly assign RNA sequences. PMID:9421498

  5. Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa.

    PubMed

    Loziuk, Philip L; Parker, Jennifer; Li, Wei; Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Sederoff, Ronald R; Chiang, Vincent L; Muddiman, David C

    2015-10-01

    Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa. PMID:26325666

  6. Effect of potential probiotic Rhodotorula benthica D30 on the growth performance, digestive enzyme activity and immunity in juvenile sea cucumber Apostichopus japonicus.

    PubMed

    Wang, Ji-hui; Zhao, Liu-qun; Liu, Jin-feng; Wang, Han; Xiao, Shan

    2015-04-01

    The effects of dietary addition of yeast Rhodotorula benthica (R. benthica) D30 which isolated from local sea mud at levels of 0 (control), 10(5), 10(6) and 10(7) CFU/g feed on the growth performance, digestive enzyme activity, immunity and disease resistance of juvenile sea cucumber Apostichopus japonicus were investigated. It was shown that dietary addition of R. benthica D30 significantly increased the growth rates of sea cucumbers (p < 0.05). The amylase activity, cellulase activity and alginase activity were increased for the animals from three probiotics treated groups. And with the supplemented concentration increased, the values of those digestive enzyme activities increased as well. Dietary addition of R. benthica D30 at the level of 10(7) CFU significantly increased the lysozyme, phagocytic and total nitric oxide synthase activity of A. japonicus (p < 0.05). While, the highest values of the phenoloxidase and alkaline phosphatase activity were found in sea cucumbers fed with R. benthica D30 at the level of 10(6) CFU. Whereas adding R. benthica D30 to diet had no significant effects on the total coelomocyte counts and acid phosphatase activity of A. japonicus (p > 0.05). It was observed that adding R. benthica D30 could significantly decrease the cumulative mortality of sea cucumbers. The present study demonstrated that dietary addition of R. benthica D30 could increase growth performance and some digestive enzyme activities, improve immunity and disease resistance of A. japonicus. And the medium (10(6) CFU) and high (10(7) CFU) additional levels showed better effects. It suggests that yeast R. benthica D30 could be a good probiotic for aquaculture. PMID:25592878

  7. Mechanism of extraordinary DNA digestion by pepsin.

    PubMed

    Zhang, Yanfang; Li, Chunchuan; Liu, Yu; Wang, Xiaoqian; Dong, Ping; Liang, Xingguo

    2016-03-25

    Recently, the protein-specific enzyme pepsin was found be capable of digesting nucleic acids unexpectedly. In this study, the effects of DNA sequence specificity, purine content (AG content), depurination and length on the nucleic acid (NA) digestion by pepsin were investigated. The results showed that pepsin functioned similar as endonuclease, and presented a moderate sequence preference compared with restriction enzymes and non-specific nuclease. The digestion was specific (sequence dependent to some extent), and pepsin preferred to cleave purine-rich sequences. The digestion of favorable sequence was dramatically accelerated when the purine base at the cleavage site was removed (created an apurinic (AP) site). However, the AP site did not help to cleave the sequence that pepsin could not cleave originally. Moreover, the results indicated that pepsin preferred to digest longer DNA (e.g. > 59 bases) than shorter one, and sequence shorter than 30 bases was barely digested. The mechanism of DNA digestion by pepsin was also discussed. PMID:26903302

  8. Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

    PubMed Central

    Bennett, C F; Chiang, M Y; Wilson-Lingardo, L; Wyatt, J R

    1994-01-01

    Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner. PMID:8065936

  9. Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

    PubMed Central

    Samanani, Nailish; Park, Sang-Un; Facchini, Peter J.

    2005-01-01

    Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of l-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type–specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type–specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types. PMID:15722473

  10. Substrate specificity determinants of the methanogen homoaconitase enzyme: structure and function of small subunit residues

    SciTech Connect

    Jeyakanthan, Jeyaraman; Drevland, Randy; Gayathri, Dasara; Velmurugan, Devadasan; Shinkai, Akeo; Graham, David E

    2010-01-01

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of -hydroxyacids to -hydroxyacids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of , -dicarboxylates with hydrophobic -chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length -carboxylate groups. These enzymes stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins leads to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, the small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between 2 and 3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence, but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These structural and kinetic results will

  11. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  12. Nutritional performance and activity of some digestive enzymes of the cotton bollworm, Helicoverpa armigera, in response to seven tested bean cultivars.

    PubMed

    Namin, Foroogh Rahimi; Naseri, Bahram; Razmjou, Jabraeil

    2014-01-01

    Nutritional performance and activity of some digestive enzymes (protease and α-amylase) of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) in response to feeding on bean (Phaseolus vulgaris L. (Fabales: Fabaceae)) cultivars (Shokufa, Akhtar, Sayyad, Naz, Pak, Daneshkadeh, and Talash) were evaluated under laboratory conditions (25 ± 1°C, 65 ± 5% RH, and a 16:8 L:D photoperiod). The highest and lowest respective values of approximate digestibility were observed when fourth, fifth, and sixth larval instar H. armigera were fed red kidney bean Akhtar and white kidney bean Daneshkadeh. The efficiency of conversion of ingested and digested food was highest when H. armigera was fed red kidney beans Akhtar and Naz and lowest when they were fed white kidney bean Pak. The highest protease activity of fifth instars was observed when they were fed red kidney bean Naz, and the highest amylase activity of fifth instars was observed when they were fed red kidney bean Sayyad. Sixth instar larvae that fed on red kidney bean Sayyad showed the highest protease activity. Larvae reared on common bean Talash and white kidney bean Pak showed the highest amylase activity. Among bean cultivars tested, red kidney bean Sayyad was the most unsuitable host for feeding H. armigera. PMID:25368049

  13. The role of lipid and carbohydrate digestive enzyme inhibitors in the management of obesity: a review of current and emerging therapeutic agents

    PubMed Central

    Tucci, Sonia A; Boyland, Emma J; Halford, Jason CG

    2010-01-01

    Obesity is a global epidemic associated with significant morbidity and mortality in adults and ill health in children. A proven successful approach in weight management has been the disruption of nutrient digestion, with orlistat having been used to treat obesity for the last 10 years. Although orlistat-induced weight loss remains modest, it produces meaningful reductions in risk factors for obesity-related conditions such as diabetes and cardiovascular disease. Moreover, this lipase inhibitor is free of the serious side effects that have dogged appetite-suppressing drugs. This success had driven investigation into new generation nutraceuticals, supplements and pharmaceutical agents that inhibit the breakdown of complex carbohydrates and fats within the gut. This review focuses on agents purported to inhibit intestinal enzymes responsible for macronutrient digestion. Except for some synthetic products, the majority of agents reviewed are either botanical extracts or bacterial products. Currently, carbohydrate digestion inhibitors are under development to improve glycemic control and these may also induce some weight loss. However, colonic fermentation induced side effects, such as excess gas production, remain an issue for these compounds. The α-glucosidase inhibitor acarbose, and the α-amylase inhibitor phaseolamine, have been used in humans with some promising results relating to weight loss. Nonetheless, few of these agents have made it into clinical studies and without any clinical proof of concept or proven efficacy it is unlikely any will enter the market soon. PMID:21437083

  14. Short-term effect of dietary yeast nucleotide supplementation on small intestinal enzyme activities, bacterial populations and metabolites and ileal nutrient digestibilities in newly weaned pigs.

    PubMed

    Sauer, N; Eklund, M; Roth, S; Rink, F; Jezierny, D; Bauer, E; Mosenthin, R

    2012-08-01

    In previous studies, dietary nucleotides have been shown to improve performance in single-stomached animals by promoting the renewal of small intestine epithelial cells and by influencing the activity and composition of the microbial community in the digestive tract. The present experiment was carried out with 12 barrows weaned at the age of 18 days and fitted with a simple T-cannula at the distal ileum. To determine short-term effects of dietary yeast nucleotides, the piglets received a grain-soybean meal-based basal diet with or without supplementation of 1 g/kg of a dried yeast product containing free nucleotides. Dietary supplementation with yeast did not affect bacterial numbers in the ileum as well as ileal concentrations of individual short-chain fatty acids (SCFA), total SCFA and total lactic acid (p > 0.05). Moreover, there was no effect of supplemental yeast nucleotides on ileal α-amylase, leucine amino peptidase, maltase and lactase activities (p > 0.05), as well as on ileal dry matter, crude protein and crude fibre digestibilities (p > 0.05). In conclusion, short-term supplementation with dietary yeast nucleotides did not affect microbial metabolite concentrations, bacterial numbers and enzyme activities in the ileal digesta as well as ileal nutrient digestibilities of newly weaned pigs. PMID:21797935

  15. Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.

    PubMed

    Botyanszki, Zsofia; Tay, Pei Kun R; Nguyen, Peter Q; Nussbaumer, Martin G; Joshi, Neel S

    2015-10-01

    Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. PMID:25950512

  16. [Activity of digestive enzymes in offspring of rats fed a ration contaminated by heavy metals during lactation].

    PubMed

    Sadykov, B A; Kuchkarova, L S; Ermatova, S U; Ergashev, N A

    2009-01-01

    Is was shown the consumption of heavy metal's salts with food by lactating rats, essential influences on formation of mechanisms of cavity and membrane digestive in offspring. This influence is shown in repression of activity of pancreatic alpha-amylase and enteral saccrase, maltase as well as in induction of lactase activity in growing rats. PMID:19514343

  17. Enzyme-gold cytochemistry of seed xyloglucans using two xyloglucan-specific hydrolases. Importance of prior heat-deactivation of the enzymes.

    PubMed

    Vian, B; Nairn, J; Reid, J S

    1991-03-01

    Two pure, homogeneous xyloglucan-hydrolyzing enzymes from germinated nasturtium seeds have been used to localize xyloglucans specifically in seed cell walls. The enzymes, a novel endo (1----4)-beta-D-glucanase which shows absolute specificity towards xyloglucans and a beta-D-galactosidase which is capable of removing galactosyl residues from polymeric xyloglucans, were used to stabilize gold sols. The complexes were applied to ultrathin sections of nasturtium (Tropaeolum majus L) and tamarind (Tamarindus indica L) seeds. The gold complexes prepared from the active enzyme proteins retained enzyme activity, and such complexes gave extremely weak section-labelling or no labelling at all. When the enzymes were subjected to heat-deactivation before being used to stabilize the gold sols, gold complexes were obtained which lacked enzyme activity, but which gave strong, specific labelling of xyloglucans in ultrathin sections. The specificity of the labelling was checked by substrate-competition, by pretreatment of sections with the active and heat-denaturated enzymes and by comparing the labelling of xyloglucan-containing storage cells with other cell types in the same section. The labelling was maximal at the pH which was optimal for the active enzyme. We conclude that the enzyme-gold complexes which retain high activity against the substrate to be localized are likely to be unsuitable as cytochemical probes because they may cause in situ substrate modification. In the case of the enzyme complexes described here the specific localization obtained with the gold complexes prepared from heat deactivated enzymes may be attributable to the retention by the heat-treated enzymatically-inactive proteins of substrate recognition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1783560

  18. The adaptation of mussels Crenomytilus grayanus to cadmium accumulation result in alterations in organization of microsomal enzyme-membrane complex (non-specific phosphatase).

    PubMed

    Zakhartsev; Chelomin; Belcheva

    2000-08-01

    The kinetic parameters (V(m), K(m) and slope) of membrane-bound microsomal non-specific phosphatase (NPase, with G6P as the substrate) from the digestive gland of unexposed and cadmium adapted (45 days for 100 µg Cd(2+)/l) mussels were investigated. In vivo and in vitro approaches were used. Adaptation of mussels (Crenomytilus grayanus) to cadmium resulted in a 1.6-fold increase in NPase activity. V(m) was increased by 1.6-fold, but K(m) was the same in terms of enzyme kinetics. This indicates that the total concentration of the enzymes in the digestive gland increased. Cd(2+) (1 mM) did not significantly alter the activity of the membrane-bound enzyme in vitro both for unexposed and for cadmium adapted mussels, meaning that cadmium ions are not a direct inhibitor of the membrane-bound enzyme in this concentration. The microsomal NPase activity in both unexposed and cadmium adapted mussels was inhibited by in vitro solubilization of microsomes with non-ionic detergent (Triton X100, 0.01%). This inhibition was uncompetitive for microsomes of unexposed mussels (K(m) decreased 3.1-fold). The most drastic events were observed in cadmium adapted mussels, where inhibition was mixed (K(m) decreased 7.2-fold). The simultaneous actions of detergent and cadmium ions did not alter NPase activity significantly in comparison with action of the detergent alone. The differences in the types and the extents of inhibition of the enzymes activity by membrane disordering agent (Triton X100) indicated that the enzyme-membrane complex (NPase) has been altered as a result of adaptation of mussels to cadmium accumulation. We conclude that the mussels produced a new enzyme-membrane complex, with the same K(m) as the previous complex, but with other detergent sensitivity and greater amounts. Thus, the adaptation capacity of this enzyme is reduced as result of adaptation of mussels to cadmium accumulation. PMID:10930649

  19. Using a fibrolytic enzyme in barley-based diets containing wheat dried distillers grains with solubles: ruminal fermentation, digestibility, and growth performance of feedlot steers.

    PubMed

    He, Z X; He, M L; Walker, N D; McAllister, T A; Yang, W Z

    2014-09-01

    Two experiments were conducted to evaluate the effects of adding an exogenous fibrolytic enzyme (FE) on ruminal pH and fermentation, digestibility, and growth performance of feedlot beef cattle fed a finishing diet containing wheat dried distillers grains with solubles (DDGS). In Exp. 1, 4 ruminally cannulated Angus heifers (average BW of 807 ± 93.9 kg) were used in a replicated 4 × 4 Latin square design. Treatments were 1) control (CON; 10% barley silage and 90% barley grain-based concentrate), 2) CON diet substituting 30% wheat DDGS for barley grain (WDG), 3) WDG diet supplemented with low FE (WDGL), and 4) WDG diet supplemented with high FE (WDGH). Heifers fed WDG had less (P = 0.01) total tract DM digestibility than heifers fed CON. Increasing FE linearly (P < 0.05) increased starch digestibility without affecting digestibility of other nutrients. Addition of FE also reduced (P = 0.03) ruminal ammonia-N (NH3-N) concentration but did not affect VFA concentration. Moreover, application of FE to wheat DDGS linearly increased in situ ruminal DM (P < 0.01) and NDF (P = 0.02) disappearance after 48 h of incubation. In Exp. 2, 160 yearling steers (initial BW = 495 ± 37.9 kg) were fed the same diets as in Exp. 1. No differences in DMI, final BW, ADG, dietary NEg, or carcass characteristics were observed among diets. However, the steers fed WDG had less (P < 0.05) G:F and greater number of (P < 0.01) abscessed livers than steers fed CON. Increasing FE application in wheat DDGS diets did not affect DMI, final BW, or ADG but tended (P < 0.09) to linearly improve feed efficiency and decreased (P = 0.03) the incidence of abscessed livers. These results demonstrated adverse effects of including wheat DDGS in finishing diets on feed digestion, feed efficiency, and animal health. Application of FE in wheat DDGS-based diets potentially improved starch digestion, protein metabolism in the rumen, feed efficiency, and animal health. PMID:24987082

  20. Study on the bio-methane yield and microbial community structure in enzyme enhanced anaerobic co-digestion of cow manure and corn straw.

    PubMed

    Wang, Xuemei; Li, Zifu; Zhou, Xiaoqin; Wang, Qiqi; Wu, Yanga; Saino, Mayiani; Bai, Xue

    2016-11-01

    The use of enzymes to improve anaerobic co-digestion (AcoD) of cow manure and corn straw was explored in this study, including cellulase pretreatment and direct additions of amylase and protease. The effects of enzymes on microbial community structure were investigated though PCR-DGGE method. Results showed that AcoD with amylase achieved the highest methane yield of 377.63ml·CH4/g·VS, which was an increase of 110.79%. The methane increment consumed the amylase of 4.18×10(-5)g/ml·CH4. Enzymes mainly affected the bacteria in the hydrolysis stage rather than the bacteria in the hydrogenesis and acetogenesis stage and the archaea in the methanogenesis stage. However, the experimental results demonstrated that enzymes had no negative influence on microbial communities; the predominant microbial communities were similar. Therefore, AcoD with amylase was an effective way to improve the bio-methane yield of cow manure and corn straw. PMID:27484671

  1. Specificity and catalysis hardwired at the RNA-protein interface in a translational proofreading enzyme

    NASA Astrophysics Data System (ADS)

    Ahmad, Sadeem; Muthukumar, Sowndarya; Kuncha, Santosh Kumar; Routh, Satya Brata; Yerabham, Antony S. K.; Hussain, Tanweer; Kamarthapu, Venu; Kruparani, Shobha P.; Sankaranarayanan, Rajan

    2015-06-01

    Proofreading modules of aminoacyl-tRNA synthetases are responsible for enforcing a high fidelity during translation of the genetic code. They use strategically positioned side chains for specifically targeting incorrect aminoacyl-tRNAs. Here, we show that a unique proofreading module possessing a D-aminoacyl-tRNA deacylase fold does not use side chains for imparting specificity or for catalysis, the two hallmark activities of enzymes. We show, using three distinct archaea, that a side-chain-stripped recognition site is fully capable of solving a subtle discrimination problem. While biochemical probing establishes that RNA plays the catalytic role, mechanistic insights from multiple high-resolution snapshots reveal that differential remodelling of the catalytic core at the RNA-peptide interface provides the determinants for correct proofreading activity. The functional crosstalk between RNA and protein elucidated here suggests how primordial enzyme functions could have emerged on RNA-peptide scaffolds before recruitment of specific side chains.

  2. A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

    SciTech Connect

    Yin, Si-Tao; Huang, Hao; Zhang, Yu-Hang; Zhou, Zi-Ren; Song, Ai-Xin; Hong, Fa-Shui; Hu, Hong-Yu

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. Black-Right-Pointing-Pointer We have established an activity assay for ubiquitin C-terminal hydrolases. Black-Right-Pointing-Pointer This assay can be applicable to other deubiquitinating enzymes. -- Abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub{sup F45W}-Xaa) and di-ubiquitin chains (Ub{sup F45W}-diUb). After removal of the intact substrate by Ni{sup 2+}-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub{sup F45W} product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

  3. Effect of method of applying fibrolytic enzymes or ammonia to Bermudagrass hay on feed intake, digestion, and growth of beef steers.

    PubMed

    Krueger, N A; Adesogan, A T; Staples, C R; Krueger, W K; Kim, S C; Littell, R C; Sollenberger, L E

    2008-04-01

    This study examined how different methods of applying a fibrolytic enzyme or ammonia affect the nutritive value of Bermudagrass hay and the performance of beef cattle. Fifty Angus x Brangus crossbred steers (mean initial BW 244 +/- 26 kg) were individually fed for ad libitum intake of a 5-wk regrowth of a mixture of Florakirk and Tifton 44 Bermudagrass [Cynodon dactylon (L.) Pers] hay for 84 d with a concentrate supplement (77% soybean hull pellets, 23% cottonseed meal (DM basis) fed at 1% of BW daily. The Bermudagrass was conserved as hay without treatment (control), with NH(3) (30 g/kg of DM), or with a fibrolytic enzyme (16.5 g/t, air-dry basis) that was applied immediately after cutting (Ec), at baling (Eb), or at feeding. Chromic oxide was dosed to steers for 10 consecutive days, and fecal Cr concentrations from the last 5 d were used to estimate apparent total tract digestibility. In situ ruminal DM degradability was measured by incubating ground (4-mm) hay samples in duplicate in each of 2 ruminally cannulated cows having ad libitum access to Bermudagrass hay and 500 g/d of soybean meal. Unlike the enzyme treatment, ammoniation increased (P < 0.001) the CP concentration and reduced (P < 0.001) NDF, hemicellulose, and lignin concentrations of hay. Total DMI was greater (P < 0.05) for steers fed hays treated with Ec or NH(3) than for those fed control hays. All additive treatments increased (P < 0.05) DM digestibility, and NH(3), Ec, and Eb treatments also increased (P < 0.01) NDF digestibility. The initial and final BW, ADG, BCS, G:F, and hip height of the steers were not affected (P > 0.05) by treatment. The wash loss fractions in hays treated with Ec and Eb were lower than that in the control hay, but the potentially degradable fraction, total degradable fraction, and the effective degradability were increased (P < 0.01) by NH(3) treatment. Application at cutting was the most promising method of enzyme treatment, and this treatment was almost as effective

  4. Effect of cooking and in vitro digestion on the stability of co-enzyme Q10 in processed meat products.

    PubMed

    Tobin, Brian D; O'Sullivan, Maurice G; Hamill, Ruth; Kerry, Joseph P

    2014-05-01

    The use of CoQ10 fortification in the production of a functional food has been demonstrated in the past but primarily for dairy products. This study aimed to determine the bio-accessibility of CoQ10 in processed meat products, beef patties and pork breakfast sausages, fortified with CoQ10. Both the patties and sausages were fortified with a micellarized form of CoQ10 to enhance solubility to a concentration of 1mg/g of sample (NovaSolQ®). An assay was developed combining in vitro digestion and HPLC analysis to quantify the CoQ10 present in fortified products (100mg/g). The cooking retention level of CoQ10 in the products was found to be 74±1.42% for patties and 79.69±0.75% for sausages. The digestibility for both products ranged between 93% and 95%, sausages did have a higher digestibility level than patties but this was not found to be significant (P<0.01). PMID:24360438

  5. Positive dermal hypersensitivity and specific antibodies in workers exposed to bio-engineered enzymes

    SciTech Connect

    Biagini, R.E.; Henningsen, G.M.; Driscoll, R.; MacKenzie, B.A.; Wilcox, T.; Scinto, J.D.; Bernstein, D.M.; Swanson, M. Mayo Clinic, Rochester, MN )

    1991-03-15

    Thirty-six employees who produced industrial enzymes from bio-engineered strains of bacteria and fungi were evaluated by skin prick testing and enzyme linked immunosorbent assays for specific IgE and IgG antibodies. The workers complained of asthma- and flu-like' symptoms which generally lessened away from work. The enzymes evaluated were {alpha}-amylase from A. niger (ind-AAN), B. licheniformis (ind-AAL) and B. subtilis (ind-AAS); purified {alpha}-amylase from B. subtilis (AAS) and A. niger (AAN); alkaline protease from B. licheniformis (ind-APL) and purified alkaline protease (APL); amylase glucosidase from A. niger (ind-AGN) and purified amylase glucosidase (AGN). Significantly positive skin tests were found for APL, AGN and ind-AAN. Significantly elevated specific IgE results were observed for AAN, AGN, and ind-AAN; elevated specific IgGs were observed for AAN, ind-AAN, ind-AAS, ind-AAL and ind-AGN. Radioimmunoassays of air filter samples (using sera with high Ab titers) for 4 of the ind-enzymes showed only ind-AAN at extremely high environmental levels. These results indicate that occupational exposure to some ind-enzymes causes immediate onset dermal hypersensitivity reactions. The results are equivocal as to whether these reactions are IgE mediated, as IgE titers were low. Contrary to this, IgG titers were extremely high and suggest that these biomarkers can be used as indicators of both individual exposure and environmental analyses.

  6. Effects of small peptides, probiotics, prebiotics, and synbiotics on growth performance, digestive enzymes, and oxidative stress in orange-spotted grouper, Epinephelus coioides, juveniles reared in artificial seawater

    NASA Astrophysics Data System (ADS)

    Wang, Tao; Cheng, Yongzhou; Chen, Xiaoyan; Liu, Zhaopu; Long, Xiaohua

    2016-04-01

    Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of diff erent dietary additives [w/w: 2% small peptides, 0.01% probiotics (Bacillus licheniformis) and 0.2% prebiotics (inulin)] on growth performance, digestive enzyme activities, and oxidative stress in juvenile Epinephelus coioides reared in artificial seawater of two salt concentrations (13.5 vs. 28.5). Weight gain rate was significantly higher in fish fed the diet supplemented with small peptides, B. licheniformis, inulin, or synbiotics than that in fish fed the basal diet; the greatest weight gain rate was found in fish fed the small peptide treatment [56.0% higher than basal diet]. Higher feed efficiency was detected in fish fed the diet supplemented with small peptides than that of fish in the other dietary treatments. Total protease activity in the stomach and intestines was highest in fish fed the small peptide-treated diet, whereas lipase activity was highest in those fed synbiotics (combination of Bacillus licheniformis and inulin) than that in fish fed the other treatments. Antioxidant enzyme (total superoxide dismutase and catalase) activities and hepatic malondialdehyde content were higher in fish receiving the dietary supplements and maintained in artificial seawater containing 13.5 salinity compared with those in the control (28.5). Hepatic catalase activity in grouper fed the diets with small peptides or synbiotics decreased significantly compared with that in control fish. Overall, the three types of additives improved growth rate of juvenile grouper and digestive enzymes activities to varying degrees but did not effectively improve antioxidant capacity under low-salinity stress conditions.

  7. Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms.

    PubMed

    Procházková, Petra; Šustr, Vladimír; Dvořák, Jiří; Roubalová, Radka; Škanta, František; Pižl, Václav; Bilej, Martin

    2013-11-01

    Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression. PMID:23999244

  8. Effect of wheat dried distillers grains with solubles and fibrolytic enzymes on ruminal fermentation, digestibility, growth performance, and feeding behavior of beef cattle.

    PubMed

    He, Z X; Walker, N D; McAllister, T A; Yang, W Z

    2015-03-01

    Two experiments were conducted to evaluate the effect of wheat dried distillers grains with solubles (DDGS) and fibrolytic enzymes (FE) on ruminal fermentation, in situ ruminal and in vivo total tract digestibility, growth performance, and feeding behavior of growing beef cattle. In Exp. 1, 6 ruminally cannulated Angus heifers (average BW of 794 ± 44.2 kg) were used in a 6 × 6 Latin square design with 2 × 3 factorial arrangement of treatments. Treatments were a control diet consisting of 50% barley silage, 10% grass hay, and 40% barley grain-based concentrate (CON) and the CON with 15% DDGS substituted for barley grain (WDG) combined with either 0, 1, or 2 mL FE/kg diet DM, respectively. Inclusion of DDGS increased total tract digestibility of CP ( < 0.01), NDF ( = 0.04), and ADF ( = 0.03). Increasing FE linearly ( = 0.03) increased CP digestibility without affecting the digestibility of other nutrients. There were no effects of DDGS inclusion or FE on ruminal pH or VFA concentration except that propionate was greater ( = 0.04) with the WDG. In situ ruminal DM and NDF disappearance of barley silage was greater ( < 0.04) in heifers fed the WDG than in heifers fed the CON after 24 h of incubation. Increasing FE linearly ( = 0.03) increased in situ NDF disappearance of barley silage after 24 h of incubation. In Exp. 2, 120 weaned steers (initial BW of 289 ± 11.0 kg) were fed diets similar to those in Exp. 1. The steers fed the WDG had greater ( < 0.01) final BW, ADG, DMI, and G:F compared with steers fed the CON. Increasing FE did not alter ADG or G:F but tended ( < 0.07) to linearly decrease DMI. There were interactions ( < 0.02) between DDGS and FE on eating rate and the time spent at the feed bunk. Supplementing FE decreased ( < 0.01) time at the bunk and increased ( < 0.01) eating rate for steers fed the WDG but not for steers fed the CON. Eating rate ( < 0.01) and meal frequency ( = 0.02) were greater but eating duration was shorter ( < 0.01) for steers fed

  9. Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

    2015-12-01

    We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

  10. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.

    PubMed

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Medina-Ruíz, Sofía; Gaytán, Paul; Carrillo-Tripp, Mauricio; Fülöp, Vilmos; Barona-Gómez, Francisco

    2013-09-01

    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism. PMID:23800623

  11. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    PubMed Central

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies. PMID:27242682

  12. Prediction and experimental validation of enzyme substrate specificity in protein structures.

    PubMed

    Amin, Shivas R; Erdin, Serkan; Ward, R Matthew; Lua, Rhonald C; Lichtarge, Olivier

    2013-11-01

    Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase-like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. PMID:24145433

  13. Prediction and experimental validation of enzyme substrate specificity in protein structures

    PubMed Central

    Amin, Shivas R.; Erdin, Serkan; Ward, R. Matthew; Lua, Rhonald C.; Lichtarge, Olivier

    2013-01-01

    Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase–like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. PMID:24145433

  14. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  15. Effect of Copper on Growth, Digestive and Antioxidant Enzyme Activities of Juvenile Qihe Crucian Carp, Carassius carassius, During Exposure and Recovery.

    PubMed

    Jiang, Hongxia; Kong, Xianghui; Wang, Shuping; Guo, Huiyun

    2016-03-01

    The toxicity of copper (Cu) on growth and activities of digestive and antioxidant enzymes in the hepatopancreas and intestine of juvenile Qihe crucian carp Carassius carassius was evaluated. The fish were exposed in different Cu solutions for 20 days, and the 0.60 mg/L group was then transferred to clean water to initiate a 20-day recovery period after Cu exposure. Results showed that all enzyme activities decreased significantly at high-concentration (0.30 and 0.60 mg/L) and long-time (20 days) Cu exposures and increased significantly at high-concentration (0.60 mg/L) and short-time Cu exposures (1 day). After the 20-day recovery period, all enzyme activities in the 0.60 mg/L group had recovered to control levels. High-concentration (0.60 mg/L) and long-time (20 days) Cu exposure markedly hindered the growth of fish, whereas the loss of fish growth can not be compensated for by a 20-day recovery period. PMID:26781633

  16. Starch digestibility, energy utilization, and growth performance of broilers fed corn-soybean basal diets supplemented with enzymes.

    PubMed

    Stefanello, C; Vieira, S L; Santiago, G O; Kindlein, L; Sorbara, J O B; Cowieson, A J

    2015-10-01

    A study was conducted to evaluate the effects of dietary α-amylase and β-xylanase supplementation of corn-soy diets, formulated with or without supplemental phytase, on growth performance, energy utilization, and starch digestibility in broiler chickens. A total of 336 slow-feathering, Cobb × Cobb 500 male broilers were randomly distributed to 6 treatments having 8 replicates of 7 birds each. Birds were fed a common starter diet to d 14 post-hatch (3,050 kcal/kg AMEn, 21.7% CP, 1.05% Ca, and 0.53% nPP). The experimental diets were provided afterwards until d 25. A 2 × 3 factorial arrangement of 2 control diets (basal = corn-soy diet without added phytase or PHY = corn-soy diet formulated with 1,000 phytase units/kg) and 3 carbohydrase supplementations (0, 80 kilo-Novo α-amylase units/kg, or 80 kilo-Novo α-amylase units/kg + 100 fungal β-xylanase units/kg) was used from d 14 to 25. Excreta were collected from 21 to 24 d and all birds were euthanized at 25 d for jejunum and ileum content collection. Samples of feed, excreta, and jejunal and ileal digesta were analyzed for determination of total tract retention and ileal apparent digestibility. No interactions between diet and carbohydrase were observed. Broilers fed diets formulated with phytase or supplemented with amylase + xylanase had higher BW gain (BWG) and lower FCR (P < 0.05) when compared with birds fed diets without carbohydrases. Relative to the basal diet, AMEn was increased (P < 0.01) by 70 kcal/kg and 99 kcal/kg when birds were fed the diet supplemented with amylase and amylase + xylanase, respectively. Starch digestibility in the jejunum and ileum was increased (P < 0.05) by 3.5% and 2.4%, respectively, when birds were fed the diet supplemented with amylase + xylanase. Results from this experiment show that corn-soy diets having phytase and supplemented with amylase and xylanase led to increased growth performance, AMEn, and starch digestibility in broilers. Furthermore, the efficacy of

  17. Functional characterization and substrate specificity of spinosyn rhamnosyltransferase by in vitro reconstitution of spinosyn biosynthetic enzymes.

    PubMed

    Chen, Yi-Lin; Chen, Yi-Hsine; Lin, Yu-Chin; Tsai, Kuo-Chung; Chiu, Hsien-Tai

    2009-03-13

    Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with d-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structure-activity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency. PMID:19126547

  18. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  19. Gamma-Glutamyl Compounds: Substrate Specificity of Gamma-Glutamyl Transpeptidase Enzymes

    PubMed Central

    Wickham, Stephanie; West, Matthew B.; Cook, Paul F.; Hanigan, Marie H.

    2011-01-01

    Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites and neuroactive compounds. Two cell surface enzymes have been identified that metabolize gamma-glutamyl compounds, gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetics analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative and is conducted at physiologic pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The Kms for reduced glutathione were 11μM for both GGT1 and GGT5. However, the Km for oxidized glutathione was 9μM for GGT1 and 43μM for GGT5. Our data show that the Kms for leukotriene C4 are equivalent for GGT1 and GGT5 at 10.8μM and 10.2μM, respectively. This assay was also used to evaluate serine-borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism and other pathways that involve gamma-glutamyl compounds. PMID:21447318

  20. Digestive capabilities reflect the major food sources in three species of talitrid amphipods.

    PubMed

    Johnston, Matthew; Johnston, Danielle; Richardson, Alastair

    2005-02-01

    Digestive enzyme activities of three talitrid amphipods were examined to investigate the relationship between their digestive capabilities and diet. Laminarinase, cellobiase, carboxymethyl-cellulase, xylanase, alpha- and beta-glucosidase and lipase were detected in all three species suggesting talitrid amphipods can readily digest dietary carbohydrate and lipid, including complex polysaccharides. Relatively high specific enzyme activity (Units (mg(-1) digestive tract protein)(-1)) of laminarinase and lipase was detected in Talorchestia marmorata, a supralittoral kelp feeder which is coherent with the digestion of lipid-esters and beta-glucans (laminarin) which are the main lipid and storage polysaccharides of brown seaweeds. Talorchestia sp., a low shore intertidal feeder, had high enzymatic activity of alpha- and beta-glucosidase, cellobiase and xylanase, which is consistent with the digestion of diatoms. Keratroides vulgaris, a forest litter feeder had a relatively low specific activity of all enzymes. It is possible that leaf litter is partially digested prior to ingestion by bacteria and fungi present in the rotting vegetation, with bacterial and fungal enzymes contributing to this species' ability to hydrolyse its diet. This study provides the first quantitative data on digestive capacity in these three talitrid amphipods and confirms the relationship between dietary preference and digestive enzyme complement. PMID:15649772

  1. Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry.

    PubMed

    Gallina, Serafina; Cunsolo, Vincenzo; Saletti, Rosaria; Muccilli, Vera; Di Francesco, Antonella; Foti, Salvatore; Lorenzten, Andrea Maria; Roepstorff, Peter

    2016-07-01

    Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. PMID:27020775

  2. Switching of self-assembly in a peptide nanostructure with a specific enzyme

    SciTech Connect

    Webber, Matthew J.; Newcomb, Christina J.; Bitton, Ronit; Stupp, Samuel I.

    2012-03-14

    Peptide self-assembly has been shown to be a useful tool for the preparation of bioactive nanostructures, and recent work has demonstrated their potential as therapies for regenerative medicine. In principle, one route to make these nanostructures more biomimetic would be to incorporate in their molecular design the capacity for biological sensing. We report here on the use of a reversible enzymatic trigger to control the assembly and disassembly of peptide amphiphile (PA) nanostructures. The PA used in these studies contained a consensus substrate sequence specific to protein kinase A (PKA), a biological enzyme important for intracellular signaling that has also been shown to be an extracellular cancer biomarker. Upon treatment with PKA, this PA molecule becomes phosphorylated causing the high aspect-ratio filamentous PA nanostructures to disassemble. Treatment with an enzyme to cleave the phosphate group results in reformation of the filamentous nanostructures. We also show that disassembly in the presence of PKA allows the enzyme-triggered release of an encapsulated cancer drug. In addition, these drug-loaded nanostructures were found to induce preferential cytotoxicity in a cancer cell line that is known to secrete high levels of PKA. This ability to control nanostructure through an enzymatic switch could allow for the preparation of highly sophisticated and biomimetic materials that incorporate a biological sensing capability to enable therapeutic specificity.

  3. Switching of Self-Assembly in a Peptide Nanostructure with a Specific Enzyme

    PubMed Central

    Webber, Matthew J.; Newcomb, Christina J.; Bitton, Ronit; Stupp, Samuel I.

    2012-01-01

    Peptide self-assembly has been shown to be a useful tool for the preparation of bioactive nanostructures, and recent work has demonstrated their potential as therapies for regenerative medicine. In principle, one route to make these nanostructures more biomimetic would be to incorporate in their molecular design the capacity for biological sensing. We report here on the use of a reversible enzymatic trigger to control the assembly and disassembly of peptide amphiphile (PA) nanostructures. The PA used in these studies contained a consensus substrate sequence specific to protein kinase A (PKA), a biological enzyme important for intracellular signaling that has also been shown to be an extracellular cancer biomarker. Upon treatment with PKA, this PA molecule becomes phosphorylated causing the high aspect-ratio filamentous PA nanostructures to disassemble. Treatment with an enzyme to cleave the phosphate group results in reformation of the filamentous nanostructures. We also show that disassembly in the presence of PKA allows the enzyme-triggered release of an encapsulated cancer drug. In addition, these drug-loaded nanostructures were found to induce preferential cytotoxicity in a cancer cell line that is known to secrete high levels of PKA. This ability to control nanostructure through an enzymatic switch could allow for the preparation of highly sophisticated and biomimetic materials that incorporate a biological sensing capability to enable therapeutic specificity. PMID:22408645

  4. Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities.

    PubMed

    Fesko, Kateryna

    2016-03-01

    Threonine aldolases have emerged as a powerful tool for asymmetric carbon-carbon bond formation. These enzymes catalyse the unnatural aldol condensation of different aldehydes and glycine to produce highly valuable β-hydroxy-α-amino acids with complete stereocontrol at the α-carbon and moderate specificity at the β-carbon. A range of microbial threonine aldolases has been recently recombinantly produced by several groups and their biochemical properties were characterized. Numerous studies have been conducted to improve the reaction protocols to enable higher conversions and investigate the substrate scope of enzymes. However, the application of threonine aldolases in organic synthesis is still limited due to often moderate yields and low diastereoselectivities obtained in the aldol reaction. This review briefly summarizes the screening techniques recently applied to discover novel threonine aldolases as well as enzyme engineering and mutagenesis studies which were accomplished to improve the catalytic activity and substrate specificity. Additionally, the results from new investigations on threonine aldolases including crystal structure determinations and structural-functional characterization are reviewed. PMID:26810201

  5. Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis.

    PubMed Central

    Wilhelm, S M; Eisen, A Z; Teter, M; Clark, S D; Kronberger, A; Goldberg, G

    1986-01-01

    Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion. Images PMID:3012533

  6. Effects of biofloc on growth performance, digestive enzyme activities and liver histology of common carp (Cyprinus carpio L.) fingerlings in zero-water exchange system.

    PubMed

    Najdegerami, Ebrahim H; Bakhshi, Farideh; Lakani, Forouzan Bagherzadeh

    2016-04-01

    Biofloc technology is considered as a method that degrades organic waste by microorganisms and produces microbial flocs. A 30-day experiment was performed to investigate the effects of partial replacement of daily feeding intake with biofloc on the growth performances, digestive enzymes activity and liver histology of the common carp Cyprinus carpio L. fingerlings. Two hundred and eight healthy fingerlings (58.6 ± 0.2 g) were randomly distributed in 12 tanks (30 L) at a density of 25.4 kg m(-3) and fed experimental treatments (100 % daily feeding rate as a control, biofloc + 75% daily feeding rate, biofloc + 50% daily feeding rate, biofloc + 25% daily feeding rate). At the end of experiment, the results indicated that the highest weight gain was observed in the fish fed BFT 75% and control which differed significantly from those fed BFT 25 % (P < 0.05). Diet BFT 75% improved total protease and pepsin activity compared with BFT 25 and 50% (P > 0.05). No significant difference was observed in case of lipase, amylase and alkaline phosphatase activity between the treatments. In the liver, histological alterations were found in the treatments, and feeding the fish with BFT 75% significantly improved hepatocellular quantification and qualification than the other groups. The results obtained in this experiment suggest that the biofloc improves growth performances, digestive enzyme activity and liver condition of the common carp fingerlings when 25% of daily feeding rate (BFT 75%) was replaced with one carbohydrate such as molasses in zero-water exchange system. PMID:26530301

  7. Effects of fish oil replacement by vegetable oil blend on digestive enzymes and tissue histomorphology of European sea bass (Dicentrarchus labrax) juveniles.

    PubMed

    Castro, Carolina; Couto, Ana; Pérez-Jiménez, Amalia; Serra, Cláudia R; Díaz-Rosales, Patricia; Fernandes, Rui; Corraze, Geneviève; Panserat, Stéphane; Oliva-Teles, Aires

    2016-02-01

    The impact of replacing circa 70% fish oil (FO) by a vegetable oil (VO) blend (rapeseed, linseed, palm oils; 20:50:30) in diets for European sea bass juveniles (IBW 96 ± 0.8 g) was evaluated in terms of activities of digestive enzymes (amylase, lipase, alkaline phosphatase, trypsin and total alkaline proteases) in the anterior (AI) and posterior (PI) intestine and tissue morphology (pyloric caeca-PC, AI, PI, distal intestine-DI and liver). For that purpose, fish were fed the experimental diets for 36 days and then liver and intestine were sampled at 2, 6 and 24 h after the last meal. Alkaline protease characterization was also done in AI and PI at 6 h post-feeding. Dietary VO promoted higher alkaline phosphatase activity at 2 h post-feeding in the AI and at all sampling points in the PI. Total alkaline protease activity was higher at 6 h post-feeding in the PI of fish fed the FO diet. Identical number of bands was observed in zymograms of alkaline proteases of fish fed both diets. No alterations in the histomorphology of PC, AI, PI or DI were noticed in fish fed the VO diets, while in the liver a tendency towards increased hepatocyte vacuolization due to lipid accumulation was observed. Overall, and with the exception of a higher intestine alkaline phosphatase activity, 70% FO replacement by a VO blend in diets for European sea bass resulted in no distinctive alterations on the postprandial pattern of digestive enzyme activities and intestine histomorphology. PMID:26364216

  8. Comparison of time-restricted and ad libitum self-feeding on the growth, feeding behavior and daily digestive enzyme profiles of Atlantic salmon

    NASA Astrophysics Data System (ADS)

    Shi, Ce; Liu, Ying; Yi, Mengmeng; Zheng, Jimeng; Tian, Huiqin; Du, Yishuai; Li, Xian; Sun, Guoxiang

    2016-07-01

    Although it has been hypothesized that a predictable feeding regime in animals allows physiological variables to be adjusted to maximize nutrient utilization and, hence, better growth performance, the assumption has rarely been tested. This study compares the Effects of time-restricted versus free access self-feeding on the growth, feeding behavior and daily digestive enzyme rhythms of Atlantic salmon (Salmo salar). In an experiment that lasted 6 weeks, fish (109.9 g) were divided into two groups: group 1 had free access to a self-feeder (FA); group 2 received three meals per day (2 h per meal) at dawn, midday and dusk via a time-restricted self-feeder (TR). At the end of the experiment, the fish were sampled every 3 h over a 24-h period. The results showed that the TR fish quickly synchronized their feeding behavior to the feeding window and their blood glucose showed a significant postprandial increase, while FA fish displayed no statistically significant rhythms (P<0.05). Pepsin activity of TR fish also showed a significant daily rhythm (P<0.05) with the acrophase at the second feed and a decrease over the next 12 h. Average daily trypsin, lipase and amylase levels of FA fish were significantly lower than those of TR fish (P<0.01); however, the growth performance of both groups was similar (P>0.05). In conclusion, the study failed to confirm a link between the entrainment of daily digestive enzyme profiles and growth performance, with the TR group showing comparatively poor blood glucose regulation.

  9. Inhibition of key digestive enzymes related to diabetes and hyperlipidemia and protection of liver-kidney functions by trigonelline in diabetic rats.

    PubMed

    Hamden, Khaled; Mnafgui, Kais; Amri, Zahra; Aloulou, Ahmed; Elfeki, Abdelfattah

    2013-03-01

    Diabetes is a serious health problem and a source of risk for numerous severe complications such as obesity and hypertension. Treatment of diabetes and its related diseases can be achieved by inhibiting key digestive enzymes related to starch and lipid digestion. The findings revealed that the administration of trigonelline to surviving diabetic rats helped to protect the pancreas β-cells from death and damage. Additionally, the supplement of trigonelline to surviving diabetic rats significantly decreased intestinal α-amylase and maltase by 36 and 52%, respectively, which led to a significant decrease in the blood glucose rate by 46%. Moreover, the administration of trigonelline to surviving diabetic rats potentially inhibited key enzymes of lipid metabolism and absorption such as lipase activity in the small intestine by 56%, which led to a notable decrease in serum triglyceride (TG) and total cholesterol (TC) rates and an increase in the HDL cholesterol level. This treatment also improved glucose, maltase, starch, and lipid oral tolerance. Trigonelline was also observed to protect the liver-kidney functions efficiently, which was evidenced by the significant decrease in the serum aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH) activities and creatinine, albumin, and urea rates. The histological analysis of the pancreas, liver, and kidney tissues further established the positive effect of trigonelline. Overall, the findings presented in this study demonstrate that the administration of trigonelline to diabetic rats can make it a potentially strong candidate for industrial application as a pharmacological agent for the treatment of hyperglycemia, hyperlipidemia, and liver-kidney dysfunctions. PMID:23641341

  10. Effect of zinc-bearing zeolite clinoptilolite on growth performance, nutrient retention, digestive enzyme activities, and intestinal function of broiler chickens.

    PubMed

    Tang, Zhigang; Wen, Chao; Li, Ping; Wang, Tian; Zhou, Yanmin

    2014-04-01

    This study was conducted to investigate the effect of zinc-bearing zeolite clinoptilolite (Zn-ZCP) on performance, growth performance, nutrient retention, digestive enzyme activities, and intestinal function in broiler chickens. A total of 180 1-day-old Arbor Acres chickens were randomly divided into three groups with six replicates of ten birds for a 21-day feeding period. Birds were fed a basal corn-soybean meal diet (29.1 mg of Zn per kilogram of diet) without supplemental zinc (control) or the same diet supplemented with 80 mg/kg zinc from ZnSO4 or Zn-ZCP. Zn-ZCP and ZnSO4 treatments had lower feed: gain ratio than that of control group (P < 0.05). Addition of Zn-ZCP increased (P < 0.05) the apparent retention of organic matter and ether extract during 14-17 days, and increased (P < 0.05) pancreatic lipase activity at 14 and 21 days as well as amylase activity at 21 days. Addition of Zn-ZCP increased the villus heights and villus height to crypt depth ratio at the duodenal (P < 0.05) and jejunal (P < 0.05) of broilers at 14 days. Broilers fed the diet supplemented with 80 mg/kg Zn from Zn-ZCP had higher villus heights and villus height to crypt depth ratio of duodenum (P < 0.05) and jejunum (P < 0.05) than those fed with control diet on day 21. Zn-ZCP treatment increased (P < 0.05) IgG and sIgA concentrations in jejunum at 21 days. The results indicated that Zn-ZCP supplementation which might have modified the release of Zn further down in the intestinal tract with the controlled-release characteristic, modulated digestive enzyme activities and intestinal structure and function, increased nutrient retention, and improved feed efficiency. PMID:24515449

  11. Chymotrypsin selectively digests β-lactoglobulin in whey protein isolate away from enzyme optimal conditions: potential for native α-lactalbumin purification.

    PubMed

    Lisak, Katarina; Toro-Sierra, Jose; Kulozik, Ulrich; Božanić, Rajka; Cheison, Seronei Chelulei

    2013-02-01

    The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7.0, 7.8 and 8.5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0.1%, 0.5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman-Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8.5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8.5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman-Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations. PMID:23317562

  12. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  13. Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes.

    PubMed

    Geurink, Paul P; van Tol, Bianca D M; van Dalen, Duco; Brundel, Paul J G; Mevissen, Tycho E T; Pruneda, Jonathan N; Elliott, Paul R; van Tilburg, Gabriëlle B A; Komander, David; Ovaa, Huib

    2016-05-01

    Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N'-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner. PMID:26996281

  14. Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

    PubMed Central

    Geurink, Paul P.; van Tol, Bianca D.M.; van Dalen, Duco; Brundel, Paul J.G.; Mevissen, Tycho E.T.; Pruneda, Jonathan N.; Elliott, Paul R.; van Tilburg, Gabriëlle B.A.; Komander, David; Ovaa, Huib

    2016-01-01

    Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N'-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner. PMID:26996281

  15. Optimization of the thermophilic anaerobic co-digestion of pig manure, agriculture waste and inorganic additive through specific methanogenic activity.

    PubMed

    Jiménez, J; Cisneros-Ortiz, M E; Guardia-Puebla, Y; Morgan-Sagastume, J M; Noyola, A

    2014-01-01

    The anaerobic co-digestion of three wastes (manure, rice straw and clay residue, an inorganic additive) at different concentration levels and their interactive effects on methanogenic activity were investigated in this work at thermophilic conditions in order to enhance hydrolytic activity and methane production. A central composite design and the response surface methodology were applied for the optimization of specific methanogenic activity (SMA) by assessing their interaction effects with a reduced number of experiments. The results showed a significant interaction among the wastes on the SMA and confirmed that co-digestion enhances methane production. Rice straw apparently did not supply a significant amount of substrate to make a difference in SMA or methane yield. On the other hand, clay residue had a positive effect as an inorganic additive for stimulating the anaerobic process, based on its mineral content and its adsorbent properties for ammonia. Finally, the optimal conditions for achieving a thermophilic SMA value close to 1.4 g CH4-COD/g VSS · d(-1) were 20.3 gVSS/L of manure, 9.8 gVSS/L of rice straw and 3.3 gTSS/L of clay. PMID:24959998

  16. Increased loading rates and specific methane yields facilitated by digesting grass silage at thermophilic rather than mesophilic temperatures.

    PubMed

    Voelklein, M A; Rusmanis, D; Murphy, J D

    2016-09-01

    This study was conducted to advance the understanding of thermophilic grass digestion. Late harvested grass silage was fermented at thermophilic conditions at increasing organic loading rates (OLR). Stable digestion took place at an OLR between 3 and 4gVSL(-1)d(-1). This enabled specific methane yields (SMY) as high as 405LCH4kgVS(-1). An accumulation of volatile fatty acids (VFA), accompanied by a gradual deterioration of pH, FOS/TAC (ratio of VFA to alkalinity) arose at an OLR between 5 and 7gVSL(-1)d(-1), yet inhibition did not occur. SMY decreased with reduced retention time ranging between 336 and 358LCH4kgVS(-1) at OLR 7 and 5gVSL(-1)d(-1) respectively. The biomethane efficiencies remained high (92-103%) at corresponding retention times. Comparative results indicated a superior performance with respect to higher loading and SMY as compared with mesophilic conditions. PMID:27268433

  17. Tissue specific response of Miscanthus×giganteus to dilute acid pretreatment for enhancing cellulose digestibility.

    PubMed

    Ji, Zhe; Zhang, Xun; Ling, Zhe; Sun, Run-Cang; Xu, Feng

    2016-12-10

    The recalcitrance in grasses varies according to cell type and tissue. In this study, dilute acid pretreatment was performed on Miscanthus×giganteus internodes that include rind and pith regions which showing heterogeneous structural and chemical changes. Pretreatment on pith effectively hydrolyzed 73.33% hemicelluloses and separated cohesive cell walls from the compound middle lamella due to lignin migration. Lignin droplets with an average diameter of 49.5±29.3nm were concurrently coalesced on wall surface, that in turn exposed more microfibrils deep in walls to be enzymatically hydrolyzed reaching 82.55%. By contrast, the rind with a relatively intergrated cell structure was covered by larger lignin droplets (101.2±44.1nm) and filled with inaccessible microfibrils limiting enzymatic sacchrification (31.50%). Taken together, the cellulose digestibility of biomass was not majorly influenced by cellulose crystallinity, while it was strongly correlated with the positive effects of hemicelluloses degradation, lignin redistribution, cellulose exposure and loosening cell wall structure. PMID:27577916

  18. Frequencies and Specificities of “Enzyme-Only” Detected Erythrocyte Alloantibodies in Patients Hospitalized in Austria: Is an Enzyme Test Required for Routine Red Blood Cell Antibody Screening?

    PubMed Central

    Habres, Claudia; Wallner, Franz; Mayr, Barbara; Halwachs-Baumann, Gabriele

    2014-01-01

    The aim of this study was to determine the frequencies and specificities of “enzyme-only” detected red blood cell (RBC) alloantibodies in the routine antibody screening and antibody identification in patients hospitalized in Austria. Routine blood samples of 2420 patients were investigated. The antibody screening was performed with a 3-cell panel in the low-ionic strength saline- (LISS-) indirect antiglobulin test (IAT) and with an enzyme-pretreated (papain) 3-cell panel fully automated on the ORTHO AutoVue Innova System. The antibody identification was carried out manually with an 11-cell panel in the LISS-IAT and with an enzyme-pretreated (papain) 11-cell panel. In total 4.05% (n = 98) of all patients (n = 2420) had a positive RBC antibody screening result. Of them 25.51% (25/98) showed “enzyme-only” detected specific or nonspecific RBC alloantibodies. Rhesus and Lewis system antibodies were found the only specificities of “enzyme-only” RBC alloantibodies: all in all 4.8% (4/98) were detected with anti-E, 3.06% (3/98) with anti-Lea, 3.06% (3/98) with anti-D after anti-D prophylaxis and 1.02% (1/98) with anti-e. In total, 14.29% (14/98) showed a nonspecific RBC alloantibody result with the enzyme test. The results of the present study demonstrate that a high number of unwanted positive reactions with the enzyme technique overshadows the detection of “enzyme-only” RBC alloantibodies. (Trial Registration: K-37-13). PMID:24790773

  19. [Detection of IgE specific for egg yolk by enzyme immunoassay. Description of a case].

    PubMed

    Carrillo Díaz, T; Cuevas Agustín, M; Moneo Goiri, I; Ibáñez Sandín, M D; Ureña Vilardell, V

    1986-01-01

    Food allergy is a common disease in our country, especially affecting atopic children. Egg-white hypersensitivity is frequently found in these patients. However, egg-yolk hypersensitivity is not usually reported in patients with egg allergy. This article describes a young patient with egg-yolk hypersensitivity, a 12 year old female patient with a medical history of contact urticaria, angioedema and severe acute bronchospasm shortly after the intake of small amounts of egg-yolk. All these episodes required treatment in emergency care units because of the severity of the symptoms. The patient did not describe any other food hypersensitivity and remained symptom-free after the intake of boiled or fried egg-white. She had clinical symptoms of grass pollen hypersensitivity and was therefore on specific immunotherapy at the time of the study. The skin prick-tests were positive to grass pollen and egg-yolk and were negative to mites, moulds, animal dander and to the common food tested (milk, fish, peanut, almond and hazel-nut). Total serum IgE was 1.160 UL/ml. The patient had a positive RAST to egg-white (0.0 PRU/ml) as well as to egg-yolk (8.6 PRU/ml). Furthermore, an indirect enzyme immunoassay as well as a reverse enzyme immunoassay also revealed the presence of specific IgE antibodies. The reverse enzyme immunoassay uses microtiter plates as a solid surface. These plates are coated with a monospecific antihuman IgE antibody. Thereafter, the serum samples are incubated overnight in the wells. After several washings, the presence of specific antibodies is revealed by means of a peroxidase conjugated allergen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3515886

  20. An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine.

    PubMed

    Kimura, Takehide; Kuwata, Hideyuki; Miyauchi, Kazuhito; Katayama, Yuki; Kayahara, Norihiko; Sugiuchi, Hiroyuki; Matsushima, Kazumi; Kondo, Yuki; Ishitsuka, Yoichi; Irikura, Mitsuru; Irie, Tetsumi

    2016-04-01

    Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice. PMID:26792376

  1. Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

    SciTech Connect

    Agarwal,R.; Burley, S.; Swaminathan, S.

    2008-01-01

    Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-{beta} and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93 angstrom resolution. The polypeptide chain adopts the typical a{beta}a PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.

  2. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. PMID:27174700

  3. Morphallactic regeneration as revealed by region-specific gene expression in the digestive tract of Enchytraeus japonensis (Oligochaeta, Annelida).

    PubMed

    Takeo, Makoto; Yoshida-Noro, Chikako; Tochinai, Shin

    2008-05-01

    Enchytraeus japonensis is a small oligochaete, which primarily reproduces asexually by fragmentation and regeneration. For precise analysis of the pattern formation during regeneration, we isolated three region-specific genes (EjTuba, mino, and horu) expressed in the digestive tract. In growing worms, the expression of EjTuba in the head and mino in the trunk region just posterior to the head were observed in defined body segments, while the expression areas of EjTuba in the trunk and horu were proportional to the total number of body segments. In the regeneration process, expression of these genes disappeared once and recovered to their original pattern by day 7. In abnormal regeneration such as a bipolar head, mino was still expressed in the region next to both the normal and the ectopic heads. These results suggest that there is morphallactic as well as epimorphic or inductive regulation of the body patterning during regeneration of E. japonensis. PMID:18393309

  4. Hypotensive effects and angiotensin-converting enzyme inhibitory peptides of reishi (Ganoderma lingzhi) auto-digested extract.

    PubMed

    Tran, Hai-Bang; Yamamoto, Atsushi; Matsumoto, Sayaka; Ito, Hisatomi; Igami, Kentaro; Miyazaki, Toshitsugu; Kondo, Ryuichiro; Shimizu, Kuniyoshi

    2014-01-01

    Reishi (Ganoderma lingzhi) has been used as a traditional medicine for millennia. However, relatively little is known about this mushroom's proteins and their bioactivities. In this study, we used reishi's own proteases to hydrolyze its protein and obtained auto-digested reishi (ADR) extract. The extract was subjected to in vitro assays and administered to spontaneous hypertensive rats (SHRs) to determine its potential for use as a hypotensive medication. Bioassay-guided fractionation and de novo sequencing were used for identifying the active compounds. After 4 h administration of ADR, the systolic pressure of SHRs significantly decreased to 34.3 mmHg (19.5% change) and the effect was maintained up to 8 h of administration, with the decrease reaching as low as 26.8 mmHg (15% reduction-compare to base line a decrease of 26.8 mmHg is less than a decrease of 34.3 mmHg so it should give a smaller % reduction). Eleven peptides were identified and four of them showed potent inhibition against ACE with IC50 values ranging from 73.1 μM to 162.7 μM. The results showed that ADR could be a good source of hypotensive peptides that could be used for antihypertensive medication or incorporation into functional foods. PMID:25178067

  5. A novel hatching enzyme from starfish Asterias amurensis: purification, characterization, and cleavage specificity.

    PubMed

    Li, Zhi Jiang; Kim, Sang Moo

    2013-02-01

    Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9 kDa of molecular weight were 449.62 U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH 8.0 and 30 °C, respectively. This enzyme was relatively stable in the range of pH 4.0-6.0 and 30-40 °C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosyl-phenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn(2+) ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca(2+), Mg(2+), and Cu(2+). Based on the results above, the starfish HE was classified as a zinc metallo- and trypsin-like serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31 mg/ml, 0.17 U/ml, and 122.70 s(-1), respectively, whereas 1.09 mg/ml, 0.12 U/ml, and 771.98 s(-1) on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen. PMID:23306897

  6. Sinapis phylogeny and evolution of glucosinolates and specific nitrile degrading enzymes.

    PubMed

    Agerbirk, Niels; Warwick, Suzanne I; Hansen, Paul R; Olsen, Carl E

    2008-12-01

    Levels of sinalbin (4-hydroxybenzylglucosinolate) and 28 other glucosinolates were determined in leaves and roots of 20 species that were either phylogenetically close to Sinapis alba, Sinapis arvensis, or Sinapis pubescens (tribe Brassiceae, Brassicaceae), or were expected to contain arylalkyl nitrilase activity. Comparison with a molecular phylogenetic tree based on ITS DNA sequences identified two separate occurrences of sinalbin. The first in a group of species related to S. alba (including members of the genera Coincya and Kremeriella); and the second in S. arvensis, nested among sinalbin deficient species. Significant 4-hydroxyphenylacetonitrile degrading enzyme activity was found in both S. alba and S. arvensis, but in S. alba the major product was the corresponding carboxylic acid, while in S. arvensis the major product was the amide. Both investigated enzyme activities, nitrilase and nitrile hydratase, were specific, accepting only certain arylacetonitriles such as 4-hydroxy and 4-methoxyphenylacetonitrile. Only the S. alba enzyme required an oxygen in para position of the substrate, as found in sinalbin. Indole-3-acetonitrile, arylcyanides, and arylpropionitriles were poor substrates. The nitrilase activity of S. alba was quantitatively comparable to that reported in the monocot Sorghum bicolor (believed to be involved in cyanogenic glycoside metabolism). Glucosinolates derived from methionine were found in all Sinapis clades. Glucosinolate patterns suggested a complex evolution of glucosinolates in the investigated species, with several apparent examples of abrupt changes in glucosinolate profiles including chain length variation and appearance of glucosinolates derived from branched-chain amino acids. NMR data for desulfated homosinalbin, 9-methylsulphonylnonylglucosinolate, 3-methylpentylglucosinolate and related glucosinolates are reported, and a facultative connection between sinalbin and specific nitrilases is suggested. PMID:18995873

  7. Optimization of ultrasound-assisted extraction of pectinase enzyme from guava (Psidium guajava) peel: Enzyme recovery, specific activity, temperature, and storage stability.

    PubMed

    Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Islam Sarker, Zaidul

    2016-01-01

    This study aimed to investigate the effects of the ultrasound-assisted extraction conditions on the yield, specific activity, temperature, and storage stability of the pectinase enzyme from guava peel. The ultrasound variables studied were sonication time (10-30 min), ultrasound temperature (30-50 °C), pH (2.0-8.0), and solvent-to-sample ratio (2:1 mL/g to 6:1 mL/g). The main goal was to optimize the ultrasound-assisted extraction conditions to maximize the recovery of pectinase from guava peel with the most desirable enzyme-specific activity and stability. Under the optimum conditions, a high yield (96.2%), good specific activity (18.2 U/mg), temperature stability (88.3%), and storage stability (90.3%) of the extracted enzyme were achieved. The optimal conditions were 20 min sonication time, 40 °C temperature, at pH 5.0, using a 4:1 mL/g solvent-to-sample ratio. The study demonstrated that optimization of ultrasound-assisted process conditions for the enzyme extraction could improve the enzymatic characteristics and yield of the enzyme. PMID:25844554

  8. Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

    PubMed

    Pati, Sarah G; Kohler, Hans-Peter E; Pabis, Anna; Paneth, Piotr; Parales, Rebecca E; Hofstetter, Thomas B

    2016-07-01

    Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants. PMID:26895026

  9. Dietary soya beans and kidney beans stimulate secretion of cholecystokinin and pancreatic digestive enzymes in 400-day-old Hooded-Lister rats but only soya beans induce growth of the pancreas.

    PubMed

    Grant, G; Alonso, R; Edwards, J E; Murray, S

    2000-04-01

    The effects of age on cholecystokinin (CCK) release, pancreatic enzyme secretion, and growth of the pancreas mediated by dietary kidney beans or soya beans were evaluated in trials with 30-, 90-, 250-, and 400-day-old rats. Soya beans increased blood CCK and caused hypersecretion of digestive enzymes and rapid pancreatic growth in all rats. Kidney beans also elevated circulating CCK and stimulated enzyme secretion. However, with 90-, 250-, and 400-day-old rats, the secretory responses were attenuated. Furthermore, kidney beans did not induce pancreatic growth in 250- and 400-day-old rats. PMID:10766458

  10. Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD

    NASA Astrophysics Data System (ADS)

    Ni, Wenqin; Lin, Melanie; Salinas, Paul; Savickas, Philip; Wu, Shiaw-Lin; Karger, Barry L.

    2013-01-01

    Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS3 (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70 %). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

  11. Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

    PubMed

    Dittrich, P

    1976-02-01

    NAD-specific "malic" enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO(4) (2-), suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn(2+), which cannot be replaced by NADP or Mg(2+). For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn(2+), and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 mumole/hr.mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO(2) for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  12. Responses of non-starch polysaccharide-degrading enzymes on digestibility and performance of growing pigs fed a diet based on corn, soya bean meal and Chinese double-low rapeseed meal.

    PubMed

    Fang, Z F; Peng, J; Liu, Z L; Liu, Y G

    2007-08-01

    This study was conducted to investigate the effect of two distinct enzyme preparations on nutrients' digestibility and growth performance of growing pigs fed diets based on corn, soya bean meal and Chinese double-low rapeseed meal (DLRM). The two enzyme preparations were Enzyme R, a preparation extracted from fermentation of a non-GMO fungus Penicillum funiculosum, developed for multi-grain and multi-animal species; and Enzyme P, a xylanase preparation from Trichoderma longibrachiatum, for pigs fed corn-based diets only. Both enzymes were tested at 0, 0.25 and 0.50 g/kg feed using 70 crossbred male pigs (Large Yorkshire x Landrace) in five dietary treatments and seven replicates in each treatment, for growth period from 27 to 68 kg live weight in 49 days. Results showed that the supplementation of both enzymes (1) increased total-tract digestibility of dietary energy from 77.5% (control) to 81.4% (Enzyme R, p < 0.05) and 81.9% (Enzyme P, p < 0.05); of neutral detergent fibre from 41.0% (control) to 57.8% (Enzyme R, p < 0.05) and 60.0% (Enzyme P, p < 0.05); (2) improved average daily gain from 786 g (control) to 829 g (Enzyme R, p < 0.05) and 846 g (Enzyme P, p < 0.05); and numerical increases in feed intake from 1.96 kg/pig/day (control) to 2.01 (Enzyme R) and 2.00 (p > 0.05) and feed conversion ratio from 2.50 (control) to 2.42 (Enzyme R) and 2.36 (Enzyme P, p < 0.05); (3) there was a dose response but no significant differences were observed in enzyme efficacy between the two enzyme preparations. The present study demonstrated beneficial effects of applying xylanase-based enzymes to improve feeding values of pig diets based on corn, soya bean meal and DLRM. PMID:17615009

  13. Long-term effects of oral tea polyphenols and Lactobacillus brevis M8 on biochemical parameters, digestive enzymes, and cytokines expression in broilers

    PubMed Central

    Li, Hua-li; Li, Zong-jun; Wei, Zhong-shan; Liu, Ting; Zou, Xiao-zuo; Liao, Yong; Luo, Yu

    2015-01-01

    This study investigates the long-term effects of oral tea polyphenols (TPs) and Lactobacillus brevis M8 (LB) on biochemical parameters, digestive enzymes, and cytokines expression in broilers. In experiment 1, 240 broiler chickens were selected to investigate the effects of 0.06 g/kg body weight (BW) TP and 1.0 ml/kg BW LB on broilers; in experiment 2, 180 broiler chickens were assigned randomly to three groups to investigate the effects of different dosages of TP (0.03, 0.06, and 0.09 g/kg BW) combined with 1.0 ml/kg BW LB on broilers; in experiment 3, 180 broiler chickens were assigned randomly to three groups to investigate the effects of different dosages of LB (0.5, 1.0, and 1.5 ml/kg BW) combined with 0.06 g/kg BW TP on broilers. The results showed that TP and LB affected serum biochemical parameters, and TP reduced serum cholesterol (CHO) and low-density lipoprotein cholesterol (LDL-C) abundances in a dosage-dependent manner (P<0.05) on Day 84. Meanwhile, broilers fed a diet supplemented with TP or LB had a lower intestinal lipase activity on Day 84 compared with the control group (P<0.05). Middle and high dosages of TP increased pancreatic lipase and proventriculus pepsin activities (P<0.05). Also middle and high dosages of LB significantly enhanced pancreatic lipase activity (P<0.05), while high LB supplementation inhibited intestinal trypsase (P<0.05) on Day 84. Furthermore, both TP and LB reduced intestinal cytokine expression and nuclear factor-κ B (NF-κB) mRNA level on Days 56 and 84. In conclusion, long-term treatment of TP and LB improved lipid metabolism and digestive enzymes activities, and affected intestinal inflammatory status, which may be associated with the NF-κB signal. PMID:26642185

  14. A Condensing Enzyme from the Seeds of Lesquerella fendleri That Specifically Elongates Hydroxy Fatty Acids1

    PubMed Central

    Moon, Hangsik; Smith, Mark A.; Kunst, Ljerka

    2001-01-01

    Lesquerella fendleri seed oil contains up to 60% hydroxy fatty acids, nearly all of which is the 20-carbon hydroxy fatty acid lesquerolic acid (d-14-hydroxyeicos-cis-11-enoic acid). Previous work suggested that lesquerolic acid in L. fendleri was formed by the elongation of the 18-carbon hydroxy fatty acid, ricinoleic acid. To identify a gene encoding the enzyme involved in hydroxy fatty acid elongation, an L. fendleri genomic DNA library was screened using the coding region of the Arabidopsis Fatty Acid Elongation1 gene as a probe. A gene, LfKCS3, with a high sequence similarity to known very long-chain fatty acid condensing enzymes, was isolated. LfKCS3 has a 2,062-bp open reading frame interrupted by two introns, which encodes a polypeptide of 496 amino acids. LfKCS3 transcripts accumulated only in the embryos of L. fendleri and first appeared in the early stages of development. Fusion of the LfKCS3 promoter to the uidA reporter gene and expression in transgenic Arabidopsis resulted in a high level of β-glucuronidase activity exclusively in developing embryos. Seeds of Arabidopsis plants transformed with LfKCS3 showed no change in their very long-chain fatty acid content. However, when these Arabidopsis plants were crossed with the transgenic plants expressing the castor oleate 12-hydroxylase, significant amounts of 20-carbon hydroxy fatty acids accumulated in the seed, indicating that the LfKCS3 condensing enzyme specifically catalyzes elongation of 18-carbon hydroxy fatty acids. PMID:11743108

  15. Altered substrate specificity of the Pterygoplichthys sp. (Loricariidae) CYP1A enzyme.

    PubMed

    Parente, Thiago E M; Urban, Philippe; Pompon, Denis; Rebelo, Mauro F

    2014-09-01

    Ethoxyresorufin is a classical substrate for vertebrate CYP1A enzymes. In Pterygoplichthys sp. (Loricariidae) this enzyme possesses 48 amino acids substitutions compared to CYP1A sequences from other vertebrate species. These substitutions or a certain subset substitution are responsible for the non-detection of the EROD reaction in this species liver microsomes. In the present study, we investigated the catalytic activity of Pterygoplichthys sp. CYP1A toward 15 potential substrates in order to understand the substrate preferences of this modified CYP1A. The fish gene was expressed in yeast and the accumulation of the protein was confirmed by both the characteristic P450-CO absorbance spectra and by detection with monoclonal antibodies. Catalytic activities were assayed with yeast microsomes and four resorufin ethers, six coumarin derivates, three flavones, resveratrol and ethoxyfluoresceinethylester. Results demonstrated that the initial velocity pattern of this enzyme for the resorufin derivatives is different from the one described for most vertebrate CYP1As. The initial velocity for the activity with the coumarin derivatives is several orders of magnitude higher than with the resorufins, i.e. the turnover number (kcat) for ECOD is 400× higher than for EROD. Nonetheless, the specificity constant (kcat/km) for EROD is only slightly higher than for ECOD. EFEE is degraded at a rate comparable to the resorufins. Pterygoplichthys sp. CYP1A also degrades 7-methoxyflavone and β-naphthoflavone but not resveratrol and chrysin. These results indicate a divergent substrate preference for Pterygoplichthys sp. CYP1A, which may be involved in the adaptation of Loricariidae fish to their particular environment and feeding habits. PMID:24911589

  16. Digestive Diseases

    MedlinePlus

    ... cells and provide energy. This process is called digestion. Your digestive system is a series of hollow ... are also involved. They produce juices to help digestion. There are many types of digestive disorders. The ...

  17. Differences in the Glucuronidation of Resveratrol and Pterostilbene: Altered Enzyme Specificity and Potential Gender Differences

    PubMed Central

    Dellinger, Ryan W.; Gomez Garcia, Angela M.; Meyskens, Frank L.

    2015-01-01

    Summary Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans. PMID:23965644

  18. An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in th nematode Caenorhabditis elegans.

    PubMed Central

    Zhen, M; Schein, J E; Baillie, D L; Candido, E P

    1996-01-01

    The ubc-2 gene in Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme (E2) homologous to yeast UBC4 and UBC5. UBC4 and UBC5 are individually dispensable class I E2 enzymes involved in the degradation of short-lived and abnormal proteins. Transgenic analysis using ubc-2-lacZ fusions and in situ immunofluorescence indicate that ubc-2 is abundantly expressed in most tissues of embryos and early larvae, but becomes specific to the nervous system in L4 larvae and adults. This suggests that the functions of this type of E2 are developmentally regulated in C.elegans. This hypothesis is supported by antisense analysis, which shows that blocking the expression of ubc-2 has a more severe effect in early developmental stages than in later stages. Through complementation of previously identified essential genes in the vicinity of ubc-2, we demonstrate that ubc-2 corresponds to let-70, a gene essential for C.elegans larval development. One let-70(ubc-2) allele contains a His75-->Tyr substitution, while another has an altered splice donor site. Images PMID:8670823

  19. Inhibition of carbohydrate and lipid digestive enzymes activities by Zygophyllum album extracts: effect on blood and pancreas inflammatory biomarkers in alloxan-induced diabetic rats.

    PubMed

    Mnafgui, Kais; Kchaou, Mouna; Hamden, Khaled; Derbali, Fatma; Slama, Sadok; Nasri, Mbarek; Salah, Hichem Ben; Allouche, Noureddine; Elfeki, Abdelfattah

    2014-03-01

    Zygophyllum album has been used as herbal medicine in Southern Tunisia to treat several diseases such as diabetes mellitus. This study is aimed to reveal the mechanisms underlying the antihyperglycemic potential, the anti-inflammatory and the protective hematological proprieties of this plant in diabetic rats. The inhibition of the α-amylase activity by different solvent-extract fractions of Z. album was tested in vitro. The fraction endowed with the powerful inhibitory activity against α-amylase was administered to surviving diabetic rats for 30 days. Data from in vitro indicated that each extract from the medicinal plant showed moderate inhibition of α-amylase enzyme except the ethyl acetate extract which was ineffective. The powerful inhibition was achieved by ethanol extract of Z. album (EZA) with an IC50 of 43.48 μg/ml as compared to acarbose (Acar) with an IC50 of 14.88 μg/ml. In vivo, the results showed that EZA decreased the α-amylase levels in serum, pancreas and intestine of diabetic rats by 40 %, 45 % and 46 %, respectively, associated with considerably reduction in blood glucose rate by 61 %. Moreover, the EZA helped to protect the structure and function of the β-cells. Interestingly, EZA had a potent anti-inflammatory effect which is manifested by decreases in CRP and TNF-α levels. Overall, a notable reduction in lipase activity both in serum and small intestine of treated diabetic rats resulted in the improvement of serum and liver lipids profile. Z. album showed a prominent antidiabetic effect via inhibition of carbohydrate and lipid digestive enzymes and ameliorated the inflammation and the disturbance of hematological biomarkers in diabetes. PMID:23996134

  20. A sensitive and specific two-site enzyme-immunoassay for human calcitonin using monoclonal antibodies.

    PubMed

    Seth, R; Motté, P; Kehely, A; Wimalawansa, S J; Self, C H; Bellet, D; Bohuon, C; MacIntyre, I

    1988-11-01

    A highly sensitive, specific and rapid two-site enzyme-immunometric assay (EIA) for the measurement of immunoreactive (ir) human calcitonin (hCT) in human plasma was developed using high-affinity monoclonal antibodies. The assay was validated in terms of sensitivity, specificity and reproducibility and its performance compared with that of a radioimmunoassay (RIA) employing a polyclonal antiserum. The sensitivity of the overnight EIA (2 pmol/l) was comparable with the long-incubation (7 days) RIA. The overnight RIA had a sensitivity of 10 pmol/l. The inter- and intra-assay variations of the EIA were less than 12%. Some related and non-related peptides were compared with synthetic hCT for cross-reactivity in the assay and were found to be negative. The mean recovery of added synthetic hCT from plasma of normal volunteers was 96%. Both RIA and EIA have been applied to the measurement of ir-hCT in normal volunteers and in patients with medullary carcinoma of the thyroid. In both groups, the level of ir-hCT measured by EIA was found to be lower than that measured by RIA, presumably due to the ability of the more specific EIA to detect only the 'mature' form of the hormone. EIA offers an attractive alternative to the more cumbersome and lengthy RIA in current usage, with the added advantage of employing a non-isotopic label. PMID:3058855

  1. Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

    PubMed

    C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

    2016-03-23

    Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality. PMID:26947758

  2. Effect of enzyme supplementation of a rye-based diet on xylanase activity in the small intestine of broilers, on intestinal crypt cell proliferation and on nutrient digestibility and growth performance of the birds.

    PubMed

    Silva, S S P; Smithard, R R

    2002-05-01

    1. A study was undertaken to investigate the susceptibility to peptic digestion of exogenous xylanase (EC 3.2.1.8) from Trichoderma longibrachiatum, added to a rye-based diet for broiler chickens, in order to elucidate its possible site of action. 2. It was also designed to investigate the effects of the enzyme (plus exogenous protease EC 3.4-24.28) when added to a rye-containing diet (60% rye/kg diet) on crypt cell proliferation in the mucosa of the small intestine, on short chain fatty acid (SCFA) concentrations in the small intestine digesta and in portal blood and on nutrient digestibilities. 3. In Experiment 1, the enzymes were added at activities 10x and 30x those recommended in commercial practice, but in Experiment 2 the activities were the recommended levels. 4. A significant proportion (estimated to be 15 to 20%) of the xylanase added at the higher concentration (15,000 and 45,000 units/kg diet) remained active in the small intestine of the growing chicken. 5. The crypt cell proliferation rate in birds fed on the control diet (45 cells/2 h) was significantly higher than in birds fed on the diets supplemented with enzyme at the higher level (29 and 33 cells/ 2 h), but there was no significant effect on SCFA. In birds fed on the diet supplemented with enzyme at the commercial level there was no clear-cut effect on crypt cell proliferation but exogenous xylanase could be detected in the small intestine. Intestinal fluid viscosity was reduced and growth performance of the birds was improved by the supplementation with exogenous enzymes. 6. Part of the improvement in growth performance could be ascribed to a 25% increase in the digestibility of nitrogen and a doubling of the digestibility of fat. PMID:12047093

  3. Changes in expression of an antimicrobial peptide, digestive enzymes, and nutrient transporters in the intestine of E. praecox-infected chickens.

    PubMed

    Yin, H; Sumners, L H; Dalloul, R A; Miska, K B; Fetterer, R H; Jenkins, M C; Zhu, Q; Wong, E A

    2015-07-01

    Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters, and an antimicrobial peptide following an Eimeria praecox challenge of chickens at days 3 and 6 post-infection. Gene expression was determined by real-time PCR and analyzed by one-way ANOVA. In the duodenum, the primary site of E. praecox infection, a number of genes were downregulated at both d3 and d6 post-infection. These genes included liver expressed antimicrobial peptide 2 (LEAP2), the cationic (CAT1), anionic (EAAT3), and L-type (LAT1) amino acid transporters, the peptide transporter PepT1 and the zinc transporter ZnT1. Other transporters were downregulated either at d3 or d6. At both d3 and d6, there was downregulation of B(o)AT and CAT1 in the jejunum and downregulation of LEAP2 and LAT1 in the ileum. LEAP2, EAAT3, and ZnT1 have been found to be downregulated following challenge with other Eimeria species, suggesting a common cellular response to Eimeria. PMID:26015586

  4. Deficiency of maize starch-branching enzyme i results in altered starch fine structure, decreased digestibility and reduced coleoptile growth during germination

    PubMed Central

    2011-01-01

    Background Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, sbe1a, had been previously observed. To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line. Results Endosperm starch granules from the sbe1a mutant were more resistant to digestion by pancreatic α-amylase, and the sbe1a mutant starch had an altered branching pattern for amylopectin and amylose. When kernels were germinated, the sbe1a mutant was associated with shorter coleoptile length and higher residual starch content, suggesting that less efficient starch utilization may have impaired growth during germination. Conclusions The present report documents for the first time a molecular phenotype due to the absence of SBEI, and suggests strongly that it is associated with altered physiological function of the starch in vivo. We believe that these results provide a plausible rationale for the conservation of SBEI in plants in both monocots and dicots, as greater seedling vigor would provide an important survival advantage when resources are limited. PMID:21599988

  5. A Comparison Effect of Copper Nanoparticles versus Copper Sulphate on Juvenile Epinephelus coioides: Growth Parameters, Digestive Enzymes, Body Composition, and Histology as Biomarkers

    PubMed Central

    Wang, Tao; Long, Xiaohua; Cheng, Yongzhou; Liu, Zhaopu; Yan, Shaohua

    2015-01-01

    Copper nanoparticles (Cu-NPs) are components in numerous commercial products, but little is known about their potential hazard in the marine environments. In this study the effects of Cu-NPs and soluble Cu on juvenile Epinephelus coioides were investigated. The fish were exposed in triplicate to control, 20 or 100 µg Cu L−1 as either copper sulphate (CuSO4) or Cu-NPs for 25 days. The growth performance decreased with increasing CuSO4 or Cu-NPs dose, more so in the CuSO4 than Cu-NPs treatment. Both forms of Cu exposure inhibited activities of digestive enzymes (protease, amylase, and lipase) found in liver, stomach, and intestine. With an increase in CuSO4 and Cu-NPs dose, crude protein and crude lipid decreased, but ash and moisture increased, more so in the CuSO4 than Cu-NPs treatment. The Cu-NPs treatment caused pathologies in liver and gills, and the kinds of pathologies were broadly of the same type as with CuSO4. With an increase in CuSO4 or Cu-NPs dose, the total polyunsaturated fatty acids decreased, but total monounsaturated fatty acids and total saturated fatty acids increased compared to control. Overall, these data showed that Cu-NPs had a similar type of toxic effects as CuSO4, but soluble Cu was more toxic than Cu-NPs. PMID:26527479

  6. Aspects of Protein Chemistry. Part I: Some Recent Insights Into Enzyme Specificity

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1976-01-01

    Describes some recent advances in enzyme structure and action, including a description of enzyme-substrate interaction. Discusses the methods for determination of amino acid sequences in proteins; the actions of chymotrypsin, trypsin, and elastase; and details of the enzyme-substrate complex derived from kinetic studies and x-ray diffraction…

  7. Influence of nutrient restriction and melatonin supplementation of pregnant ewes on maternal and fetal pancreatic digestive enzymes and insulin-containing clusters.

    PubMed

    Keomanivong, F E; Lemley, C O; Camacho, L E; Yunusova, R; Borowicz, P P; Caton, J S; Meyer, A M; Vonnahme, K A; Swanson, K C

    2016-03-01

    Primiparous ewes (n=32) were assigned to dietary treatments in a 2×2 factorial arrangement to determine effects of nutrient restriction and melatonin supplementation on maternal and fetal pancreatic weight, digestive enzyme activity, concentration of insulin-containing clusters and plasma insulin concentrations. Treatments consisted of nutrient intake with 60% (RES) or 100% (ADQ) of requirements and melatonin supplementation at 0 (CON) or 5 mg/day (MEL). Treatments began on day 50 of gestation and continued until day 130. On day 130, blood was collected under general anesthesia from the uterine artery, uterine vein, umbilical artery and umbilical vein for plasma insulin analysis. Ewes were then euthanized and the pancreas removed from the ewe and fetus, trimmed of mesentery and fat, weighed and snap-frozen until enzyme analysis. In addition, samples of pancreatic tissue were fixed in 10% formalin solution for histological examination including quantitative characterization of size and distribution of insulin-containing cell clusters. Nutrient restriction decreased (P⩽0.001) maternal pancreatic mass (g) and α-amylase activity (U/g, kU/pancreas, U/kg BW). Ewes supplemented with melatonin had increased pancreatic mass (P=0.03) and α-amylase content (kU/pancreas and U/kg BW). Melatonin supplementation decreased (P=0.002) maternal pancreatic insulin-positive tissue area (relative to section of tissue), and size of the largest insulin-containing cell cluster (P=0.04). Nutrient restriction decreased pancreatic insulin-positive tissue area (P=0.03) and percent of large (32 001 to 512 000 µm2) and giant (⩾512 001 µm2) insulin-containing cell clusters (P=0.04) in the fetus. Insulin concentrations in plasma from the uterine vein, umbilical artery and umbilical vein were greater (P⩽0.01) in animals receiving 100% requirements. When comparing ewes to fetuses, ewes had a greater percentage of medium insulin-containing cell clusters (2001 to 32 000 µm2) while fetuses

  8. [Value of prostate-specific antigen measurements with newly developed enzyme immunoassay (MARKIT-M PA)].

    PubMed

    Arai, Y; Onishi, H; Oishi, K; Takeuchi, H; Yoshida, O

    1992-10-01

    Serum prostate-specific antigen (PSA) levels in patients with prostate cancer and benign prostate hypertrophy (BPH) were investigated with a newly developed enzyme immunoassay (MARKIT-M PA, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). Sensitivity of the assay system is 0.5 ng/ml and the detection range is 0.5-100 ng/ml. There was a high linear correlation (r = 0.987) between the assay and MARKIT-F PA, and values obtained with the assay were almost equal to those yielded by MARKIT-F PA assay. Using the BPH group as a negative control, the upper cut-off value in BPH patients was determined to be 3.6 ng/ml. Of the 48 patients with untreated prostate cancer, 77% was detectable by means of MARKIT-M PA assay. Using the BPH group as a negative control, specificity and efficiency were 93% and 86%, respectively. In another group of 27 BPH patients whose blood samples were taken immediately after digital prostatic examination, PSA was elevated in 15%. During follow-up of prostate cancer patients, PSA was elevated in 82% at the time of clinically detectable progression. In 15 patients whose disease was clinically well controlled, all levels of PSA were observed to be negative. These findings suggests that detection of serum PSA with this assay is of great use both in the diagnosis and monitoring of prostate cancer patients. PMID:1282772

  9. Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

    PubMed

    Thomas, N E; Bramson, H N; Miller, W T; Kaiser, E T

    1987-07-14

    As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3663600

  10. Blocking c-myc and stat3 by E. coli expressed and enzyme digested siRNA in mouse melanoma

    SciTech Connect

    Hong Jie; Zhao Yingchun; Huang Weida . E-mail: whuang@fudan.edu.cn

    2006-09-22

    Tumour cells often show alteration in the signal-transduction pathways, leading to proliferation in response to external signals. Oncogene overexpression and constitutive expression is a common phenomenon in the development and progression of many human cancers. Therefore oncogenes provide potential targets for cancer therapy. RNA interference (RNAi), mediated by small interfering RNA (siRNA), silences genes with a high degree of specificity and potentially represents a general approach for molecularly targeted anti-cancer therapy. The data presented in this report evaluated the method of systemically administering combined esiRNAs to multiple targets as compared with the method of using a single kind of esiRNA to a single target. Our experimental data revealed that the mixed treatment of esiC-MYC and esiSTAT3 had a better inhibition effect than the single treatment of esiC-MYC or esiSTAT3 on mouse B16 melanoma.

  11. Effects of exogenous enzyme supplementation to corn- and soybean meal-based or complex diets on growth performance, nutrient digestibility, and blood metabolites in growing pigs.

    PubMed

    Jo, J K; Ingale, S L; Kim, J S; Kim, Y W; Kim, K H; Lohakare, J D; Lee, J H; Chae, B J

    2012-09-01

    Two experiments were conducted to determine the effects of dietary supplementation of exogenous enzymes on growth performance, apparent total tract digestibility (ATTD) of energy and nutrients, blood metabolites, fecal VFA, and fecal ammonia-N in growing pigs (Sus scrofa) fed a corn (Zea mays L.)- and soybean [Glycine max (L.) Merr.] meal (SBM)-based diet. In Exp. 1, 240 growing barrows (initial BW: 55.6 ± 0.9 kg) were randomly allotted to 5 treatments on the basis of BW. There were 4 replicates in each treatment with 12 pigs per replicate. The 5 treatments consisted of a corn-SBM-based control diet and 4 additional diets were similar to the control diet, with the exception that 0.05% β-mannanase (M), α-amylase + β-mannanase (AM), β-mannanase + protease (MPr), or α-amylase + β-mannanase + protease (AMP) was added to the diets, which were fed for 28 d. Pigs fed the AM, MPr, or AMP diet had greater (P < 0.05) ADG than pigs fed the control diet. Pigs fed the AMP diet also had greater (P < 0.05) ADG than pigs fed the M, AM, or MPr diet. Pigs fed the AMP diet had greater (P < 0.05) G:F than pigs fed the control diet. The G:F of the pigs fed the M, AM, or MPr diet were not different (P > 0.05) from the G:F in pigs fed the AMP or control diet. The ADFI, ATTD of nutrients, blood metabolites, and fecal VFA and ammonia-N concentrations were not different among treatments. In Exp. 2, 192 growing barrows (initial BW: 56.9 ± 1.0 kg) were allotted to 4 treatments. There were 4 replicates in each treatment with 12 pigs per replicate. Pigs were fed a corn-SBM-based diet (CSD) or a complex diet (CD) that contained corn, SBM, 3% rapeseed (Brassica napus L.) meal, 3% copra (Cocos nucifera L.) meal, and 3% palm (Elaeis guineensis Jacq.) kernel meal. Each diet was prepared without exogenous enzymes or with 0.05% AMP and all diets were fed for 28 d. The ADG and G:F of pigs fed the CSD were greater (P < 0.05) than pigs fed the CD. However, the type of diet had no effect on the

  12. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

    USGS Publications Warehouse

    Alcorn, S.W.; Pascho, R.J.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibaclerium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

  13. Glucose-Specific Enzyme IIA Has Unique Binding Partners in The Vibrio cholerae Biofilm

    PubMed Central

    Pickering, Bradley S.; Smith, Daniel R.; Watnick, Paula I.

    2012-01-01

    ABSTRACT Glucose-specific enzyme IIA (EIIAGlc) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIAGlc activates transcription of the genes required for Vibrio cholerae biofilm formation. While EIIAGlc modulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIAGlc activates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIAGlc in both planktonic and biofilm cells. A surprising number of novel EIIAGlc binding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIAGlc, such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog of Escherichia coli CsrD, was found to be a dominant binding partner of EIIAGlc. Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIAGlc. To explore the existence of multiprotein complexes centered on EIIAGlc, we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIAGlc, this analysis yielded many of the same proteins that copurified with EIIAGlc. We hypothesize that EIIAGlc serves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners. PMID:23131828

  14. Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

    PubMed

    Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

    2016-08-23

    Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists. PMID:27501351

  15. Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

    PubMed Central

    Aguayo, M. Francisca; Cáceres, Juan Carlos; Fuentealba, Matías; Muñoz, Rodrigo; Stange, Claudia; Cabrera, Ricardo; Handford, Michael

    2015-01-01

    Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. Nicotinamide adenine dinucleotide+-dependent SORBITOL DEHYDROGENASE (SDH, E. C. 1.1.1.14) from Arabidopsis thaliana L. sorbitol dehydrogenase (AtSDH) is capable of oxidizing several polyols including sorbitol, ribitol, and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S) and C4 (R) polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling, and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn.) (Hemiptera: Aleyrodidae) SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S) and C4 (R). PMID:25755662

  16. Development of an enzyme-linked immunosorbent assay specific to Sudan red I.

    PubMed

    Xu, Ting; Wei, Ke Yi; Wang, Jia; Eremin, Sergei A; Liu, Shang Zhong; Li, Qing X; Li, Ji

    2010-10-01

    To obtain antibodies to develop an enzyme-linked immunosorbent assay (ELISA) for the analysis of Sudan red I, haptens were designed and synthesized via four different strategies: (i) attachment of a spacer at the para position of the benzene ring, (ii) attachment of a spacer at the naphthol part, (iii) attachment of a spacer at the hydroxyl group of the Sudan red I molecule, and (iv) use of a fragment of the target molecule. A total of 10 haptens were used to generate immunogens, coating antigens, and polyclonal antibodies. One of the heterologous ELISAs developed exhibited an IC(50) of 1.6 ng/ml, a limit of detection (LOD) of 0.03 ng/ml, and a dynamic range between 0.1 and 14 ng/ml. The assay had 13% cross-reactivity with Para red and negligible cross-reactivity with other structure-related compounds. This ELISA was much more specific than those published previously. This assay was used to determine Sudan red I residues in tomato sauce and chili powder samples after simple pretreatment. The results were validated by comparison with high-performance liquid chromatography (HPLC). The average recoveries of Sudan red I by ELISA and HPLC were in ranges of 70-97% and 82-114%, respectively, indicating suitability of the developed ELISA for screening of Sudan red I in foods. PMID:20522332

  17. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

    PubMed

    Alcorn, S W; Pascho, R J

    2000-05-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g. PMID:10826838

  18. A novel pH–enzyme-dependent mesalamine colon-specific delivery system

    PubMed Central

    Jin, Lei; Ding, Yi-cun; Zhang, Yu; Xu, Xiao-qing; Cao, Qin

    2016-01-01

    The aim of the present study was to design a new pH–enzyme double-dependent mesalamine colon-specific delivery system. The drug release behaviors in vitro and pharmacokinetics and biodistribution in vivo were further evaluated. The mean particle diameters of mesalamine-coated microparticles were 312.2 µm. In vitro, a small amount of mesalamine was released in HCl at a pH of 1.2 and PBS medium at a pH of 7.4 for 5 hours, and 71% of the entrapped mesalamine was further released during the subsequent 20 hours of incubation. A greater area under the plasma concentration–time curve (AUC)0–t was obtained for the coated microparticles (1.9-fold) compared to the suspensions group, which indicated that the encapsulated mesalamine had mostly been absorbed in rats over the period of 12 hours. The AUC0–t of the coated microparticles in colon was 2.63-fold higher compared to the suspensions (P<0.05). Hence, mesalamine-coated microparticles are considered to maintain the drug concentration within target ranges for a long period of time. PMID:27382255

  19. Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii.

    PubMed

    Asaikkutti, Annamalai; Bhavan, Periyakali Saravana; Vimala, Karuppaiya; Karthik, Madhayan; Cheruparambath, Praseeja

    2016-05-01

    The green synthesized Mn3O4 nanoparticles (manganese-oxide nanoparticles) using Ananas comosus (L.) peel extract was characterized by various techniques. HR-SEM photograph showed that manganese-oxide nanoparticles (Mn-oxide NPs) were spherical in shape, with an average size of 40-50 nm. The Zeta potential revealed the surface charge of Mn-oxide NPs to be negative. Further, the Mn-oxide NPs were dietary supplemented for freshwater prawn Macrobrachium rosenbergii. The experimental basal diets were supplemented with Mn-oxide NPs at the rates of 0 (control), 3.0, 6.0, 9.0, 12, 15 and 18 mg/kg dry feed weight. The as-supplemented Mn-oxide NPs were fed in M. rosenbergii for a period of 90 days. The experimental study demonstrated that prawns fed with diet supplemented with 3-18 mg Mn-oxide NPs/kg shows enhanced (P<0.05) growth performance, including final weight and weight gain (WG). Significant differences (P<0.05) in feed conversion ratio (FCR) were observed in prawn fed with different diets. Additionally, prawns fed with 3.0-18 mg/kg Mn-oxide NPs supplemented diets achieved significant (P<0.05) improvement in growth performance, digestive enzyme activities and muscle biochemical compositions, while, the prawns fed with 16 mg/kg of Mn-oxide NPs showed enhanced performance. Prawns fed on diet supplemented with 16 mg/kg Mn-oxide NPs showed significantly (P<0.05) higher total protein level. The antioxidants enzymatic activity (SOD and CAT) metabolic enzymes status in muscle and hepatopancreas showed no significant (P>0.05) alterations in prawns fed with 3.0-18 mg/kg of Mn-oxide NPs supplemented diets. Consequently, the present work proposed that 16 mg/kg of Mn-oxide NPs could be supplemented for flexible enhanced survival, growth and production of M. rosenbergii. Therefore, the data of the present study recommend the addition of 16 mg/kg of Mn-oxide NPs diet to developed prawn growth and antioxidant defense system. PMID:27049122

  20. A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

    SciTech Connect

    Tasayco, M.L.; Prestwich, G.D. )

    1990-02-25

    Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.

  1. Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

    PubMed Central

    Grabarczyk, Daniel B.; Chappell, Paul E.; Johnson, Steven; Stelzl, Lukas S.; Berks, Ben C.

    2015-01-01

    The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a “suicide enzyme” strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein–protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway. PMID:26655737

  2. Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of intestinal alpha-glucosidases and pancreatic alpha-amylases is an approach to controlling blood glucose and serum insulin levels in individuals with Type II diabetes. The two human intestinal glucosidases are maltase-glucoamylase and sucrase-isomaltase. Each incorporates two family 31 ...

  3. Digestive diseases

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007447.htm Digestive diseases To use the sharing features on this page, please enable JavaScript. Digestive diseases are disorders of the digestive tract, which ...

  4. Moving college students to a better understanding of substrate specificity of enzymes through utilizing multimedia pre-training and an interactive enzyme model

    NASA Astrophysics Data System (ADS)

    Saleh, Mounir R.

    Scientists' progress in understanding enzyme specificity uncovered a complex natural phenomenon. However, not all of the currently available biology textbooks seem to be up to date on this progress. Students' understanding of how enzymes work is a core requirement in biochemistry and biology tertiary education. Nevertheless, current pre-college science education does not provide students with enough biochemical background to enable them to understand complex material such as this. To bridge this gap, a multimedia pre-training presentation was prepared to fuel the learner's prior knowledge with discrete facts necessary to understand the presented concept. This treatment is also known to manage intrinsic cognitive load during the learning process. An interactive instructional enzyme model was also built to motivate students to learn about substrate specificity of enzymes. Upon testing the effect of this combined treatment on 111 college students, desirable learning outcomes were found in terms of cognitive load, motivation, and achievement. The multimedia pre-training group reported significantly less intrinsic cognitive load, higher motivation, and demonstrated higher transfer performance than the control and post-training groups. In this study, a statistical mediation model is also proposed to explain how cognitive load and motivation work in concert to foster learning from multimedia pre-training. This type of research goes beyond simple forms of "what works" to a deeper understanding of "how it works", thus enabling informed decisions for multimedia instructional design. Multimedia learning plays multiple roles in science education. Therefore, science learners would be some of the first to benefit from improving multimedia instructional design. Accordingly, complex scientific phenomena can be introduced to college students in a motivating, informative, and cognitively efficient learning environment.

  5. Programming of enzyme specificity by substrate mimetics: investigations on the Glu-specific V8 protease reveals a novel general principle of biocatalysis.

    PubMed

    Wehofsky, N; Bordusa, F

    1999-01-25

    In this paper the universal validity of the substrate mimetic concept in enzymatic C-N ligations was expanded to anionic leaving groups based on the specificity determinants of Glu-specific endopeptidase from Staphylococcus aureus (V8 protease). In an empirical way a specific mimetic moiety was designed from simple structure-function relationship studies. The general function of the newly developed substrate mimetics to serve as an artificial recognition site for V8 protease have been examined by hydrolysis kinetic studies. Enzymatic peptide syntheses qualify the strategy of substrate mimetics as a powerful concept for programming the enzyme specificity in the direction of a more universal application of enzymes in the general area of biocatalysis. PMID:9989609

  6. Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients

    PubMed Central

    Hoffmann, Julia; Marsh, Leigh M.; Pieper, Mario; Stacher, Elvira; Ghanim, Bahil; Kovacs, Gabor; König, Peter; Wilkens, Heinrike; Haitchi, Hans Michael; Hoefler, Gerald; Klepetko, Walter; Olschewski, Horst; Olschewski, Andrea

    2015-01-01

    Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis. PMID:25840998

  7. Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

    PubMed Central

    Selvig, Kyla; Ballou, Elizabeth R.; Nichols, Connie B.

    2013-01-01

    Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species. PMID:24014765

  8. Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells

    SciTech Connect

    Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao ); Wongsasant, Budsaba )

    1991-12-01

    The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

  9. Compartment-specific expression of collagens and their processing enzymes in intrapulmonary arteries of IPAH patients.

    PubMed

    Hoffmann, Julia; Marsh, Leigh M; Pieper, Mario; Stacher, Elvira; Ghanim, Bahil; Kovacs, Gabor; König, Peter; Wilkens, Heinrike; Haitchi, Hans Michael; Hoefler, Gerald; Klepetko, Walter; Olschewski, Horst; Olschewski, Andrea; Kwapiszewska, Grazyna

    2015-05-15

    Alterations in extracellular matrix (ECM) have been implicated in the pathophysiology of pulmonary hypertension. Here, we have undertaken a compartment-specific study to elucidate the expression profile of collagens and their processing enzymes in donor and idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries. Predominant intimal, but also medial and perivascular, remodeling and reduced lumen diameter were detected in IPAH pulmonary arteries. Two-photon microscopy demonstrated accumulation of collagen fibers. Quantification of collagen in pulmonary arteries revealed collagen accumulation mainly in the intima of IPAH pulmonary arteries compared with donors. Laser capture-microdissected pulmonary artery profiles (intima+media and perivascular tissue) were analyzed by real-time PCR for ECM gene expression. In the intima+media of IPAH vessels, collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) 33 were higher expressed, whereas MMP10, ADAM17, TIMP1, and TIMP3 were less abundant. Localization of COLXVIII, its cleavage product endostatin, and MMP10, ADAM33, and TIMP1 was confirmed in pulmonary arteries by immunohistochemistry. ELISA for collagen XVIII/endostatin demonstrated significantly elevated plasma levels in IPAH patients compared with donors, whereas circulating MMP10, ADAM33, and TIMP1 levels were similar between the two groups. Endostatin levels were correlated with pulmonary arterial wedge pressure, and established prognostic markers of IPAH, right atrial pressure, cardiac index, 6-min walking distance, NH2-terminal pro-brain natriuretic peptide, and uric acid. Expression of unstudied collagens, MMPs, ADAMs, and TIMPs were found to be significantly altered in IPAH intima+media. Elevated levels of circulating collagen XVIII/endostatin are associated with markers of a poor prognosis. PMID:25840998

  10. Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

    PubMed

    Magden, J; Takeda, N; Li, T; Auvinen, P; Ahola, T; Miyamura, T; Merits, A; Kääriäinen, L

    2001-07-01

    Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families. PMID:11413290

  11. A Study in Enzyme Kinetics Using an Ion-Specific Electrode.

    ERIC Educational Resources Information Center

    Turchi, Sandra; And Others

    1989-01-01

    Describes an undergraduate biochemistry laboratory experiment on enzyme kinetics using the D-amino acid oxidase system and an ammonia electrode. Preparation of an ammonia standard curve, a sample preparation, and inhibition studies are discussed. (YP)

  12. Biochemical characterization of plasmepsin V from Plasmodium vivax Thailand isolates: Substrate specificity and enzyme inhibition.

    PubMed

    Sappakhaw, Khomkrit; Takasila, Ratchaneekorn; Sittikul, Pichamon; Wattana-Amorn, Pakorn; Assavalapsakul, Wanchai; Boonyalai, Nonlawat

    2015-12-01

    Plasmepsin V (PMV) is a Plasmodium aspartic protease responsible for the cleavage of the Plasmodium export element (PEXEL) motif, which is an essential step for export of PEXEL containing proteins and crucial for parasite viability. Here we describe the genetic polymorphism of Plasmodium vivax PMV (PvPMV) Thailand isolates, followed by cloning, expression, purification and characterization of PvPMV-Thai, presenting the pro- and mature-form of PvPMV-Thai. With our refolding and purification method, approximately 1mg of PvPMV-Thai was obtained from 1g of washed inclusion bodies. Unlike PvPMV-Ind and PvPMV-Sal-1, PvPMV-Thai contains a four-amino acid insertion (SVSE) at residues 246-249. We have confirmed that this insertion did not interfere with the catalytic activity as it is located in the long loop (R241-E272) pointing away from the substrate-binding pocket. PvPMV-Thai exhibited similar activity to PfPMV counterparts in which PfEMP2 could be hydrolyzed more efficiently than HRPII. Substrate specificity studies at P1' showed that replacing Ser by Val or Glu of the PfEMP2 peptide markedly reduced the enzyme activity of PvPMV similar to that of PfPMV whereas replacing His by Val or Ser of the HRPII peptide increased the cleavage activity. However, the substitution of amino acids at the P2 position with Glu dramatically reduced the cleavage efficiency by 80% in PvPMV in contrast to 30% in PfPMV, indicating subtle differences around the S2 binding pocket of both PfPMV and PvPMV. Four inhibitors were also evaluated for PvPMV-Thai activity including PMSF, pepstatin A, nelfinavir, and menisporopsin A-a macrocyclic polylactone. We are the first to show that menisporopsin A partially inhibits the PvPMV-Thai activity at high concentration. Taken together, these findings provide insights into recombinant production, substrate specificity and inhibition of PvPMV-Thai. PMID:26795263

  13. Plant rhizosphere species-specific stoichiometry and regulation of extracellular enzyme and microbial community structure

    NASA Astrophysics Data System (ADS)

    Bell, C. W.; Calderon, F.; Pendall, E.; Wallenstein, M. D.

    2012-12-01

    control soil samples) were collected on day 28, 78, and 148 (N = 4 /sample period/species). Microbial community structure was quantified using the barcoded pyrosequencing protocols. We measured the potential activity of seven hydrolytic soil enzymes to represent the degradation of C, N, and P-rich substrates. Soil microbial C:N biomass responses to specific plant rhizospheres (MBC and MBN) were measured using the chloroform fumigation extraction method followed by DOC & N analysis. Fourier Transform Infrared Spectroscopy was used to assess differences in plant and soil C chemistry. We found that species specific rhizospheres are characteristic of very different soil chemical, edaphic, and microbial properties. These plant species act as gateways that introduce variability into soil C, N, and P ecosystem functional dynamics directly facilitated by rhizosphere - microbe associations. Our results suggest that nutrient stoichiometry within plant species' rhizospheres is a useful tool for identifying intra-ecosystem functional patterns. By identifying what and how specific species rhizospheres differ among the overall plant community, we can better predict how below-ground microbial community function and subsequent ecosystem processes can be influenced by alterations in plant community shifts based on the rhizosphere effects.

  14. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    PubMed

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1). PMID:26196069

  15. Relationships among measures of growth performance and efficiency with carcass traits, visceral organ mass, and pancreatic digestive enzymes in feedlot cattle.

    PubMed

    Mader, C J; Montanholi, Y R; Wang, Y J; Miller, S P; Mandell, I B; McBride, B W; Swanson, K C

    2009-04-01

    Ninety-three crossbred steer calves (BW+/-SD=385+/-50 kg) were used (n=48 steers in yr 1, n=45 steers in yr 2) to examine the relationship among carcass traits, lean, bone, and fat proportions, visceral tissue weights, and pancreatic digestive enzyme activity with DMI, ADG, G:F, and residual feed intake. Calves were progeny from crossbred dams predominantly of Angus and Simmental breeding and were sired by Angus, Simmental, crossbred (predominantly of Angus and Simmental breeding), Charolais, or Piedmontese bulls. Steers were fed a high-moisture corn-based diet for an average of 112 d. Partial correlation analysis accounting for year, pen within year, week of slaughter within year, and sire breed was conducted. Gain:feed was negatively correlated (P 0.10) between performance measures and the pancreatic proportional content of alpha-amylase and trypsin activity (units/kg of BW). These data indicate that carcass fatness traits and changes in the

  16. Donor substrate specificity of 4-alpha-glucanotransferase of porcine liver glycogen debranching enzyme and complementary action to glycogen phosphorylase on debranching.

    PubMed

    Watanabe, Yumiko; Makino, Yasushi; Omichi, Kaoru

    2008-03-01

    Glycogen debranching enzyme (GDE) has both 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Here, we examined 4-alpha-glucanotransferase action of porcine liver GDE on four 6(4)-O-alpha-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6(4)-O-alpha-glucosyl-PA-maltooctaose (B4/81) was liberated from 6(4)-O-alpha-maltopentaosyl-PA-maltooctaose (B4/85), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84) and 6(4)-O-alpha-maltotriosyl-PA-maltooctaose (B4/83), whereas 6(4)-O-alpha-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-alpha-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-alpha-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-alpha-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-alpha-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the alpha-1,6-linked glucosyl residue is retained. PMID:18174188

  17. Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

    SciTech Connect

    Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong

    2014-07-03

    AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves DNA containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~ 70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~ 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

  18. Identification of testis-specific ubiquitin-conjugating enzyme in the ascidian Ciona intestinalis.

    PubMed

    Yokota, Naoto; Harada, Yoshito; Sawada, Hitoshi

    2010-07-01

    The ubiquitin-proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin-conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole-mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. PMID:20578064

  19. Specific Enzymatic Halogenation-From the Discovery of Halogenated Enzymes to Their Applications In Vitro and In Vivo.

    PubMed

    Weichold, Veit; Milbredt, Daniela; van Pée, Karl-Heinz

    2016-05-23

    During the last 20 years, focus has shifted from haloperoxidases to flavin-dependent and non-heme-iron halogenases because of their proven involvement in the biosynthesis of halogenated metabolites in different organisms and the regioselectivity of their reactions. During the first 10-12 years, the main research topics were the detection of halogenases as well as the elucidation of three-dimensional structures and reaction mechanisms. This Review mainly deals with studies on halogenating enzymes published between 2010 and 2015. It focusses on the elucidation of the involvement of halogenating enzymes in halometabolite biosynthesis, application of halogenases in in vivo and in vitro systems, in vivo modification of biosynthetic pathways in bacteria and plants, improvement of enzyme stability, broadening of substrate specificity, and the combination of biocatalysis with chemical synthesis to produce new compounds. PMID:27059664

  20. Rhamnogalacturonan alpha-L-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin.

    PubMed Central

    Mutter, M; Beldman, G; Schols, H A; Voragen, A G

    1994-01-01

    Two alpha-L-rhamnohydrolases with different substrate specificities were isolated from a commercial preparation produced by Aspergillus aculeatus. The first rhamnohydrolase was active toward p-nitrophenyl-alpha-L- rhamnopyranoside, naringin, and hesperidin and was termed p-nitrophenyl-alpha-L-rhamnopyranohydrolase (pnp-rhamnohydrolase). From the data collected, the enzyme seemed specific for the alpha-1,2- or alpha-1,6-linkage to beta-D-glucose. The pnp-rhamnohydrolase had a molecular mass of 87 kD (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH optimum of 5.5 to 6, a temperature optimum of 60 degrees C, and a specific activity toward pnp-alpha-L-rhamnopyranoside (pnp-Rha) of 13 units mg-1 protein. The second rhamnohydrolase, on the contrary, was active toward rhamnogalacturonan (RG) fragments, releasing Rha, and was therefore termed RG-rhamnohydrolase. The RG-rhamnohydrolase had a molecular mass of 84 kD, a pH optimum of 4, a temperature optimum of 60 degrees C, and a specific activity toward RG oligomers of 60 units mg-1 protein. The RG-rhamnohydrolase liberated Rha from the nonreducing end of the RG chain and appeared specific for the alpha-1,4-linkage to alpha-D-galacturonic acid. The enzyme was hindered when this terminal Rha residue was substituted at the 4-position by a beta-D-galactose. The results so far obtained did not indicate particular preference of the enzyme for low or high molecular mass RG fragments. From the results it can be concluded that a new enzyme, an RG alpha-L-rhamnopyranohydrolase, has been isolated with high specificity toward RG regions of pectin. PMID:7972516

  1. Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon.

    PubMed

    Yamada, M; Saier, M H

    1987-04-25

    The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene. PMID:3553176

  2. LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspens...

  3. Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine.

    PubMed Central

    Kelln, R A; Kinahan, J J; Foltermann, K F; O'Donovan, G A

    1975-01-01

    The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'-diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded by pyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual ribo- or deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered. PMID:1102530

  4. NADP-malic enzyme from the C4 plant Flaveria bidentis: nucleotide substrate specificity.

    PubMed

    Ashton, A R

    1997-09-15

    NADP-malic enzyme (NADP-ME, EC 1.1.1.40) was purified to near-homogeneity from leaves of the C4 dicot Flaveria bidentis and shown to possess intrinsic NAD-dependent malic enzyme activity. The NAD-dependent activity is optimal at pH 7.5 and in the presence of Mn2+. The Km for NAD is very high (20 mM), while the Vmax is 50% greater than the Vmax with NADP under the same conditions. The NAD-dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (Ki approximately 2 microM). This very low Ki reflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, the Km for NADP is 1.5 microM while the Ki for NADPH is 2 microM. Chicken liver NADP-ME also has NAD-dependent activity that is inhibited by low concentrations of NADPH. These results indicate that the NAD- and NADP-dependent activities are likely catalyzed by the same active site. The use of NAD as an alternative coenzyme revealed interactions between the binding of coenzyme and metal ions on the Km values of each of the other participants in the malic enzyme reaction. Thus, the affinity of malic enzyme for the divalent metal ions Mg2+ and particularly Mn2+ as well as the other substrate L-malate is also dependent on the nucleotide coenzyme substrate. In turn, the divalent metal ion influences the affinity of the enzyme for the coenzyme as well as L-malate. With NADP as substrate the Km for Mn2+ is 4 microM, whereas with NAD the Km is 300 microM. The relatively high affinity of the enzyme for Mn2+ and low affinity for NAD required the use of metal ion buffers when determining these values because of the substantial depletion of free Mn2+ caused by binding to NADP. PMID:9308897

  5. Nitric oxide inhibits specific enzymes in the Krebs cycle and the respiratory chain of rat hepatocyte mitochondria

    SciTech Connect

    Stadler, J.; Billiar, T.R.; Curran, R.D.; Kim, R.; Simmons, R.L. )

    1990-02-26

    Nitric oxide (NO) is a highly-reactive molecule produced from L-arginine as recently described. In macrophages and tumor cells, NO inhibits specific mitochondrial enzymes presumably by attacking their intrinsic 4Fe-4S centers. The susceptible enzymes include aconitase of the Krebs cycle and oxidoreductase (complex II) of the electron transport chain. The authors have recently demonstrated that hepatocytes (HC) produce NO in large amounts in response to endotoxin and inflammatory cytokines. To determine whether HC suffer a similar enzyme inhibition, the authors exposed rat HC to increasing concentrations of NO solutions for 5 minutes. The activity of aconitase, complex 1, complex 2, and complex 4 (cytochrome oxidase) was determined by measuring O{sub 2} consumption after addition of enzyme-specific substrates. An NO concentration-dependent inhibition of aconitase, complex 1, and complex 2 was measured. After exposure to 0.6 mM solution, the activity of aconitase was blocked to non-measurable values while complex 1 was reduced to 11 + 8%, and complex 2 to 36 + 2% of the activity of control HC. Complex 4 of the respiratory chain remained intact at 100 + 8%. These data indicate that HC, like other cell types, are susceptible to inhibition of important steps of energy production by NO. As NO is produced in response to septic stimuli, this mechanism may play a role in the metabolic dysfunction of HC in sepsis.

  6. DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI

    PubMed Central

    Smith, Rachel M.; Diffin, Fiona M.; Savery, Nigel J.; Josephsen, Jytte; Szczelkun, Mark D.

    2009-01-01

    LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide). PMID:19808936

  7. Release of angiotensin converting enzyme-inhibitor peptides during in vitro gastrointestinal digestion of Parmigiano Reggiano PDO cheese and their absorption through an in vitro model of intestinal epithelium.

    PubMed

    Basiricò, L; Catalani, E; Morera, P; Cattaneo, S; Stuknytė, M; Bernabucci, U; De Noni, I; Nardone, A

    2015-11-01

    The occurrence of 8 bovine casein-derived peptides (VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP, and HLPLP) reported as angiotensin converting enzyme-inhibitors (ACE-I) was investigated in the 3-kDa ultrafiltered water-soluble extract (WSE) of Parmigiano Reggiano (PR) cheese samples by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry via an electrospray ionization source. Only VPP, IPP, LHLPLP, and HLPLP were revealed in the WSE, and their total amount was in the range of 8.46 to 21.55 mg/kg of cheese. Following in vitro static gastrointestinal digestion, the same ACE-I peptides along with the newly formed AYFYPEL and AYFYPE were found in the 3 kDa WSE of PR digestates. Digestates presented high amounts (1,880-3,053 mg/kg) of LHLPLP, whereas the remaining peptides accounted for 69.24 to 82.82 mg/kg. The half-maximal inhibitory concentration (IC50) values decreased from 7.92 ± 2.08 in undigested cheese to 3.20 ± 1.69 after in vitro gastrointestinal digestion. The 3-kDa WSE of digested cheeses were used to study the transport of the 8 ACE-I peptides across the monolayers of the Caco-2 cell culture grown on a semipermeable membrane of the transwells. After 1h of incubation, 649.20 ± 148.85 mg/kg of LHLPLP remained in the apical compartment, whereas VPP, IPP, AYFYPEL, AYFYPE, and HLPLP accounted in total for less than 36.78 mg/kg. On average, 0.6% of LHLPLP initially present in the digestates added to the apical compartment were transported intact to the basolateral chamber after the same incubation time. Higher transport rate (2.9%) was ascertained for the peptide HLPLP. No other intact ACE-I peptides were revealed in the basolateral compartment. For the first time, these results demonstrated that the ACE-I peptides HLPLP and LHLPLP present in the in vitro digestates of PR cheese are partially absorbed through an in vitro model of human intestinal epithelium. PMID:26364103

  8. Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

    PubMed

    Libantová, Jana; Kämäräinen, Terttu; Moravcíková, Jana; Matusíková, Ildikó; Salaj, Jan

    2009-05-01

    The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated. PMID:18437530

  9. Retrostatin, a new specific enzyme inhibitor against avian myeloblastosis virus reverse transcriptase.

    PubMed

    Nishio, M; Kuroda, A; Suzuki, M; Ishimaru, K; Nakamura, S; Nomi, R

    1983-07-01

    A novel enzyme inhibitor against RNA-directed DNA polymerase of avian myeloblastosis virus was produced by an isolate of a new streptomycete for which the name Streptomyces retrostaticus is proposed. This enzyme inhibitor, which was named retrostatin, did not inhibit DNA-directed DNA polymerase of Escherichia coli and DNA-directed RNA polymerase of Ehrlich ascites tumor cells. Retrostatin was produced by the microorganism together with streptonigrin. These two substances were extracted from the culture broth with ethyl acetate at acidic pH. Retrostatin is an acidic pH indicator and the free acid was recovered as a red powder. Retrostatin had weak antibiotic activities against Gram-positive bacteria and yeasts. PMID:6193091

  10. An improved method for specificity annotation shows a distinct evolutionary divergence among the microbial enzymes of the cholylglycine hydrolase family.

    PubMed

    Panigrahi, Priyabrata; Sule, Manas; Sharma, Ranu; Ramasamy, Sureshkumar; Suresh, C G

    2014-06-01

    Bile salt hydrolases (BSHs) are gut microbial enzymes that play a significant role in the bile acid modification pathway. Penicillin V acylases (PVAs) are enzymes produced by environmental microbes, having a possible role in pathogenesis or scavenging of phenolic compounds in their microbial habitats. The correct annotation of such physiologically and industrially important enzymes is thus vital. The current methods relying solely on sequence homology do not always provide accurate annotations for these two members of the cholylglycine hydrolase (CGH) family as BSH/PVA enzymes. Here, we present an improved method [binding site similarity (BSS)-based scoring system] for the correct annotation of the CGH family members as BSH/PVA enzymes, which along with the phylogenetic information incorporates the substrate specificity as well as the binding site information. The BSS scoring system was developed through the analysis of the binding sites and binding modes of the available BSH/PVA structures with substrates glycocholic acid and penicillin V. The 198 sequences in the dataset were then annotated accurately using BSS scores as BSH/PVA enzymes. The dataset presented contained sequences from Gram-positive bacteria, Gram-negative bacteria and archaea. The clustering obtained for the dataset using the method described above showed a clear distinction in annotation of Gram-positive bacteria and Gram-negative bacteria. Based on this clustering and a detailed analysis of the sequences of the CGH family in the dataset, we could infer that the CGH genes might have evolved in accordance with the hypothesis stating the evolution of diderms and archaea from the monoderms. PMID:24644246

  11. Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34 ▿

    PubMed Central

    Sadowski, Martin; Suryadinata, Randy; Lai, Xianning; Heierhorst, Jörg; Sarcevic, Boris

    2010-01-01

    Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination. PMID:20194622

  12. Stimulus specificity of prostaglandin inhibition of rabbit polymorphonuclear leukocyte lysosomal enzyme release and superoxide anion production.

    PubMed Central

    Fantone, J. C.; Marasco, W. A.; Elgas, L. J.; Ward, P. A.

    1984-01-01

    Prostaglandins (PGs) of the E series and PGI2 have been shown to inhibit acute inflammatory reactions in vivo and polymorphonuclear leukocyte (PMN), chemotaxis, lysosomal enzyme release, and superoxide anion (O-2) production in vitro. This inhibition of neutrophil stimulation by PGEs and PGI2 has been correlated with their ability to increase intracellular cyclic adenosine monophosphate (cAMP) levels. However, the mechanism(s) by which PGEs and PGI2 alter the complex biochemical and biophysical events associated with stimulus-response coupling in the neutrophil are not clear. It is reported here that both PGEs and PGI2 in micromolar concentrations inhibit formyl-methionyl-leucyl-phenylalanine (FMLP)- and zymosan-induced lysosomal enzyme secretion and superoxide anion production in a dose-dependent manner. No preincubation time of PMNs with the prostaglandins is required for inhibition. Addition of PGEs 10 seconds or later after FMLP stimulation does not alter the biologic response of the neutrophils to the stimulus, suggesting that the prostaglandin inhibition effects early events associated with stimulus-response coupling in the neutrophil. Prostaglandin inhibition of lysosomal enzyme release by the calcium ionophore A23187 was overcome by increasing the extracellular ionophore and/or calcium concentration, suggesting that PGs may modulate intracellular free calcium levels in a manner similar to that observed with platelets. Inhibition of phorbol myristate acetate (PMA)-induced neutrophil lysosomal enzyme secretion by PGEs and PGI2 was overcome by increasing concentrations of PMA. However, neither PGEs nor PGI2 altered O-2 production by PMA-treated neutrophils. These data indicate a dissociation between PMA-stimulated O-2 production and lysosomal enzyme release. These findings are consistent with the hypothesis that inhibition of neutrophil stimulation by PGEs and PGI2 is a result of increased intracellular cyclic AMP levels and modulation of calcium

  13. Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex Trunculus digestive cells

    PubMed Central

    2011-01-01

    Background Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. Results The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. Conclusion The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids. PMID:21631952

  14. Enzyme-synthesized highly branched maltodextrins have slow glucose generation at the mucosal alpha-glucosidase level and are slowly digestible "in vivo"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For digestion of starch in humans, alpha-amylase first hydrolyzes starch molecules to produce alpha-limit dextrins, followed by complete hydrolysis to glucose by the mucosal alpha-glucosidases in the small intestine. It is known that alpha-1,6 linkages in starch are hydrolyzed at a lower rate than a...

  15. Digestive System

    MedlinePlus

    ... is about 30 feet long. continue How Digestion Works Digestion Begins in the Mouth The process of ... a noncongenital condition. Esophagitis is usually caused by gastroesophageal reflux disease (GERD) , a condition in which the esophageal ...

  16. Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

    PubMed Central

    Cambra, Inés; Hernández, David; Diaz, Isabel; Martinez, Manuel

    2012-01-01

    C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed. PMID:22615948

  17. A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.

    PubMed

    Do, Viet Ha; Tran, Phuong Lan; Ni, Li; Park, Kwan Hwa

    2016-01-01

    A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched β-cyclodextrin (Glcn-β-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis. PMID:26403601

  18. Development of a Highly Sensitive and Specific Blastomycosis Antibody Enzyme Immunoassay Using Blastomyces dermatitidis Surface Protein BAD-1

    PubMed Central

    Smedema, Melinda L.; Durkin, Michelle M.; Brandhorst, T. Tristan; Hage, Chadi A.; Connolly, Patricia A.; Leland, Diane S.; Davis, Thomas E.; Klein, Bruce S.; Wheat, L. Joseph

    2014-01-01

    Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis. PMID:24285817

  19. Determination of Mineral-Specific Clumped Isotope Acid Digestion Fractionation Factors Using Heating Experiments and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Henry, D.; Tang, J.; Mosenfelder, J. L.; Eagle, R.; Tripati, A.

    2014-12-01

    Clumped isotope thermometry involves the determination of formation temperatures of carbonates from the fraction of isotopologues containing multiple rare isotopes (oxygen-18 and carbon-13). At high temperatures, the abundance of these isotopologues should be stochastic. At lower temperatures, there is a tendency for heavy isotopes to form bonds with each other. However, spectroscopic determination of isotope ratios with high precision is difficult in solids, and so 13C-18O bond abundance is not measured in the solid phase. Instead, analysis of carbonates is performed using gas source mass spectrometry, by reacting the carbonate samples with phosphoric acid and measuring the evolved CO2 gas. As an oxygen atom is lost during the conversion of CO32- groups to CO2, this reaction is hypothesized to result in mineral and acid digestion temperature-dependent fractionation. In order to quantify this fractionation between CO32- and CO2, this experiment seeks to determine acid fractionation factors for carbonate samples of varying composition by randomizing samples through intense heating and comparing analyte CO2 measured composition to the expected composition for a stochastically distributed sample. From this analysis, future carbonate measurements can be calibrated to account for acid digestion fractionation.

  20. Turnaround of axoplasmic transport of selected particle-specific enzymes at an injury in control and diisopropylphosphorofluoridate-treated rats.

    PubMed

    Schmidt, R E; Yu, M J; McDougal, D B

    1980-09-01

    Reversal of direction (turnaround) of axonal transport of particle-specific enzyme activities was studied at a ligature placed on rat sciatic nerve. In the principal experiment, the ligature remained on the nerve in vivo several hours, allowing enzyme activities (acetylcholinesterase, acid phosphatase, and monoamine oxidase) to accumulate immediately proximal to the tie. The nerve was then tied a second time, proximal to the first tie, and incubated in vitro for several more hours. Accumulation of enzyme activities just distal to the second tie was measured. This second accumulation, of activities traveling in the retrograde direction, was shown to be the result of turnaround in several ways. (1) The increase in activity distal to the second tie was equal to the decrease in activity proximal to the first. (2) The increase in enzyme activities distal to the second tie was greatly reduced when the accumulation proximal to the first tie was trapped by placing a third tie between the first and second ties. (3) It was shown that the activity that accumulated distal to the second tie could not have been in retrograde motion at the time of the first tie. (4) Accumulation distal to the second tie was not a function of the length of nerve segment included between the two ties. In contrast to the consistent occurrence of turnaround of orthograde flow, turnaround of retrograde flow could not be demonstrated. Turnaround transport was blocked by incubation in the cold and in the presence of NaCN or vinblastine. The turnaround process operated on all three enzymes studied, suggesting that it operates on lysosomes and mitochondria, as well as on the endoplasmic reticulum-like material bearing acetylcholinesterase. Evidence for the participation of the transport process in the renewal of AChE in the distal portions of the axon was obtained in experiments using diisopropylphosphorofluoridate and cycloheximide. PMID:6161227

  1. A potent specific inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans and of certain other fungal enzymes.

    PubMed

    Niehaus, W G; Flynn, T

    1993-09-01

    A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases from Cryptococcus neoformans and Schizophyllum commune and glucose-6-phosphate dehydrogenase from Schizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than the Cryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent. PMID:8302365

  2. Molecular co-evolution of a protease and its substrate elucidated by analysis of the activity of predicted ancestral hatching enzyme

    PubMed Central

    2013-01-01

    Background Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. We have comprehensively studied the molecular mechanisms of the enzyme action to its substrate egg envelope, and determined the gene/protein structure and phylogenetic relationships. Because the hatching enzyme must have evolved while maintaining its ability to digest the egg envelope, the hatching enzyme-egg envelope protein pair is a good model for studying molecular co-evolution of a protease and its substrate. Results Hatching enzymes from medaka (Oryzias latipes) and killifish (Fundulus heteroclitus) showed species-specific egg envelope digestion. We found that by introducing four medaka-type residue amino acid substitutions into recombinant killifish hatching enzyme, the mutant killifish hatching enzyme could digest medaka egg envelope. Further, we studied the participation of the cleavage site of the substrate in the species-specificity of hatching enzyme. A P2-site single amino acid substitution was responsible for the species-specificity. Estimation of the activity of the predicted ancestral enzymes towards various types of cleavage sites along with prediction of the evolutionary timing of substitutions allowed prediction of a possible evolutionary pathway, as follows: ancestral hatching enzyme, which had relatively strict substrate specificity, developed broader specificity as a result of four amino acid substitutions in the active site cleft of the enzyme. Subsequently, a single substitution occurred within the cleavage site of the substrate, and the recent feature of species-specificity was established in the hatching enzyme-egg envelope system. Conclusions The present study clearly provides an ideal model for protease-substrate co-evolution. The evolutionary process giving rise to species-specific egg envelope digestion of hatching enzyme was initiated by amino acid substitutions in the enzyme, resulting in altered substrate specificity, which later

  3. Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction.

    PubMed Central

    Gangloff, A; Garneau, A; Huang, Y W; Yang, F; Lin, S X

    2001-01-01

    Human oestrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17beta-HSD1 at 37 degrees C and pH 7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 microM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v=V.[s]/[K(m)+[s](1+[s]/K(i))] gave a K(m) of 0.07+/-0.01 microM, a k(cat) (for the dimer) of 1.5+/-0.1 s(-1), a specificity of 21 microM(-1) x s(-1) and a K(i) of 1.3 microM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42+/-0.07 microM for the K(m), 0.8+/-0.04 s(-1) for the k(cat) and 1.9 microM(-1) x s(-1) for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (K(m)=24+/-4 microM; k(cat)=0.47+/-0.06 s(-1); specificity=0.002 microM(-1) x s(-1)) or DHT (K(m)=26+/-6 microM; k(cat)=0.2+/-0.02 s(-1); specificity=0.0008 microM(-1) x s(-1)) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17beta-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate. PMID:11336660

  4. Magnetic enzyme-linked immunosorbent assay (MELISA) for determination of specific IgG in paracoccidioidomycosis.

    PubMed

    de Camargo, Z P; Guesdon, J L; Drouhet, E; Improvisi, L

    1984-01-01

    A magnetic solid phase enzyme-linked immunosorbent assay (MELISA) for quantification of IgG antibodies to somatic and metabolic antigens of Paracoccidioides brasiliensis was developed. Activation of magnetic polyacrylamide agarose beads with concanavalin A was superior to glutaraldehyde activation, and test sensitivity was higher for somatic than for metabolic antigens. Comparative MELISA, counterimmunoelectrophoresis and erythroimmunoassay tests with sera from 33 proven cases of paracoccidioidomycosis, 14 cases of histoplasmosis and 20 normal human sera showed the MELISA could distinguish antibody levels in paracoccidioidomycosis from those in normal sera; however two sera from histoplasmosis cases cross-reacted in the MELISA. MELISA is a rapid test (5-6 h) and the results suggest it has considerable potential value for assay of anti-P. brasiliensis antibodies. PMID:6438813

  5. [Classification of alcohol metabolizing enzymes and polymorphisms--specificity in Japanese].

    PubMed

    Harada, S

    2001-04-01

    Multiple forms and gene loci of human alcohol dehydrogenase (ADH EC: 1.2.1.3) and aldehyde dehydrogenase (ALDH, EC: 1.2.1.3) in the major pathway of alcohol metabolism have been found and characterized in the last two decades. With the coenzyme NAD, these enzymes catalyze the reversible conversion of organic alcohols to ketones or aldehydes, and aldehyde to acetic acid. The ADH genes are mapped to chromosome 4p21-25, but the ALDH genes are localized at different chromosomes. The cytochrome P450 2E1 (CYP2E1) gene, which is mapped to chromosome 10q24.3-qter contributes also the conversion of ethanol to acetaldehyde. Genetic polymorphisms have been reported in these alcohol metabolizing enzymes. The metabolisms of alcohol and acetaldehyde in liver and blood after drinking alcohol are thought to be influenced by the interactive action of these enzymes. Amongst the five major classes of the ADH subunits (alpha, beta, gamma, pi, chi, sigma), beta and gamma subunits show genetic polymorphisms. Recently a new nomenclature for ALDH genes has been recommend based on divergent evolution and chromosomal mapping. Two major isoforms designated as cytosolic ALDH1 and mitochondrial ALDH2 can be distinguished by their electrophoretic and kinetic properties as well as by their subcellular localization. Mitochondrial ALDH2 is a major enzyme in the oxidation of acetaldehyde derived from ethanol metabolism. The catalytic deficiency of ALDH2 isozyme is responsible for flushing and other vasomotor symptoms caused by higher acetaldehyde levels after alcohol intake. So far, frequencies of the two alleles of ALDH2 in Mongoloid have been reported in the different population groups. The catalytic deficiency of ALDH2 is caused by a structural point mutation at amino acid position 487, where a substitution of Glu to Lys resulting from a transition of G (C) to A (T) at 1510 nucleotide from the initiation codon has occurred. Individuals deficient in ALDH2 activity refrain from excessive drinking

  6. Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.

    PubMed

    Pattanayak, Vikram; Guilinger, John P; Liu, David R

    2014-01-01

    The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this chapter, we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~10(12)-membered) libraries of potential nuclease substrates. PMID:25398335

  7. Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products.

    PubMed

    Hellmuth, Hendrik; Wittrock, Sabine; Kralj, Slavko; Dijkhuizen, Lubbert; Hofer, Bernd; Seibel, Jürgen

    2008-06-24

    Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency. PMID:18512955

  8. Variability in intra-specific and monosporous isolates of Volvariella volvacea based on enzyme activity, ITS and RAPD.

    PubMed

    Ahlawat, O P; Gupta, P; Kamal, S; Dhar, B L

    2010-06-01

    Two parents and 15 monosporous isolates were morphologically characterized and were found to vary in their growth characteristics on malt extract agar medium. The isolates also varied in enzymes activity profile with respect to exoglucanase, endoglucanase, xylanase, laccase and polyphenol oxidase. Further in polymerase chain reaction (PCR) amplification of Internal Transcribed Spacer (ITS) region of 5.8S rDNA, an amplicon of same length (720 bp) was amplified from two parents and all the monosporous isolates, which revealed that all belong to the same species. The combined phylogenetic analysis of random amplified polymorphic DNA (RAPD) profiles obtained with five decamer primers (operon kit B) series primers also revealed intra-specific variation of 60% with in the two parent strains and their single spore isolates (SSIs). However, the variations between the parent strains and their SSIs were lesser and it was 50 and 32% in parent strains, OE-210 and OE-12, respectively. Based upon phylogenetic analysis, the isolates of parent strain, OE-210 formed 7 distinct phylogenetic clades, while of strain OE-12 formed 4 clades. The study elucidates that isolates showing variations in morphological growth characteristics and enzymes activity also showed variations in their RAPD profiles, revealed through phylogenetic analysis of RAPD profiles. It is also evident from the study that morphological characterization along with enzymes activity assay of strains is essential before their use in yield evaluation trials with final authentication from molecular analysis. PMID:23100827

  9. Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate*

    PubMed Central

    Withers, Saunia; Lu, Fachuang; Kim, Hoon; Zhu, Yimin; Ralph, John; Wilkerson, Curtis G.

    2012-01-01

    Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be “monomer” precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters. PMID:22267741

  10. Specific capture of the hydrolysate on magnetic beads for sensitive detecting plant vacuolar processing enzyme activity.

    PubMed

    Zhou, Jun; Cheng, Meng; Zeng, Lizhang; Liu, Weipeng; Zhang, Tao; Xing, Da

    2016-05-15

    Conventional plant protease detection always suffers from high background interference caused by the complex coloring metabolites in plant cells. In this study, a bio-modified magnetic beads-based strategy was developed for sensitive and quantitative detection of plant vacuolar processing enzyme (VPE) activity. Cleavage of the peptide substrate (ESENCRK-FITC) after asparagine residue by VPE resulted in the 2-cyano-6-amino-benzothiazole (CABT)-functionalized magnetic beads capture of the severed substrate CRK-FITC via a condensation reaction between CABT and cysteine (Cys). The catalytic activity was subsequently obtained by the confocal microscopy imaging and flow cytometry quantitative analysis. The sensor system integrated advantages of (i) the high efficient enrichment and separation capabilities of magnetic beads and (ii) the catalyst-free properties of the CABT-Cys condensation reaction. It exhibited a linear relationship between the fluorescence signal and the concentration of severed substrate in the range of 10-600 pM. The practical results showed that, compared with normal growth conditions, VPE activity was increased by 2.7-fold (307.2 ± 25.3 μM min(-1)g(-1)) upon cadmium toxicity stress. This platform effectively overcame the coloring metabolites-caused background interference, showing fine applicability for the detection of VPE activity in real samples. The strategy offers great sensitivity and may be further extended to other protease activity detection. PMID:26797250

  11. Specific activities of antioxidative enzymes in the cochlea of guinea pigs at different stages of development.

    PubMed

    Zelck, U; Nowak, R; Karnstedt, U; Koitschev, A; Käcker, N

    1993-01-01

    Significant activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were found in the cochleas of guinea pigs of different ages. The specific activities of SOD and GSH-Px (expressed as units/mg protein) increased significantly from fetal animals to animals 2 days old and then to 6-month-old animals. PMID:8369116

  12. Effects of Protease, Phytase and a Bacillus sp. Direct-Fed Microbial on Nutrient and Energy Digestibility, Ileal Brush Border Digestive Enzyme Activity and Cecal Short-Chain Fatty Acid Concentration in Broiler Chickens

    PubMed Central

    Murugesan, Ganapathi R.; Romero, Luis F.; Persia, Michael E.

    2014-01-01

    Two experiments were conducted to determine the effects of protease and phytase (PP) and a Bacillus sp. direct-fed microbial (DFM) on dietary energy and nutrient utilization in broiler chickens. In the first experiment, Ross 308 broiler chicks were fed diets supplemented with PP and DFM in a 2×2 factorial arrangement. The 4 diets (control (CON), CON + PP, CON + DFM, and CON + PP + DFM) were fed from 15–21 days of age. In Experiment 1, significant interaction (P≤0.01) between PP and DFM on the apparent ileal digestibility coefficient for starch, crude protein, and amino acid indicated that both additives increased the digestibility. Both additives increased the nitrogen retention coefficient with a significant interaction (P≤0.01). Although no interaction was observed, significant main effects (P≤0.01) for nitrogen-corrected apparent ME (AMEn) for PP or DFM indicated an additive response. In a follow-up experiment, Ross 308 broiler chicks were fed the same experimental diets from 1–21 days of age. Activities of ileal brush border maltase, sucrase, and L-alanine aminopeptidase were increased (P≤0.01) by PP addition, while a trend (P = 0.07) for increased sucrase activity was observed in chickens fed DFM, in Experiment 2. The proportion of cecal butyrate was increased (P≤0.01) by DFM addition. Increased nutrient utilization and nitrogen retention appear to involve separate but complementary mechanisms for PP and DFM, however AMEn responses appear to have separate and additive mechanisms. PMID:25013936

  13. Characterization of enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked immunosorbent assay.

    PubMed Central

    Van Tassell, R L; Lyerly, D M; Wilkins, T D

    1994-01-01

    Within the past decade, certain strains of Bacteroides fragilis have been associated with diarrhea in humans and cytotoxic activity on certain colon carcinoma cell lines. An enzyme-linked immunosorbent assay (ELISA) for detecting the enterotoxin of B. fragilis in cultures and stools was developed by using high-titer monospecific goat and rabbit antitoxins in an indirect format. The lower limit of detection for purified toxin was approximately 0.05 micrograms/ml; the linear range was from 0.05 to 10 microgram/ml. Using the ELISA to screen cultures of toxigenic and nontoxigenic strains of B. fragilis, we observed 100% correlation with 16 known toxigenic strains which had various cytotoxic activities on HT-29 cells. In addition, we found 6 of 62 previously untested strains also to be positive in both assays. Stability studies revealed that although the cytotoxic activities of crude and purified toxin preparations incubated at elevated temperatures were rapidly lost, the ELISA responses were not significantly reduced. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis showed that the purified toxin autodigested to several stable peptides. Studies on partially purified membranes from the toxigenic strains revealed the presence of several membrane-associated components which were noncytotoxic but strongly immunoreactive in the ELISA. Preliminary studies with spiked feces indicated that the ELISA may be useful for screening not only cultures for the enterotoxigenic B. fragilis but also stool specimens. Ongoing studies are focusing on determining the nature of the toxin's apparent proteolytic capabilities and investigating the feasibility of using the ELISA on stool specimens from healthy and diarrheic humans. Images PMID:8556504

  14. The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity.

    PubMed

    de Groot, Rens; Lane, David A; Crawley, James T B

    2010-10-21

    ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (k(cat)/K(m) reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1' M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1' amino acid specificity, we hypothesize, by shaping the S1' pocket. It is concluded that 2 subsites, D187-R193 and D252-P256, in the metalloprotease domain play an important role in cleavage efficiency and site specificity. PMID:20647566

  15. Tumor-Specific Formation of Enzyme-Instructed Supramolecular Self-Assemblies as Cancer Theranostics.

    PubMed

    Huang, Peng; Gao, Yuan; Lin, Jing; Hu, Hao; Liao, Hsien-Shun; Yan, Xuefeng; Tang, Yuxia; Jin, Albert; Song, Jibin; Niu, Gang; Zhang, Guofeng; Horkay, Ferenc; Chen, Xiaoyuan

    2015-10-27

    Despite the effort of developing various nanodelivery systems, most of them suffer from undesired high uptakes by the reticuloendothelial system, such as liver and spleen. Herein we develop an endogenous phosphatase-triggered coassembly strategy to form tumor-specific indocyanine green (ICG)-doped nanofibers (5) for cancer theranostics. Based on coordinated intermolecular interactions, 5 significantly altered near-infrared absorbance of ICG, which improves the critical photoacoustic and photothermal properties. The phosphatase-instructed coassembly process, as well as its theranostic capability, was successfully conducted at different levels ranging from in vitro, living cell, tissue mimic, to in vivo. Specifically, the tumor uptake of ICG was markedly increased to 15.05 ± 3.78%ID/g, which was 25-fold higher than that of free ICG (0.59 ± 0.24%ID/g) at 4 h after intravenous injection. The resulting ultrahigh T/N ratios (>15) clearly differentiated tumors from the surrounding normal tissue. Complete tumor elimination with high therapeutic accuracy has been successfully achieved upon laser irradiation (0.8 W/cm(2), 5 min) within 24-48 h postinjection. As the first example, in vivo formation of tumor-specific ICG-doped nanofiber for PTT theranostics owns the immense potential for clinical translation of personalized nanomedicine with targeted drug delivery as well as for cancer theranostics. PMID:26301492

  16. Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

    SciTech Connect

    Kino, Kuniki . E-mail: kkino@waseda.jp; Kuratsu, Shoko; Noguchi, Atsushi; Kokubo, Masahiro; Nakazawa, Yuji; Arai, Toshinobu; Yagasaki, Makoto; Kirimura, Kohtaro

    2007-01-12

    Glutathione (GSH) is synthesized by {gamma}-glutamylcysteine synthetase ({gamma}-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed {gamma}-GCS-GS catalyzing both {gamma}-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the {gamma}-GCS activity, S. agalactiae {gamma}-GCS-GS had different substrate specificities from those of Escherichia coli {gamma}-GCS. Furthermore, S. agalactiae {gamma}-GCS-GS synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-X{sub aa}-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding {gamma}-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae {gamma}-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed {gamma}-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-Cys-X{sub aa}. Whereas the substrate specificities of {gamma}-GCS domain protein and GS domain protein of S. agalactiae {gamma}-GCS-GS were the same as those of S. agalactiae {gamma}-GCS-GS.

  17. Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots †

    PubMed Central

    Levanony, Hanna; Bashan, Yoav; Kahana, Zvi E.

    1987-01-01

    The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 105 CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 108 CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd. PMID:16347284

  18. Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

    PubMed Central

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa. PMID:25393679

  19. Prolonged ingestion of prehydrolyzed whey protein induces little or no change in digestive enzymes, but decreases glutaminase activity in exercising rats.

    PubMed

    Nery-Diez, Ana Cláudia C; Carvalho, Iara R; Amaya-Farfán, Jaime; Abecia-Soria, Maria Inés; Miyasaka, Célio K; Ferreira, Clécio da S

    2010-08-01

    Because consumption of whey protein hydrolysates is on the increase, the possibility that prolonged ingestion of whey protein hydrolysates affect the digestive system of mammals has prompted us to evaluate the enzymatic activities of pepsin, leucine-aminopeptidase, chymotrypsin, trypsin, and glutaminase in male Wistar rats fed diets containing either a commercial whey isolate or a whey protein hydrolysate with medium degree of hydrolysis and to compare the results with those produced by physical training (sedentary, sedentary-exhausted, trained, and trained-exhausted) in the treadmill for 4 weeks. The enzymatic activities were determined by classical procedures in all groups. No effect due to the form of the whey protein in the diet was seen in the activities of pepsin, trypsin, chymotrypsin, and leucine-aminopeptidase. Training tended to increase the activity of glutaminase, but exhaustion promoted a decrease in the trained animals, and consumption of the hydrolysate decreased it even further. The results are consistent with the conclusion that chronic consumption of a whey protein hydrolysate brings little or no modification of the proteolytic digestive system and that the lowering of glutaminase activity may be associated with an antistress effect, counteracting the effect induced by training in the rat. PMID:20482282

  20. Effects of Dietary Pb and Cd and Their Combination on Glutathion-S-Transferase and Catalase Enzyme Activities in Digestive Gland and Foot of the Green Garden Snail, Cantareus apertus (Born, 1778).

    PubMed

    Mleiki, Anwar; Marigómez, Ionan; El Menif, Najoua Trigui

    2015-06-01

    The present study was focused on the assessment of glutathion-S-transferase (GST) and catalase (CAT) activities in the digestive gland and foot of the land snail, Cantareus apertus (Born, 1778), exposed to different nominal dietary concentrations of Pb (25 and 2500 mg Pb/Kg), Cd (5 and 100 mg Cd/Kg) and their combination (25 mg Pb + 5 mg Cd/Kg and 2500 mg Pb + 100 mg Cd/Kg) for 7 and 60 days. GST activity was significantly increased after 7 and 60 days exposure to the highest concentration of Pb, Cd and their combination. The levels of CAT activity were different in the two studied organs but in both cases it resulted increased after 7 and 60 days of exposure, which varied significantly between metals and dietary concentrations. Therefore, it can be concluded that GST and CAT enzymes in digestive gland and foot of C. apertus are responsive to Cd, Pb and their combination, whereby they are suitable to be included in a battery of biomarkers for ecosystem health assessment in metal polluted soils using this species as sentinel. PMID:25899572

  1. Nicotinamide Adenine Dinucleotide-specific “Malic” Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism 1

    PubMed Central

    Dittrich, Peter

    1976-01-01

    NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO42−, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn2+, which cannot be replaced by NADP or Mg2+. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn2+, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO2 for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  2. Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets

    PubMed Central

    Flierman, Dennis; van der Heden van Noort, Gerbrand J.; Ekkebus, Reggy; Geurink, Paul P.; Mevissen, Tycho E.T.; Hospenthal, Manuela K.; Komander, David; Ovaa, Huib

    2016-01-01

    Summary Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents. PMID:27066941

  3. Safety evaluation of phytase 50104 enzyme preparation (also known as VR003), expressed in Pseudomonas fluorescens, intended for increasing digestibility of phytate in monogastrics.

    PubMed

    Krygier, Scott; Solbak, Arne; Shanahan, Diane; Ciofalo, Vince

    2014-11-01

    Phytase 50104 enzyme (also known as VR003) can be added to swine and poultry diets to catalyze the hydrolysis of phosphate from phytic acid, thereby increasing phosphorus bioavailability in these animals. This enzyme was produced from a Pseudomonas fluorescens (P. fluorescens) production strain and was tested in acute, subchronic and genotoxicity studies. Dosages of the test article preparation ranged from 5000μg/plate for in vitro toxicity studies to 2000mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000mg/kg/day) resulted in a safety margin of 5870 based on TOS and a conservative estimate of total poultry consumption at the highest inclusion rate. There was no toxicity reported for any of these studies or in the following additional safety studies: eye irritation, dermal irritation, and delayed hypersensitivity studies. A review of the public literature indicated that P. fluorescens fulfilled the recognized safety criteria pertinent to microbial production strains used in the manufacture of food/feed enzyme preparations. The results of the toxicity studies presented herein attest to the safety of phytase 50104 enzyme for its intended use. PMID:24945743

  4. Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME".

    PubMed

    Barbas, Ana; Matos, Rute G; Amblar, Mónica; López-Viñas, Eduardo; Gomez-Puertas, Paulino; Arraiano, Cecília M

    2009-07-31

    RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3'-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme." PMID:19458082

  5. G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

    PubMed

    Hirschi, Alexander; Martin, William J; Luka, Zigmund; Loukachevitch, Lioudmila V; Reiter, Nicholas J

    2016-08-01

    Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. PMID:27277658

  6. [Fibrinogen/fibrin-specific enzymes from copperhead (Agkistrodon halys halys) and cobra (Naja oxiana eichwald) snake venoms].

    PubMed

    Yunusova, E S; Sadykov, E S; Sultanalieva, N M; Shkinev, A V

    2016-03-01

    Ability of fractions of cobra's (Naja oxiana Eichwald) and copperhead snake's (Agkistrodon halys halys) venoms to hydrolyze fibrinogen/fibrin was studied. In cobra's snake a component with molecular mass of nearly 60 kDa was found to hydrolyze a-chain of fibrinogen but failed to hydrolyze casein/azocasein and fibrin. A fibrinogen-specific metalloproteinase, the enzyme was inhibited by EDTA. Cobra's venom reduced the mass of donor's fresh blood clots. The copperhead snake's venom and the fractions obtained by gel-filtration (HW-50) and ion exchange chromatography (DEAE-650) were found to hydrolyze casein/azocasein, a- and b-chains of fibrinogen/fibrin and donor's blood clots. The results from the study of the venom and proteolytically active fractions are the evidence for a thrombolytic potential in a copperhead snake's venom. PMID:27420616

  7. Conservation, fiber digestibility, and nutritive value of corn harvested at 2 cutting heights and ensiled with fibrolytic enzymes, either alone or with a ferulic acid esterase-producing inoculant.

    PubMed

    Lynch, J P; Baah, J; Beauchemin, K A

    2015-02-01

    did not improve corn silage fermentation or its nutritive value and resulted in some negative effects on these parameters. The effects of using a fibrolytic enzyme product at ensiling, either alone or in combination with a ferulic acid esterase-producing inoculant, did not differ between corn harvested at either NC or HC. Silage made from HC had a greater starch content and lower fiber content than NC silage, whereas cutting height did not affect the in vitro digestibility indices. PMID:25483202

  8. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  9. Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species. PMID:23114700

  10. Evaluation of Enzyme-Linked Immunosorbent Assays for Detection of Mycoplasma bovis-Specific Antibody in Bison Sera

    PubMed Central

    Sacco, Randy E.; Olsen, Steven C.

    2013-01-01

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. A method for the detection of M. bovis-specific serum antibodies is needed in order to establish prevalence and transmission patterns. Enzyme-linked immunosorbent assays (ELISAs) validated for the detection of M. bovis-specific serum IgG in cattle are commercially available, but their suitability for bison sera has not been determined. A collection of bison sera, most from animals with a known history of infection or vaccination with M. bovis, was tested for M. bovis-specific IgG using commercially available kits as well as an in-house ELISA in which either cattle or bison M. bovis isolates were used as a source of antigen. Comparison of the results demonstrates that ELISAs optimized for cattle sera may not be optimal for the identification of bison seropositive for M. bovis, particularly those with low to moderate antibody levels. The reagent used for the detection of bison IgG and the source of the antigen affect the sensitivity of the assay. Optimal performance was obtained when the capture antigen was derived from bison isolates rather than cattle isolates and when a protein G conjugate rather than an anti-bovine IgG conjugate was used for the detection of bison IgG. PMID:23843427

  11. Glial high-affinity binding site with specificity for angiotensin II not angiotensin III: a possible N-terminal-specific converting enzyme

    SciTech Connect

    Printz, M.P.; Jennings, C.; Healy, D.P.; Kalter, V.

    1986-01-01

    Anomalous binding properties of angiotensin II to fetal rat brain primary cultures suggested a possible contribution from contaminating glia. To investigate this possibility, cultures of C6 glioma, a clonal rat cell line, were examined for the presence of angiotensin II receptors. A specific high-affinity site for (/sup 125/I)angiotensin II was measured both by traditional methodology using whole cells and by autoradiography. This site shared properties similar to that found with the brain cells, namely low ligand internalization and markedly decreased affinity for N-terminal sarcosine or arginine-angiotensin analogs. The competition rank order was angiotensin II much greater than (Sar1,Ile8)angiotensin II greater than or equal to des(Asp1,Arg2)angiotensin II. Angiotensin III did not compete for binding to the site. High-pressure liquid chromatography analysis indicated that the ligand either in the incubation or bound to the site was stable at 15 degrees C, but there was very rapid and extensive degradation by the C6 glioma cells at 37 degrees C. It is concluded that the site exhibits unusual N-terminal specificity for angiotensin with nanomolar affinity for angiotensin II. If angiotensin III is an active ligand in the brain, the site may have a converting enzyme function. Alternatively, it may form the des-Asp derivatives of angiotensin for subsequent degradation by other enzymatic pathways. Either way, it is proposed that the site may modulate the brain-angiotensin system.

  12. Influence of different length linker containing DHEA-7-CMO-enzyme conjugates on sensitivity and specificity of DHEA-17-CMO-antibody.

    PubMed

    Shrivastav, Tulsidas G; Chaube, Shail K; Kariya, Kiran P; Singh, Rita; Kumar, Dinesh; Pandit, Deepa; Ujawane, Pragati; Kumari, Poonam; Pandey, Bhavana

    2011-01-01

    Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates. PMID:21728820

  13. Specific serum immunoglobulin D, detected by antibody capture enzyme-linked immunosorbent assay (ELISA), in cytomegalovirus infection.

    PubMed Central

    Mortensen, J; Nielsen, S L; Sørensen, I; Andersen, H K

    1989-01-01

    An antibody capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of immunoglobulin D (IgD) antibodies to cytomegalovirus (CMV) in sera from blood donors and various groups of patients infected with CMV. This method has previously been found especially valuable in detecting specific antibodies of the IgM, IgE, IgA and IgG class in patients with CMV infection. Specific CMV IgD antibodies were found in 37% of CMV seropositive blood donors and in 47 (88%) of the 53 patients investigated, including bone marrow transplant and renal allograft transplant patients, patients with CMV mononucleosis, neonates with CMV infection and AIDS patients with CMV infection. The highest IgD reactivity was found in patients having either a primary post-transplant CMV infection or CMV mononucleosis. The IgD reactivity in patients with AIDS and in neonates was low. It was also found that in the acute phase of CMV infection the development of CMV antibodies of the IgD class was similar to the development of antibodies of the other classes. The maintenance of IgD activity in some patients together with the presence of CMV IgD antibodies in a great proportion of the blood donors indicates that the development of CMV IgD antibodies resembles that of the IgG class. Determination of specific IgD antibodies offered no advantage over determination of specific antibodies of the IgM, IgE and IgA classes in the diagnosis of CMV infection. PMID:2539278

  14. Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme.

    PubMed

    Araujo, M C; Melo, R L; Cesari, M H; Juliano, M A; Juliano, L; Carmona, A K

    2000-07-25

    Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains. PMID:10913258

  15. The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II.

    PubMed

    Bharati, Akhilendra Pratap; Singh, Neha; Kumar, Vikash; Kashif, Md; Singh, Amit Kumar; Singh, Priyanka; Singh, Sudhir Kumar; Siddiqi, Mohammad Imran; Tripathi, Timir; Akhtar, Md Sohail

    2016-01-01

    RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription. PMID:27503426

  16. Phloem-specific expression of Yang cycle genes and identification of novel Yang cycle enzymes in Plantago and Arabidopsis.

    PubMed

    Pommerrenig, Benjamin; Feussner, Kirstin; Zierer, Wolfgang; Rabinovych, Valentyna; Klebl, Franz; Feussner, Ivo; Sauer, Norbert

    2011-05-01

    The 5-methylthioadenosine (MTA) or Yang cycle is a set of reactions that recycle MTA to Met. In plants, MTA is a byproduct of polyamine, ethylene, and nicotianamine biosynthesis. Vascular transcriptome analyses revealed phloem-specific expression of the Yang cycle gene 5-METHYLTHIORIBOSE KINASE1 (MTK1) in Plantago major and Arabidopsis thaliana. As Arabidopsis has only a single MTK gene, we hypothesized that the expression of other Yang cycle genes might also be vascular specific. Reporter gene studies and quantitative analyses of mRNA levels for all Yang cycle genes confirmed this hypothesis for Arabidopsis and Plantago. This includes the Yang cycle genes 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1. We show that these two enzymes are sufficient for the conversion of methylthioribose-1-phosphate to 1,2-dihydroxy-3-keto-5-methylthiopentene. In bacteria, fungi, and animals, the same conversion is catalyzed in three to four separate enzymatic steps. Furthermore, comparative analyses of vascular and nonvascular metabolites identified Met, S-adenosyl Met, and MTA preferentially or almost exclusively in the vascular tissue. Our data represent a comprehensive characterization of the Yang cycle in higher plants and demonstrate that the Yang cycle works primarily in the vasculature. Finally, expression analyses of polyamine biosynthetic genes suggest that the Yang cycle in leaves recycles MTA derived primarily from polyamine biosynthesis. PMID:21540433

  17. The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II

    PubMed Central

    Bharati, Akhilendra Pratap; Singh, Neha; Kumar, Vikash; Kashif, Md.; Singh, Amit Kumar; Singh, Priyanka; Singh, Sudhir Kumar; Siddiqi, Mohammad Imran; Tripathi, Timir; Akhtar, Md. Sohail

    2016-01-01

    RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription. PMID:27503426

  18. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  19. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  20. Peptides present in the non-digestible fraction of common beans (Phaseolus vulgaris L.) inhibit the angiotensin-I converting enzyme by interacting with its catalytic cavity independent of their antioxidant capacity.

    PubMed

    Luna-Vital, Diego A; González de Mejía, Elvira; Mendoza, Sandra; Loarca-Piña, Guadalupe

    2015-05-01

    The aim was to evaluate the angiotensin-I converting enzyme (ACE) inhibitory potential and the antioxidant capacity of pure synthesized peptides (GLTSK, LSGNK, GEGSGA, MPACGSS and MTEEY) originally identified in the non-digestible fraction (NDF) of common beans (P. vulgaris L.) that had previously demonstrated antiproliferative activity against human colorectal cancer cells. The five peptides were able to inhibit ACE with half maximal inhibitory concentration (IC50) values ranging from 65.4 (GLTSK) to 191.5 μM (MPACGSS). The combination of GLTSK and MTEEY increased the ACE inhibition by 30% compared to equieffective doses of the single peptides. According to molecular docking analysis, the five peptides had lower estimated free energy values (-6.47 to -9.34 kcal mol(-1)) when they interacted with the catalytic site of ACE than that of the substrate hippuryl-histidyl-leucine (-5.41 kcal mol(-1)), thus inhibiting the enzymatic activity. According to molecular docking analysis, the five peptides interacted with four (His353, Ala354, Glu411 and Tyr523) out of 6 catalytic residues. Moreover, MPACGSS had the highest antioxidant activity according to Ferric reducing antioxidant power (FRAP) (421.58 μmol FeSO4 mg(-1)), Fe(2+) chelation (2.01 μmol Na2EDTA mg(-1)) assays, and also in DPPH (748.39 μmol Trolox per mg of dry peptide) and ABTS (561.42 μmol Trolox mg(-1)) radical scavenging assays. The results support the hypothesis that peptides present in the non-digestible fraction of common beans (Phaseolus vulgaris L.) may exert their physiological benefits independent of their antioxidant capacity, by ACE inhibition through interaction with its catalytic cavity. PMID:25881860

  1. Abnormally high digestive enzyme activity and gene expression explain the contemporary evolution of a Diabrotica biotype able to feed on soybeans.

    PubMed

    Curzi, Matías J; Zavala, Jorge A; Spencer, Joseph L; Seufferheld, Manfredo J

    2012-08-01

    Western corn rootworm (Diabrotica virgifera) (WCR) depends on the continuous availability of corn. Broad adoption of annual crop rotation between corn (Zea mays) and nonhost soybean (Glycine max) exploited WCR biology to provide excellent WCR control, but this practice dramatically reduced landscape heterogeneity in East-central Illinois and imposed intense selection pressure. This selection resulted in behavioral changes and "rotation-resistant" (RR) WCR adults. Although soybeans are well defended against Coleopteran insects by cysteine protease inhibitors, RR-WCR feed on soybean foliage and remain long enough to deposit eggs that will hatch the following spring and larvae will feed on roots of planted corn. Other than documenting changes in insect mobility and egg laying behavior, 15 years of research have failed to identify any diagnostic differences between wild-type (WT)- and RR-WCR or a mechanism that allows for prolonged RR-WCR feeding and survival in soybean fields. We documented differences in behavior, physiology, digestive protease activity (threefold to fourfold increases), and protease gene expression in the gut of RR-WCR adults. Our data suggest that higher constitutive activity levels of cathepsin L are part of the mechanism that enables populations of WCR to circumvent soybean defenses, and thus, crop rotation. These new insights into the mechanism of WCR tolerance of soybean herbivory transcend the issue of RR-WCR diagnostics and management to link changes in insect gut proteolytic activity and behavior with landscape heterogeneity. The RR-WCR illustrates how agro-ecological factors can affect the evolution of insects in human-altered ecosystems. PMID:22957201

  2. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

    PubMed

    Chauvigné, François; Verdura, Sara; Mazón, María José; Boj, Mónica; Zanuy, Silvia; Gómez, Ana; Cerdà, Joan

    2015-09-15

    In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes. PMID:25449660

  3. Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

    PubMed

    López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

    2007-11-01

    An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen. PMID:17998551

  4. Influence of different length linker containing DHEA-7-cmo-enzyme conjugates on sensitivity and specificity of DHEA-3-hs-antibody.

    PubMed

    Shrivastav, Tulsidas G; Chaube, Shail K; Kariya, Kiran P; Singh, Rita; Kumar, Dinesh; Jain, Parul; Karwar, Suryakant; Uyake, Jyoti; Deshmukh, Bhavana

    2012-01-01

    The introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced different homobifunctional spacers having varying atomic length (3 to 10) between enzyme and dehydroepiandrosterone (DHEA) moiety and studied their effects on functional parameters such as sensitivity and specificity of DHEA enzyme immunoassays. DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-carboxymethyloxime (DHEA-7-CMO) as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as an enzyme label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that DHEA moiety having a 3-hemisuccinate carboxyl arm that is hydrophilic in nature and spacer arm urea that is also hydrophilic in nature when used for the link to the protein carrier and enzyme for the preparation of immunogen and enzyme conjugate respectively resulted in development of assay having comparable sensitivity and lowest ED(50) as compared to other spacers. Thus sensitivity and ED(50) of the assay depend partly on the nature of the steroid and spacer arm link to the carrier protein and the enzyme. PMID:22181816

  5. Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma.

    PubMed

    Greifenhagen, Uta; Nguyen, Viet Duc; Moschner, Johann; Giannis, Athanassios; Frolov, Andrej; Hoffmann, Ralf

    2015-02-01

    Glycation refers to a nonenzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end products (AGEs), such as N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML). Although CEL and CML are considered to be markers of arteriosclerosis, diabetes mellitus, and aging, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and a lack of enrichment strategies. Here, we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML, m/z 142.1 and 187.1; CEL, m/z 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding m/z values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC-MS/MS. The same strategy revealed 21 CML sites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus. PMID:25423611

  6. Action pattern, specificity, lytic activities, and physiological role of five digestive beta-glucanases isolated from Periplaneta americana.

    PubMed

    Genta, Fernando A; Terra, Walter R; Ferreira, Clélia

    2003-11-01

    Three laminarinases (LAM, LIC 1, and LIC 2) and two cellulases (CEL 1 and CEL 2) were purified to homogeneity from Periplaneta americana midguts. These beta-glucanases are secreted by salivary glands, stabilized by calcium ions, and have pH optima around 6. LAM (46 kDa) is active only on laminarin, native or with oxidized ends, and so it is an endo-beta-1,3-glucanase (EC 3.2.1.39). It processively releases mainly glucose from laminarin and shows lytic activity on fungal cells. LIC 1 (25 kDa) is an endo-beta-1,3(4)-glucanase (EC 3.2.1.6.), because it cleaves internal bonds on both laminarin and lichenin. LIC 1 lyses fungal cells and apparently have high affinity for sequences of cellotetraoses linked by beta-1,3 links, releasing cellotetraose from lichenin. The reaction catalyzed by LIC 1 is not in rapid equilibrium, as suggested by activity-pH data. These data also showed that a group in LIC 1 with pK=4.9 is necessary for substrate binding. LIC 2 (23 kDa) seems to be similar to LIC 1. The laminarinases are inactivated by carbodiimide, suggesting the presence of a carboxyl group involved in catalysis. LAM and LIC 2 are inhibited by excess laminarin as substrate. CEL 1 (72 kDa) and CEL 2 (73 kDa) quickly decrease the molecular weight of lichenin used as substrate. Therefore, they are endo-beta-1,4-glucanases (EC 3.2.1.4). Both CEL 1 and CEL 2 are also active on crystalline cellulose. The specificities of P. americana beta-glucanases agree with the omnivorous detritus-feeding habit of this insect, as they are able to attack plant (CEL 1, CEL 2, LIC 1 and LIC 2) and fungal (LIC 1 and LAM) cell walls. PMID:14563360

  7. CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

    PubMed Central

    Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L.; Precht, Patricia; Mughal, Mohamed R.; Wood, William H.; Zhang, Yonqing; Becker, Kevin G.; Mattson, Mark P.; Pazin, Michael J.

    2011-01-01

    CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease. PMID:21931736

  8. Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition.

    PubMed

    Zeng, Haopeng; Chen, Jiahong; Zhang, Chijian; Huang, Xin-An; Sun, Yuanming; Xu, Zhenlin; Lei, Hongtao

    2016-04-01

    On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens. PMID:26976361

  9. Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains

    SciTech Connect

    Choi, Ji-Hye; Lee, Heeseob; Kim, Young-Wan; Park, Jong-Tae; Woo, Eui-Jeon; Kim, Myo-Jeong; Lee, Byong-Hoon

    2009-01-09

    A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both {alpha}-(1,6)- and {alpha}-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with {sup 14}C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-{alpha}-(1,6)-{beta}-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

  10. Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

    PubMed Central

    Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

    2015-01-01

    The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

  11. Development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses.

    PubMed

    Kooijman, Lotte J; Mapes, Samantha M; Pusterla, Nicola

    2016-07-01

    Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations. PMID:27216723

  12. An Enzyme-Mediated Methodology for the Site-Specific Radiolabeling of Antibodies Based on Catalyst-Free Click Chemistry

    PubMed Central

    Zeglis, Brian M.; Davis, Charles B.; Aggeler, Robert; Kang, Hee Chol; Chen, Aimei; Agnew, Brian J.; Lewis, Jason S.

    2013-01-01

    An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal 89Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with 89Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing 89Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to high selective tumor uptake (67.5 ± 5.0 %ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic. PMID:23688208

  13. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins.

    PubMed

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2016-01-01

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC50 values for aflatoxins G₁ and G₂ were 17.18 ng·mL(-1) and 19.75 ng·mL(-1), respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL(-1). To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi. PMID:26729164

  14. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins

    PubMed Central

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2015-01-01

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G1 and G2. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G1 and G2, and did not cross-react with aflatoxins B1, B2, or M1. Its IC50 values for aflatoxins G1 and G2 were 17.18 ng·mL−1 and 19.75 ng·mL−1, respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL−1. To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G1 and G2 and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B1-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi. PMID:26729164

  15. Development of a highly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples.

    PubMed

    Han, Dan; Yu, Meng; Knopp, Dietmar; Niessner, Reinhard; Wu, Mei; Deng, Anping

    2007-08-01

    A highly selective and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for Sudan I was developed. Two hapten derivatives with different lengths of carboxylic spacer at the azo-bound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OA) conjugates were applied as coating antigens. The antisera which were obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions it was found that IC50 and LOD values of seven pairs based on four antisera and two coating antigens were in the range of 0.3-2 ng/mL and 0.02-0.1 ng/mL, respectively. The most sensitive ELISA could be established with Sudan I-propionic acid-OA coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with six edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of three replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r = 0.9851, n = 7), demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples. PMID:17622156

  16. Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

    PubMed

    Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-08-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

  17. Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

    PubMed Central

    Prieto, José M.; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E.; Garrido, Joseba M.; Juste, Ramon A.

    2014-01-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

  18. Extracellular enzyme kinetics scale with resource availability

    EPA Science Inventory

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  19. Enzyme Therapy: Current Perspectives.

    PubMed

    UmaMaheswari, Thiyagamoorthy; Hemalatha, Thiagarajan; Sankaranarayanan, Palavesam; Puvanakrishnan, Rengarajulu

    2016-01-01

    Enzymes control all metabolic processes in human system from simple digestion of food to highly complex immune response. Physiological reactions occuring in healthy individuals are disturbed when enzymes are deficient or absent. Enzymes are administered for normalizing biological function in certain pathologies. Initially, crude proteolytic enzymes were used for the treatment of gastrointestinal disorders. Recent advances have enabled enzyme therapy as a promising tool in the treatment of cardiovascular, oncological and hereditary diseases. Now, a spectrum of other diseases are also covered under enzyme therapy. But, the available information on the use of enzymes as therapeutic agents for different diseases is scanty. This review details the enzymes which have been used to treat various diseases/disorders. PMID:26891548

  20. Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

    SciTech Connect

    Bhushan, Bharat; Halasz, Annamaria; Hawari, Jalal . E-mail: jalal.hawari@nrc.ca

    2005-12-02

    A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24 nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393 Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1 Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D{sup -}) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.

  1. A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.

    PubMed

    Maeda, M; Murayama, N; Ishii, H; Uryu, N; Ota, M; Tsuji, K; Inoko, H

    1989-11-01

    The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing. PMID:2576477

  2. Pretreatment of coconut mill effluent using celite-immobilized hydrolytic enzyme preparation from Staphylococcus pasteuri and its impact on anaerobic digestion.

    PubMed

    Kanmani, Palanisamy; Kumaresan, Kuppamuthu; Aravind, Jeyaseelan

    2015-01-01

    Biological treatment of oil and grease (O&G)-containing industrial effluents has long been a challenging issue. Practically feasible avenues to bring down their O&G load and enhance treatability are desired. In one such endeavour, the partially purified lipase from Staphylococcus pasteuri COM-4A was immobilized on celite carrier and applied for the enzymatic hydrolysis of unsterilized coconut oil mill effluent. In batch hydrolysis experiments, optimum conditions of 1% (w/v) immobilized lipase beads, one in four effluent dilution, and a contact time of 30 h resulted in 46% and 24% increase in volatile fatty acids and long-chain fatty acids and a concomitant 52% and 32% decrease in O&G and chemical oxygen demand (COD) levels, respectively. Batch anaerobic biodegradation trials with this prehydrolyzed effluent showed 89%, 91%, and 90% decrease in COD, proteins, and reducing sugars, respectively. These results were validated in a hybrid stirred tank--upflow anaerobic sludge blanket reactor. Average COD and O&G reductions effected by the hybrid reactor were found to be 89% and 88%, whereas that by the control reactor without enzymatic hydrolysis were only 60% and 47%, respectively. A maximum of 0.86 L methane gas was generated by the hybrid reactor per gram of VS added. Hence, this celite-immobilized crude lipase, sourced from a native laboratory isolate, seems to be a workable alternative to commercial enzyme preparations for the management of lipid-rich industrial effluents. PMID:26033963

  3. Effects of Industrial Heating Processes of Milk-Based Enteral Formulas on Site-Specific Protein Modifications and Their Relationship to in Vitro and in Vivo Protein Digestibility.

    PubMed

    Wada, Yasuaki; Lönnerdal, Bo

    2015-08-01

    Heat treatments are applied to milk and dairy products to ensure their microbiological safety and shelf lives. Types of heating processes may have different effects on protein modifications, leading to different protein digestibility. In this study, milk-based liquid nutritional formulas (simulating enteral formulas) were subjected to steam injection ultra-high-temperature treatment or in-can sterilization, and the formulas were investigated by proteomic methods and in vitro and in vivo digestion assays. Proteomic analyses revealed that in-can sterilization resulted in higher signals for N(ε)-carboxymethyllysine and dephosphorylation of Ser residues in major milk proteins than in steam-injected formula, reflecting the more severe thermal process of in-can sterilization. In vitro and in vivo digestion assays indicated that steam injection improved protein digestibility, supposedly by denaturation, while the improvement seemed to be overwhelmed by formation of aggregates that showed resistance to digestion in in-can sterilized formula. Adverse effects of heat treatment on protein digestibility are more likely to be manifested in milk-based formulas than in cow's milk. Although the differences might be of limited significance in terms of amino acid bioavailability, these results emphasize the importance of protein quality of raw materials and selection of heating processes. PMID:26161498

  4. The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

    SciTech Connect

    Paxman, Jason J.; Borg, Natalie A.; Horne, James; Thompson, Philip E.; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S.; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M.; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P.; Rossjohn, Jamie; Scanlon, Martin J.

    2010-09-07

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  5. Sediment distribution, hydrolytic enzyme profiles and bacterial activities in the guts of Oneirophanta mutabilis, Psychropotes longicauda and Pseudostichopus villosus: what do they tell us about digestive strategies of abyssal holothurians?

    NASA Astrophysics Data System (ADS)

    Roberts, D.; Moore, H. M.; Berges, J.; Patching, J. W.; Carton, M. W.; Eardly, D. F.

    This paper describes inter-specific differences in the distribution of sediment in the gut compartments and in the enzyme and bacterial profiles along the gut of abyssal holothurian species - Oneirophanta mutabilis, Psychropotes longicauda and Pseudostichopus villosus sampled from a eutrophic site in the NE Atlantic at different times of the year. Proportions of sediments, relative to total gut contents, in the pharynx, oesophagus, anterior and posterior intestine differed significantly in all the inter-species comparisons, but not between inter-seasonal comparisons. Significant differences were also found between the relative proportions of sediments in both the rectum and cloaca of Psychropotes longicauda and Oneirophanta mutabilis. Nineteen enzymes were identified in either gut-tissue or gut-content samples of the holothurians studied. Concentrations of the enzymes in gut tissues and their contents were highly correlated. Greater concentrations of the enzymes were found in the gut tissues suggesting that they are the main source of the enzymes. The suites of enzymes recorded were broadly similar in each of the species sampled collected regardless of the time of the year, and they were similar to those described previously for shallow-water holothurians. Significant inter-specific differences in the gut tissue concentrations of some of the glycosidases suggest dietary differences. For example, Psychropotes longicauda and Pseudostichopus villosus contain higher levels of chitobiase than Oneirophanta mutabilis. There were no seasonal changes in bacterial activity profiles along the guts of O. mutabilis and Pseudostichopus villosus. In both these species bacterial activity and abundance declined between the pharynx/oesophagus and anterior intestine, but then increased along the gut and became greatest in the rectum/cloaca. Although the data sets were more limited for Psychropotes longicauda, bacterial activity increased from the anterior to the posterior intestine

  6. Glutathione-dependent detoxifying enzymes in rainbow trout liver: Search for specific biochemical markers of chemical stress

    SciTech Connect

    Petrivalsky, M.; Machala, M.; Nezveda, K.; Piacka, V.; Svobodova, Z. |; Drabek, P.

    1997-07-01

    Activities of trout liver microsomal glutathione S-transferase (GST) and a series of cytosolic glutathione-dependent detoxifying enzymes were determined after a single intraperitoneal treatment with phenobarbital, 2,2-bis (p-chlorophenyl)-1,1-dichloroethane (p,p{prime}-DDE), 2,3-dimethoxynaphthoquinone (NQ), or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study aimed to find xenobiotic-specific parameters applicable as biochemical markers of the impacts of the prototypal xenobiotics. The effects of xenobiotics on cytosolic GST activities were substrate dependent. The rate of conjugation of p-nitrobenzyl chloride was significantly induced by higher doses of p,p{prime}-DDE or NQ. The conjugation of ethacrynic acid was enhanced by phenobarbital, p,p{prime}-DDE, and NQ. The GST activity against 1,2-epoxy-3-(p-nitrophenoxy)propane was induced only by phenobarbital and by lower doses of p,p{prime}-DDE. The cytosolic GST activity, measured with 1-chloro-2,4-dinitrobenzene as a substrate, was only weakly increased by phenobarbital, TCDD, higher doses of p,p{prime}-DDE, or by NQ at the lowest dose of 1 mg/kg. Although the latter activity is frequently used as a biomarker in ecotoxicology, various factors (including its weak inducibility) indicate that this biochemical parameter is probably not a suitable indicator of contamination in fish. Similarly, cytosolic glutathione peroxidase was not affected by the prototypal xenobiotics and appeared to be an unsuitable bioindicator of oxidative impacts of the tested compounds. On the other hand, microsomal GST activity was nonspecifically increased by phenobarbital, NQ, TCDD, and high doses of p,p{prime}-DDE. Glutathione reductase, another potential biomarker of oxidative stress, was induced by phenobarbital, NQ, and, to a lesser extent, p,p{prime}-DDE; therefore it appeared to be a less sensitive indicator to the exposure to prototypal xenobiotics than the microsomal GST.

  7. Enzyme-free detection of sequence-specific microRNAs based on nanoparticle-assisted signal amplification strategy.

    PubMed

    Li, Ru-Dong; Wang, Qian; Yin, Bin-Cheng; Ye, Bang-Ce

    2016-03-15

    Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA-Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe-miRNA-CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu(2+)). Cu(2+) could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA-Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu(2+), which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5 pM to 1000 pM with a detection limit of ~0.73 pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit. PMID:26547010

  8. Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

    PubMed Central

    Callahan, Scott J.; Luyten, Yvette A.; Gupta, Yogesh K.; Wilson, Geoffrey G.; Roberts, Richard J.; Morgan, Richard D.; Aggarwal, Aneel K.

    2016-01-01

    The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others. PMID:27082731

  9. Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, de...

  10. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis.

    PubMed

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the -2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the -1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  11. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis

    PubMed Central

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  12. The role of specific Smad linker region phosphorylation in TGF-β mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle.

    PubMed

    Rostam, Muhamad A; Kamato, Danielle; Piva, Terence J; Zheng, Wenhua; Little, Peter J; Osman, Narin

    2016-08-01

    Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis. PMID:27153775

  13. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors.

    PubMed

    Jian, Hui; Wang, Yingwu; Bai, Yan; Li, Rong; Gao, Renjun

    2016-01-01

    Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. PMID:27409601

  14. Digestibility of gluten proteins is reduced by baking and enhanced by starch digestion

    PubMed Central

    Pan, Xiaoyan; Bellido, Vincent; Toole, Geraldine A.; Gates, Fred K.; Wickham, Martin S. J.; Shewry, Peter R.; Bakalis, Serafim; Padfield, Philip; Mills, E. N. Clare

    2015-01-01

    Scope Resistance of proteins to gastrointestinal digestion may play a role in determining immune‐mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood. Methods and results Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS‐PAGE and immunoblotting using monoclonal antibodies specific for celiac‐toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases. Conclusion The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten‐starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices. PMID:26202208

  15. Rapid Online Non-Enzymatic Protein Digestion Combining Microwave Heating Acid Hydrolysis and Electrochemical Oxidation

    PubMed Central

    Basile, Franco; Hauser, Nicolas

    2010-01-01

    We report an online non-enzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision induced dissociation (CID) tandem mass spectrometry (MS/MS). The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical (microwave/echem) digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel non-enzymatic digestion method, when analyzed by ESI-MS, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two non-enzymatic methods overcomes shortcomings with each individual method in that: i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids, and ii) the inability of the electrochemical-cleavage method to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min digestion time) on a series of standard peptides and proteins as well as an E. coli protein extract. PMID:21138252

  16. Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes.

    PubMed

    Hay, Sam; Johannissen, Linus O; Hothi, Parvinder; Sutcliffe, Michael J; Scrutton, Nigel S

    2012-06-13

    The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions. PMID:22632111

  17. Modifying crops to increase cell wall digestibility.

    PubMed

    Jung, Hans-Joachim G; Samac, Deborah A; Sarath, Gautam

    2012-04-01

    Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants is highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-digestible. Digestibility of grasses is slowed severely by lignification of most tissues, but these cell walls remain largely digestible. Cell wall lignification creates an access barrier to potentially digestible wall material by rumen bacteria if cells have not been physically ruptured. Traditional breeding has focused on increasing total dry matter digestibility rather than cell wall digestibility, which has resulted in minimal reductions in cell wall lignification. Brown midrib mutants in some annual grasses exhibit small reductions in lignin concentration and improved cell wall digestibility. Similarly, transgenic approaches down-regulating genes in monolignol synthesis have produced plants with reduced lignin content and improved cell wall digestibility. While major reductions in lignin concentration have been associated with poor plant fitness, smaller reductions in lignin provided measurable improvements in digestibility without significantly impacting agronomic fitness. Additional targets for genetic modification to enhance digestibility and improve roughages for use as biofuel feedstocks are discussed; including manipulating cell wall polysaccharide composition, novel lignin structures, reduced lignin/polysaccharide cross-linking, smaller lignin polymers, enhanced development of non-lignified tissues, and targeting specific cell types. Greater tissue specificity of transgene expression will be needed to maximize benefits while avoiding negative impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promoter. PMID:22325867

  18. Comparative Digestive Physiology

    PubMed Central

    Karasov, William H.; Douglas, Angela E.

    2015-01-01

    In vertebrates and invertebrates, morphological and functional features of gastrointestinal (GI) tracts generally reflect food chemistry, such as content of carbohydrates, proteins, fats, and material(s) refractory to rapid digestion (e.g., cellulose). The expression of digestive enzymes and nutrient transporters approximately matches the dietary load of their respective substrates, with relatively modest excess capacity. Mechanisms explaining differences in hydrolase activity between populations and species include gene copy number variations and single-nucleotide polymorphisms. Transcriptional and posttranscriptional adjustments mediate phenotypic changes in the expression of hydrolases and transporters in response to dietary signals. Many species respond to higher food intake by flexibly increasing digestive compartment size. Fermentative processes by symbiotic microorganisms are important for cellulose degradation but are relatively slow, so animals that rely on those processes typically possess special enlarged compartment(s) to maintain a microbiota and other GI structures that slow digesta flow. The taxon richness of the gut microbiota, usually identified by 16S rRNA gene sequencing, is typically an order of magnitude greater in vertebrates than invertebrates, and the interspecific variation in microbial composition is strongly influenced by diet. Many of the nutrient transporters are orthologous across different animal phyla, though functional details may vary (e.g., glucose and amino acid transport with K+ rather than Na+ as a counter ion). Paracellular absorption is important in many birds. Natural toxins are ubiquitous in foods and may influence key features such as digesta transit, enzymatic breakdown, microbial fermentation, and absorption PMID:23720328

  19. Enzyme array-amperometric detection in carbohydrate analysis

    SciTech Connect

    Sun, M.; Lee, C.S.

    1998-03-05

    The introduction of an enzyme array-electrochemical detection method for carbohydrate analysis is demonstrated by using two complex and one high mannose N-linked oligosaccharides. Instead of measuring the remaining uncleaved oligosaccharides in enzymatic digestion, released monosaccharides are directly quantified by pulsed amperometric detection at a gold electrode. The measured monosaccharide concentrations in combination with the enzyme array analysis provide structural characterization of oligosaccharides. The enzyme array-electrochemical detection method does not require any separation procedure or any prior labeling of oligosaccharides. However, this method is limited by the use of purified oligosaccharide samples and the nature of the enzyme array. The development of more sophisticated enzyme arrays relies upon the introduction of a bank of highly specific (bond, arm, aglycon) exoglycosidases.

  20. Crystal structures of the Helicobacter pylori MTAN enzyme reveal specific interactions between S-adenosylhomocysteine and the 5'-alkylthio binding subsite.

    PubMed

    Mishra, Vidhi; Ronning, Donald R

    2012-12-01

    The bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadenosine (5'-DOA), and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for Helicobacter pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and the 5'-alkylthiol binding site of MTAN have never been resolved. We have determined crystal structures of an inactive mutant form of H. pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH, allowing the determination of the structure of a ternary enzyme-product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5'-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori. PMID:23148563

  1. Mi2β Shows Chromatin Enzyme Specificity by Erasing a DNase I-hypersensitive Site Established by ACF*S⃞

    PubMed Central

    Ishii, Haruhiko; Du, Hansen; Zhang, Zhaoqing; Henderson, Angus; Sen, Ranjan; Pazin, Michael J.

    2009-01-01

    ATP-dependent chromatin-remodeling enzymes are linked to changes in gene expression; however, it is not clear how the multiple remodeling enzymes found in eukaryotes differ in function and work together. In this report, we demonstrate that the ATP-dependent remodeling enzymes ACF and Mi2β can direct consecutive, opposing chromatin-remodeling events, when recruited to chromatin by different transcription factors. In a cell-free system based on the immunoglobulin heavy chain gene enhancer, we show that TFE3 induces a DNase I-hypersensitive site in an ATP-dependent reaction that requires ACF following transcription factor binding to chromatin. In a second step, PU.1 directs Mi2β to erase an established DNase I-hypersensitive site, in an ATP-dependent reaction subsequent to PU.1 binding to chromatin, whereas ACF will not support erasure. Erasure occurred without displacing the transcription factor that initiated the site. Other tested enzymes were unable to erase the DNase I-hypersensitive site. Establishing and erasing the DNase I-hypersensitive site required transcriptional activation domains from TFE3 and PU.1, respectively. Together, these results provide important new mechanistic insight into the combinatorial control of chromatin structure. PMID:19158090

  2. Molecular Characterization of an rsmD-Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme

    PubMed Central

    Rana, Ajay Kumar; Chandra, Sharat; Siddiqi, Mohammad Imran

    2013-01-01

    The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi, its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi. The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC50], 0.32 μM), followed by NSC-658343 (IC50, 4.13 μM) and NSC-657589 (IC50, 7.5 μM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors. PMID:23733469

  3. Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

    PubMed

    Itoh, Nobuya; Kazama, Miki; Takeuchi, Nami; Isotani, Kentaro; Kurokawa, Junji

    2016-06-01

    Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies. PMID:27419059

  4. Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

    PubMed Central

    Berg Miller, Margret E.; Antonopoulos, Dionysios A.; Rincon, Marco T.; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M.; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J.; Bayer, Edward A.; White, Bryan A.

    2009-01-01

    Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has

  5. Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

    PubMed

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2014-11-01

    There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap. PMID:25259405

  6. [Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

    PubMed

    Vitkova, O N; Kapustina, T P; Mikhailova, V V; Safonov, G A; Vlasova, N N; Belousova, R V

    2015-01-01

    The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases. PMID:27024917

  7. Detection of Specific Antibodies against Tembusu Virus in Ducks by Use of an E Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Yin, Xiuchen; Lv, Rang; Chen, Xiaodan; Hua, Ronghong

    2013-01-01

    We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks. PMID:23616462

  8. Crystal Structures of the Helicobacter pylori MTAN Enzyme Reveal Specific Interactions between S-Adenosylhomocysteine and the 5'-Alkylthio Binding Subsite

    SciTech Connect

    Mishra, Vidhi; Ronning, Donald R.

    2012-11-13

    The bacterial 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5'-methylthioadenosine (MTA), 5'-deoxyadenosine (5'-DOA), and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for Helicobacter pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and the 5'-alkylthiol binding site of MTAN have never been resolved. We have determined crystal structures of an inactive mutant form of H. pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH, allowing the determination of the structure of a ternary enzyme–product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5'-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori.

  9. Screening of white-rot fungi manganese peroxidases: a comparison between the specific activities of the enzyme from different native producers

    PubMed Central

    2012-01-01

    In this study manganese peroxidase (MnP) enzymes from selected white-rot fungi were isolated and compared for potential future recombinant production. White-rot fungi were cultivated in small-scale in liquid media and a simplified process was established for the purification of extracellular enzymes. Five lignin degrading organisms were selected (Bjerkandera sp., Phanerochaete (P.) chrysosporium, Physisporinus (P.) rivulosus, Phlebia (P.) radiata and Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). In total 8 different MnP isoforms were purified. Results of this study reinforce the conception that MnPs from distinct organisms differ substantially in their properties. Production of the extracellular enzyme in general did not reach a substantial level. This further suggests that these native producers are not suitable for industrial scale production of the enzyme. The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa. PMID:23190610

  10. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland

    PubMed Central

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Čapek, Petr; Kaiser, Christina; Torsvik, Vigdis L.; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation (“buried topsoils”), resulting from a decrease in fungal abundance compared to recent (“unburied”) topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation. PMID

  11. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland.

    PubMed

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Capek, Petr; Kaiser, Christina; Torsvik, Vigdis L; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation ("buried topsoils"), resulting from a decrease in fungal abundance compared to recent ("unburied") topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation. PMID:25360132

  12. Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle*

    PubMed Central

    DeBalsi, Karen L.; Wong, Kari E.; Koves, Timothy R.; Slentz, Dorothy H.; Seiler, Sarah E.; Wittmann, April H.; Ilkayeva, Olga R.; Stevens, Robert D.; Perry, Christopher G. R.; Lark, Daniel S.; Hui, Simon T.; Szweda, Luke; Neufer, P. Darrell; Muoio, Deborah M.

    2014-01-01

    Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIPSKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIPSKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability. PMID:24482226

  13. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII.

    PubMed Central

    Summers, J

    1975-01-01

    Digestion of polyoma viral DNA with a restriction enzyme from Haemophilus aegyptius generates at least 22 unique fragments. The fragments have been characterized with respect to size and physical order on the polyoma genome, and the 5' to 3' orientation of the (+) and (-) strands has been determined. A method for specific radiolabeling of adjacent fragments was employed to establish the fragment order. This technique may be useful for ordering the fragments produced by digestion of complex DNAs. Images PMID:163927

  14. Influence of sporophore development, damage, storage, and tissue specificity on the enzymic formation of volatiles in mushrooms (Agaricus bisporus).

    PubMed

    Combet, Emilie; Henderson, Janey; Eastwood, Daniel C; Burton, Kerry S

    2009-05-13

    The enzymic oxidation of the polyunsaturated fatty acid-linoleic acid leads, in fungi, to the formation of a unique class of nonconjugated hydroperoxides, which are cleaved to form eight-carbon volatiles characteristic of mushroom and fungal flavor. However, the enzymes involved in this biosynthetic pathway, the bioavailability of the fatty acid substrate, and the occurrence of the reaction products (hydroperoxides and eight-carbon volatiles) are not fully understood. This study investigated the lipids, fatty acids, and hydroperoxide levels, as well as eight-carbon volatile variations in the fungal model Agaricus bisporus, according to four parameters: sporophore development, postharvest storage, tissue type, and damage. Eight-carbon volatiles were measured using solid phase microextraction and gas chromatography-mass spectrometry. Tissue disruption had a major impact on the volatile profile, both qualitatively and quantitatively; 3-octanone was identified as the main eight-carbon volatile in whole and sliced sporophore, an observation overlooked in previous studies due to the use of tissue disruption and solvent extraction for analysis. Fatty acid oxidation and eight-carbon volatile emissions decreased with sporophore development and storage, and differed according to tissue type. The release of 1-octen-3-ol and 3-octanone by incubation of sporophore tissue homogenate with free linoleic acid was inhibited by acetylsalicylic acid, providing evidence for the involvement of a heme-dioxygenase in eight-carbon volatile production. PMID:19326947

  15. [Characteristics of proteinase digestive function in invertebrates--inhabitants of cold seas].

    PubMed

    Mukhin, V A; Smirnova, E B; Novikov, V Iu

    2007-01-01

    Digestive proteinases of various taxa of invertebrates of the Northern seas have been studied: crustaceans Paralithodes camtchaticus, Pandalus borealis; molluscs Chlamys islandicus, Buccinum undatum, Serripes groenlandicus, and echinoderms Strongylocentrotus droebachiensis, Cucumaria frondosa, Asterias rubens, and Grossaster papposus. The presence of two proteolytic activity peaks in the acid (pH 2.5-3.5) and low alkaline zones (pH 7.5-8.5) and a similar proteinase spectrum have been revealed in digestive organs of the studied animals. The proteolytic activity in digestive organs of the Barents Sea invertebrates exceeds significantly that of terrestrial homoiothermal animals, which seems to be an extensive compensation for poor differentiation of the digestive system and for low substrate specificity of the enzymes as well as for cold conditions of the habitat. The principal qualitative difference between vertebrates and invertebrates consists in that the latter have no pepsin activity, but do have the cathepsin activity that is absent in vertebrate digestive organs. Contribution to the acid proteolysis is made by lysosomal cathepsins, rather than by pepsins. Activity in the alkaline and neutral pH zones is provided by serine proteinases. In digestive cavities of invertebrates, hydrolysis of proteins and mechanical processing of food occur only in the low alkaline zone, whereas acid proteolysis has intracellular lysosomal localization. PMID:18038635

  16. A rapid DNA digestion system.

    PubMed

    Fu, Lung-Ming; Lin, Che-Hsin

    2007-04-01

    This paper presents a novel microfluidic DNA digestion system incorporating a high performance micro-mixer. Through the appropriate control of fixed and periodic switching DC electric fields, electrokinetic forces are established to mix the DNA and restriction enzyme samples and to drive them through the reaction column of the device. The experimental and numerical results show that a mixing performance of 98% can be achieved within a mixing channel of length 1.6 mm when a 150 V/cm driving voltage and a 5 Hz switching frequency are applied. The relationship between the mixing performance, switching frequency, and main applied electric field is derived. It is found that the optimal switching frequency depends upon the magnitude of the main applied electric field. The successful digestion of lambda-DNA using Eco RI restriction enzyme is demonstrated. The DNA-enzyme reaction is completed within 15 min in the proposed microfluidic system, compared to 50 min in a conventional large-scale system. Hence, the current device provides a valuable tool for rapid lambda-DNA digestion, while its mixer system delivers a simple yet effective solution for mixing problems in the micro-total-analysis-systems field. PMID:17195107

  17. A localized tolerance in the substrate specificity of the fluorinase enzyme enables "last-step" 18F fluorination of a RGD peptide under ambient aqueous conditions.

    PubMed

    Thompson, Stephen; Zhang, Qingzhi; Onega, Mayca; McMahon, Stephen; Fleming, Ian; Ashworth, Sharon; Naismith, James H; Passchier, Jan; O'Hagan, David

    2014-08-18

    A strategy for last-step (18)F fluorination of bioconjugated peptides is reported that exploits an "Achilles heel" in the substrate specificity of the fluorinase enzyme. An acetylene functionality at the C-2 position of the adenosine substrate projects from the active site into the solvent. The fluorinase catalyzes a transhalogenation of 5'-chlorodeoxy-2-ethynyladenosine (ClDEA) to 5'-fluorodeoxy-2-ethynyladenosine (FDEA). Extending a polyethylene glycol linker from the terminus of the acetylene allows the presentation of bioconjugation cargo to the enzyme for (18)F labelling. The method uses an aqueous solution (H2(18)O) of [(18)F]fluoride generated by the cyclotron and has the capacity to isotopically label peptides of choice for positron emission tomography (PET). PMID:24989327

  18. Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

    SciTech Connect

    Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

    2006-03-01

    Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.

  19. PpASCL, a moss ortholog of anther-specific chalcone synthase-like enzymes, is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway.

    PubMed

    Colpitts, Che C; Kim, Sung Soo; Posehn, Sarah E; Jepson, Christina; Kim, Sun Young; Wiedemann, Gertrud; Reski, Ralf; Wee, Andrew G H; Douglas, Carl J; Suh, Dae-Yeon

    2011-12-01

    Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes. We analyzed available P.patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P.patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity. Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl α-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL. These results suggest that ASCL functions together with other sporophyte-specific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants. PMID:21883237

  20. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    EPA Science Inventory

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  1. The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

    PubMed

    Reddi, Ravikumar; Singarapu, Kiran Kumar; Pal, Debnath; Addlagatta, Anthony

    2016-07-19

    It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell. PMID:27225936

  2. Dengue virus serotyping based on envelope and membrane and nonstructural protein NS1 serotype-specific capture immunoglobulin M enzyme-linked immunosorbent assays.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Su, Chien-Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2004-06-01

    Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping. PMID:15184425

  3. Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

    PubMed Central

    Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders. PMID:27006517

  4. Cell-Specific Expression of Homospermidine Synthase, the Entry Enzyme of the Pyrrolizidine Alkaloid Pathway in Senecio vernalis, in Comparison with Its Ancestor, Deoxyhypusine Synthase1

    PubMed Central

    Moll, Stefanie; Anke, Sven; Kahmann, Uwe; Hänsch, Robert; Hartmann, Thomas; Ober, Dietrich

    2002-01-01

    Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-β-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis. PMID:12226485

  5. Engineering Digestion: Multiscale Processes of Food Digestion.

    PubMed

    Bornhorst, Gail M; Gouseti, Ourania; Wickham, Martin S J; Bakalis, Serafim

    2016-03-01

    Food digestion is a complex, multiscale process that has recently become of interest to the food industry due to the developing links between food and health or disease. Food digestion can be studied by using either in vitro or in vivo models, each having certain advantages or disadvantages. The recent interest in food digestion has resulted in a large number of studies in this area, yet few have provided an in-depth, quantitative description of digestion processes. To provide a framework to develop these quantitative comparisons, a summary is given here between digestion processes and parallel unit operations in the food and chemical industry. Characterization parameters and phenomena are suggested for each step of digestion. In addition to the quantitative characterization of digestion processes, the multiscale aspect of digestion must also be considered. In both food systems and the gastrointestinal tract, multiple length scales are involved in food breakdown, mixing, absorption. These different length scales influence digestion processes independently as well as through interrelated mechanisms. To facilitate optimized development of functional food products, a multiscale, engineering approach may be taken to describe food digestion processes. A framework for this approach is described in this review, as well as examples that demonstrate the importance of process characterization as well as the multiple, interrelated length scales in the digestion process. PMID:26799793

  6. Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

    PubMed Central

    van Tiel, F H; Boere, W A; Vinjé, J; Harmsen, T; Benaissa-Trouw, B J; Kraaijeveld, C A; Snippe, H

    1984-01-01

    Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes. PMID:6386855

  7. Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027.

    PubMed

    Van Lanen, Steven G; Lin, Shuangjun; Dorrestein, Pieter C; Kelleher, Neil L; Shen, Ben

    2006-10-01

    C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically. PMID:16887797

  8. Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestion and selected MS/MS ion monitoring.

    PubMed

    Carrera, Mónica; Cañas, Benito; López-Ferrer, Daniel; Piñeiro, Carmen; Vázquez, Jesús; Gallardo, José M

    2011-07-15

    A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only 11 peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers. PMID:21627098

  9. Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers.

    PubMed

    Lei, Zhao; Shao, Yuxin; Yin, Xiaonan; Yin, Dafei; Guo, Yuming; Yuan, Jianmin

    2016-06-22

    Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers. PMID:27285356

  10. Copper-modified gold electrode specific for monosaccharide detection Use in amperometric determination of phenylmercury based on invertase enzyme inhibition.

    PubMed

    Mohammadi, H; Amine, A; El Rhazi, M; Brett, C M A

    2004-04-19

    The electrochemical oxidation of mono- and disaccharides at various copper-modified electrodes is reported: glassy carbon modified at open circuit or by electrochemical deposition of copper, gold modified by electrochemical deposition, and at bulk copper electrodes. A comparative study of these four electrodes was made by linear sweep voltammetry and amperometry. The maximum oxidation peak separation between disaccharides and monosaccharides is about 200mV. After optimization, amperometric determination of monosaccharides was done at +0.30 versus Ag/AgCl in 0.15M NaOH at the copper-modified gold electrode. Using the developed method, the enzymatic activities of invertase and beta-galactosidase were determined through their reaction with sucrose and lactose, respectively. Validation was carried out by a spectrophotometric method based on 3,5-dinitrosalicylic acid, and it was shown that the proposed electrochemical method is more sensitive. The analytical utility of the copper-modified gold electrode was tested for the determination of organic mercury. Addition of phenylmercury standards to the invertase solution caused a decrease in the enzyme activity, and allowed the determination of phenylmercury in pharmaceutical samples. The concentration has been determined in the 10-55ngml(-1) range. PMID:18969385

  11. Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

    PubMed

    Studt, Lena; Janevska, Slavica; Niehaus, Eva-Maria; Burkhardt, Immo; Arndt, Birgit; Sieber, Christian M K; Humpf, Hans-Ulrich; Dickschat, Jeroen S; Tudzynski, Bettina

    2016-03-01

    Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety. PMID:26662839

  12. A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

    PubMed Central

    Yuan, Jing; Hohn, Michael J.; Sherrer, R. Lynn; Palioura, Sotiria; Su, Dan; Söll, Dieter

    2010-01-01

    The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNACys (Sep-tRNACys) into Cys-tRNACys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase (SepSecS) converts O-phosphoseryl-tRNASec (Sep-tRNASec) to selenocysteinyl-tRNASec (Sec-tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that SeptRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNASec to Cys-tRNASec, and that Sep is crucial for SepCysS recognition. PMID:20493852

  13. Functional Utrastructure of Genlisea (Lentibulariaceae) Digestive Hairs

    PubMed Central

    Płachno, Bartosz Jan; Kozieradzka-Kiszkurno, Małgorzata; Świątek, Piotr

    2007-01-01

    Background and Aims Digestive structures of carnivorous plants produce external digestive enzymes, and play the main role in absorption. In Lentibulariaceae, the ultrastructure of digestive hairs has been examined in some detail in Pinguicula and Utricularia, but the sessile digestive hairs of Genlisea have received very little attention so far. The aim of this study was to fill this gap by expanding their morphological, anatomical and histochemical characterization. Methods Several imaging techniques were used, including light, confocal and electron microscopy, to reveal the structure and function of the secretory hairs of Genlisea traps. This report demonstrates the application of cryo-SEM for fast imaging of whole, physically fixed plant secretory structures. Key Results and Conclusion The concentration of digestive hairs along vascular bundles in subgenus Genlisea is a primitive feature, indicating its basal position within the genus. Digestive hairs of Genlisea consist of three compartments with different ultrastructure and function. In subgenus Tayloria the terminal hair cells are transfer cells, but not in species of subgenus Genlisea. A digestive pool of viscous fluid occurs in Genlisea traps. In spite of their similar architecture, the digestive-absorptive hairs of Lentibulariaceae feature differences in morphology and ultrastructure. PMID:17550910

  14. Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme*

    PubMed Central

    Dhindwal, Sonali; Patil, Dipak N.; Mohammadi, Mahmood; Sylvestre, Michel; Tomar, Shailly; Kumar, Pravindra

    2011-01-01

    Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphBB-356), the crystal structures of the apo-enzyme, the binary complex with NAD+, and the ternary complexes with NAD+-2,3-dihydroxybiphenyl and NAD+-4,4′-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphBB-356 converts the dihydrodiol metabolites of 3,3′-dichlorobiphenyl, 2,4,4′-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphBB-356 elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls. PMID:21880718

  15. The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Specificity and immobilization of aminopeptidase.

    PubMed

    Vosbeck, K D; Greenberg, B D; Awad, W M

    1975-05-25

    We recently described the purification of two aminopeptidases from Streptomyces griseus (Vosbeck, K.D., Chow, K.-F., and Awad, W.M., Jr. (1973) J. Biol. Chem. 248, 6329-6034). An analysis of the amino acid composition reveals very little differences in the two proteins. Each protein has alanine as the NH2-terminal residue. The aminopeptidases were treated separately with acetic anhydride; as noted in the past, the presence of glycerol is required to achieve excellent yields of acetylated active derivatives (Siegel, S., and Awad, W.M., Jr. (1973 J. Biol. Chem. 248, 3233-3240). However, in the present case much higher concentrations of glycerol (50%) are needed during acetylation to obtain derivatives with completely reacted NH2-terminal residues. The epsilon-amino groups were not completely acetylated. In contrast to the native enzymes, the acetylated derivatives show an affinity for DEAE-cellulose, a property consonant with the changes in net charge. The kinetic constants for each enzyme against L-leucine-p-nitroanilide do not change significantly after acetylation. The specificities of the two aminopeptidases were examined extensively on a semiquantitative basis. The activities are not restricted by the length of substrate chains. Each enzyme shows a preference for hydrophobic residues at the ultimate and penultimate positions. Charge residues are released a slower rates. No prolidase activity is demonstrable even at high enzyme to substrate ratios; however, NH2-terminal proline residues are released readily. D-Amino acid residues at the ultimate or penultimate position substantially reduce the rate of hydrolysis; D-leucyl-D-leucine is not hydrolyzed... PMID:805135

  16. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-Specific antibody in bison sera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. This report demonstrates that ELISAs for detection of M. bovis-specific antibody in cattle are not optimal for identification of seropositive bison. An ELISA optimized for use with bison sera is ...

  17. Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.

    PubMed Central

    Howard, T; Balogh, R; Overbeek, P; Bernstein, K E

    1993-01-01

    The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-