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Sample records for specific enzyme digestion

  1. Amazing pancreas: specific regulation of pancreatic secretion of individual digestive enzymes in rats.

    PubMed

    Maouyo, D; Morisset, J

    1995-02-01

    We investigated the effects of somatostatin (SMS)-201-995, atropine, and MK-329 on the role of cholinergic- and cholecystokinin-related systems and on the secretory relationship between five pancreatic digestive enzymes in rats. Animals kept in restraint cages and provided with pancreatic, biliary, duodenal, and jugular vein cannulas were treated as follows: 1) 0.25 micrograms.kg-1.h-1 caerulein alone, 2) both 0.25 micrograms.kg-1.h-1 caerulein and 100 micrograms.kg-1.h-1 atropine, 3) both caerulein and 5 micrograms.kg-1.h-1 SMS, 4) 91.3 micrograms.kg-1.h-1 carbachol alone, 5) both carbachol and 0.5 mg.kg-1.h-1 MK-329, and 6) both carbachol and 5 micrograms.kg-1.h-1 SMS, respectively. Food, but not water, was denied rats starting 10 h before the experiment and throughout the 6-h experimental period. The secretory patterns over the 6-h experimental period showed noticeably independent regulation of pancreatic secretion of individual digestive enzymes. The relationship between paired enzymes significantly varied according to the treatment. The correlation between chymotrypsinogen and the other enzymes was markedly modulated by MK-329. Our results suggest that SMS is a major "gate-keeper" in the regulation of exocrine pancreatic secretion and that the secretion of each digestive enzyme is individually regulated. Furthermore, they suggest that cholecystokinin and acetylcholine and their respective agonists are essentially initiators of secretory processes of the pancreas. Therefore, the paradigms of the regulation of pancreatic secretion heretofore accepted should be reexamined.

  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. Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes.

    PubMed

    Baeumlisberger, Dominic; Rohmer, Marion; Arrey, Tabiwang N; Mueller, Benjamin F; Beckhaus, Tobias; Bahr, Ute; Barka, Guenes; Karas, Michael

    2011-06-03

    The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.

  4. Digestive Enzyme Replacement Therapy: Pancreatic Enzymes and Lactase.

    PubMed

    Felicilda-Reynaldo, Rhea Faye D; Kenneally, Maria

    2016-01-01

    Maldigestion occurs when digestive enzymes are lacking to help break complex food components into absorbable nutrients within the gastrointestinal tract. Education is needed to help patients manage the intricacies of digestive enzyme replacement therapies and ensure their effectiveness in reducing symptoms of maldigestion.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase.

    PubMed

    Jones, Kyra; Sim, Lyann; Mohan, Sankar; Kumarasamy, Jayakanthan; Liu, Hui; Avery, Stephen; Naim, Hassan Y; Quezada-Calvillo, Roberto; Nichols, Buford L; Pinto, B Mario; Rose, David R

    2011-07-01

    Inhibition of intestinal α-glucosidases and pancreatic α-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 glycoside hydrolases responsible for the final step of starch hydrolysis. Here we compare the inhibition profiles of the individual N- and C-terminal catalytic subunits of both glucosidases by clinical glucosidase inhibitors, acarbose and miglitol, and newly discovered glucosidase inhibitors from an Ayurvedic remedy used for the treatment of Type II diabetes. We show that features of the compounds introduce selectivity towards the subunits. Together with structural data, the results enhance the understanding of the role of each catalytic subunit in starch digestion, helping to guide the development of new compounds with subunit specific antidiabetic activity. The results may also have relevance to other metabolic diseases such as obesity and cardiovascular disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

    PubMed

    Metes, D; Gambotto, A A; Nellis, J; Ruscin, A; Stewart-Akers, A M; Morel, P A; Rao, A S

    2001-12-01

    We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.

  8. Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.

    PubMed

    Amary, Maria Fernanda C; Pauwels, Patrick; Meulemans, Els; Roemen, Guido M; Islam, Lily; Idowu, Bernadine; Bousdras, Konstantinos; Diss, Timothy C; O'Donnell, Paul; Flanagan, Adrienne M

    2007-09-01

    Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

  9. Digestive enzymes of human and nonhuman primates.

    PubMed

    Janiak, Mareike Cora

    2016-09-01

    All living organisms need to consume nutrients to grow, survive, and reproduce, making the successful acquisition of food resources a powerful selective pressure. However, acquiring food is only part of the challenge. While all animals spend much of their daily activity budget hunting, searching for, or otherwise procuring food, a large part of what is involved in overall nutrition occurs once the meal has been swallowed. Most nutritional components are too complex for immediate use and must be broken down into simpler compounds, which can then be absorbed by the body. This process, digestion, is catalyzed by enzymes that are either endogenous or produced by the host's microbial population.(1) Research shows that the nutritional value of food is partially constrained by the digestive abilities of the microbial community present in the host's gut and that these microbes rapidly adapt to changes in diet and other environmental pressures.(2) An accumulating body of evidence suggests that endogenously produced digestive enzymes also have been, and still are, common targets of natural selection, further cementing their crucial role in an organism's digestive system.(3-5). © 2016 Wiley Periodicals, Inc.

  10. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  12. Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes.

    PubMed

    Boros, Eszter; Szabó, András; Zboray, Katalin; Héja, Dávid; Pál, Gábor; Sahin-Tóth, Miklós

    2017-02-17

    Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

  13. 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.

  14. Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus.

    PubMed

    Perera, Erick; Moyano, F J; Díaz, M; Perdomo-Morales, R; Montero-Alejo, V; Alonso, E; Carrillo, O; Galich, G S

    2008-07-01

    We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

  15. Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue.

    PubMed

    Gass, Jonathan; Bethune, Michael T; Siegel, Matthew; Spencer, Andrew; Khosla, Chaitan

    2007-08-01

    Celiac sprue is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Proteolytically resistant, proline- and glutamine-rich gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible persons. We investigated a new combination enzyme product, consisting of a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata), for its ability to digest gluten under gastric conditions. The ability of this combination enzyme to digest and detoxify whole-wheat bread gluten was investigated. In vitro and in vivo (rat) experimental systems were developed to simulate human gastric digestion, and the resulting material was analyzed by high-performance liquid chromatography, enzyme-linked immunoabsorbent assay, and patient-derived T-cell proliferation assays. The analysis revealed that EP-B2 extensively proteolyzes complex gluten proteins in bread, whereas SC PEP rapidly detoxifies the residual oligopeptide products of EP-B2 digestion. In vitro dose variation data suggests that an approximate 1:1 weight ratio of the 2 enzymes should maximize their synergistic potential. The efficacy of this 2-enzyme glutenase was verified in a rat model of gastric gluten digestion. By combining 2 enzymes with gastric activity and complementary substrate specificity, it should be possible to increase the safe threshold of ingested gluten, thereby ameliorating the burden of a highly restricted diet for patients with celiac sprue.

  16. Susceptibility of glutinous rice starch to digestive enzymes.

    PubMed

    Guo, Li; Zhang, Juanjuan; Hu, Jian; Li, Xueling; Du, Xianfeng

    2015-09-05

    To understand the susceptibility of glutinous rice starch to digestive enzymes and its potential impact on glycemic response, enzyme kinetics and in vitro digestibility of the native and gelatinized starches were investigated. The results showed that the Km values of the native and gelatinized starch were 10.35 mg/mL and 9.92 mg/mL, respectively. The digestion rate coefficients k values of the native and gelatinized starches were 2.0 × 10(-3)min(-1) and 1.1 × 10(-2)min(-1), respectively. The contents of rapid digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) in native glutinous rice starch were 8.92%, 21.52% and 69.56%, respectively. After gelatinization, the amounts of RDS, SDS and RS were 18.47%, 29.75% and 51.78%, respectively. The native and gelatinized glutinous rice starches were 10.34% and 14.07% for hydrolysis index (HI), as well as 43.14% and 45.92% for glycemic index (GI), respectively. During the in vitro digestion, the crystallinity of native glutinous rice starch was increased from 34.7% to 35.8% and 38.4% after 20 and 120 min, respectively.

  17. 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

  18. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. 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.

  20. [Effects of starvation on digestive enzyme activities of Monopterus albus].

    PubMed

    Yang, Dai-qin; Chen, Fang; Ruan, Guo-liang; Hu, Cheng-wen; Cao, Sheng-huan

    2007-05-01

    Starvation is a major environmental stress, which has a broad effect on the physiology and ecology of aquatic animals. In this study, Monopterus albus was starved for 30 days at (20 +/- 0.5) degrees C, and the activities of protease, trypsin, amylase and lipase in its digestive organs were measured on the 0, 3rd, 5th, 10th, 15th, 20th, and 30th day of starvation. The results showed that starvation had definite effects on the activities of all test enzymes. With the prolongation of starvation, the activities of test enzymes decreased, which was most significant when the fish was starved for 5-10 days. After 10 days of starvation, the decreasing trend of the enzyme activities became less obvious.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. Digestive enzyme and alkaline phosphatase activities during the early stages of Silurus soldatovi development.

    PubMed

    Liu, Wei; Zhang, Xiu-Mei; Wang, Li-Bo

    2010-12-01

    To provide a theoretical basis to improve the survival and growth rate and optimize diet of sheatfish (Silurus soldatovi), the activities of certain digestive enzymes and alkaline phosphatases were investigated during larval development of one-ten day old individuals. Results indicated that sheatfish larva (~ three days after hatching) had high levels of alkaline protease activity, which peaked at five days old and dipped by eight days old, although the trend was generally upward. Acid protease activity at one-eight days old was low, after which it increased rapidly. Amylase activity reached the highest value at five days old, after which it began to decline. Lipase activity fluctuated markedly and showed two peaks at three-four days old and six-eight days old. Larval digestive enzyme activity and alkaline phosphatase activity were higher when fed live food than when fed an artificial diet. Throughout the early development process, alkaline protease activity was higher than acid protease, alkaline protease and amylase specific activity decreased significantly for eight-day-old transition larvae, while acid protease activity increased rapidly. These results indicate that the changes in digestive enzyme activity were relevant to digestive function conversion during fish larvae development. Alkaline phosphatase activity showed an upward trend over the first ten days of life, which indicated that the gastrointestinal function of sheatfish improved gradually.

  3. The effect of enzyme digestion time on the detection of diatom species.

    PubMed

    Yu, Zhengliang; Liu, Chao; Wang, Huijun; Chen, Ling; Hu, Sunlin; Zhao, Jian

    2014-05-01

    This study is aimed at detecting diatom in lung, liver and kidney tissues using PCR - DHPLC technology after different periods of enzyme digestion to assess the effect of enzyme digestion on the detection of diatom species. Twenty Randomly selected experimental rabbits were drowned at the same place. Their liver, kidney, and lung tissues were removed for sampling. After the extraction of DNA from the samples, amplification was conducted with specific primers of the SSU gene of diatom. Then, an analysis was performed with agarose gel electrophoresis and DHPLC. Within 2 h-8 h, the amount of the diatom species found in the lung gradually increased over time and was statistically significant <. After 8 h, with enzyme digestion, the amount of the diatom species found in lung showed no significant increase (>). However, as for the liver and kidney, within 2h-6h, the amount of the diatom species gradually increased over time and was statistically significant <. After 6h, the fig. did not present significant growth (>). The amount of the diatom species found in the organs after different periods of digestion time had significant differences, which provides a reference for the detection of diatoms and also, has a good application prospect in the forensic identification of drowning.

  4. 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.

  5. In vitro digestion of gliadin by gastrointestinal enzymes and by pyrrolidonecarboxylate peptidase.

    PubMed

    Caldwell, K A

    1980-02-01

    The enzymatic hydrolysis of whole gliadin has been studied in vitro using sequential treatments by pepsin, trypsin, and pancreatin. Amino-terminal pyroglutamic acid-peptides were formed at each stage of the digestion process and the concentration of these peptides increased as the hydrolysis proceeded. Digests were further fractionated on columns of AG 50W-X8 or SE-Sephadex. Enzymic digests and selected column fractions were analyzed with pyrrolidonecarboxylate peptidase. Each digest or fraction was degraded further by this peptidase. Enzyme activity was greatest towards peptic-tryptic-pancreatic digests, peptic-tryptic digests, peptic digests, and undigested gliadin, in that order. The stability of lysosomal membranes to synthetic L-pyroglutamic acid, L-pyroglutamyl-L-alanine, L-pyroglutamyl-L-proline, and to selected fractions of the enzymic digests was tested. Each treatment ruptured lysosomal membranes. Findings are discussed in relation to the normal catabolism of gliadin and the alterations that may occur in certain pathological states.

  6. 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.

  7. Modulation of digestive enzyme activities during ontogeny of Labeo rohita larvae fed ascorbic acid enriched zooplankton.

    PubMed

    Mitra, Gopa; Mukhopadhyay, P K; Ayyappan, S

    2008-04-01

    The effect of supplementation of ascorbic acid through enriched zooplankton [10%, 20% and 30% ascorbyl palmitate (AP) inclusion in diet of zooplankton] on different digestive enzyme activities during ontogeny of Labeo rohita larvae was studied from 4 day to 15 day post hatch. Ascorbic acid (AA) content in different groups of unenriched (8.6+/-0.71) and enriched zooplankton were, 750+/-29.3, 1409.1+/-45.5, 2009.21+/-199.2 mug/g respectively on dry matter basis with differences (P<0.05) between the treatments. A difference (P<0.05) was found in tissue AA level in different dietary groups. Low amylase, protease, lipase and alkaline phosphatase activities were present in rohu larvae from the mouth opening stage which showed increasing trend with the age of larvae and increasing dietary AA content. A clear dose-dependent modulation of digestive enzyme activities in response to 10%, 20% and 30% AP enriched zooplankton feeding was evidenced from positive correlations between dietary AA content with magnitude of elevation of enzyme activity in different groups. There were 57, 55, 29.2 and 2 fold increases in amylase activity; 7.35, 7.02, 4.43 and 2.73 fold increases in protease activity; 45.636, 41.50, 19.83 and 13.69 fold increases in lipase activity and 6, 5, 3, and 2 fold increases in alkaline phosphatase activity observed in the 15th day post hatch larvae fed 20%, 30%, 10%AP enriched and normal zooplankton respectively, than 4-day post hatch larvae of the respective groups. Enzyme activities were also positively correlated with specific growth rates of wet weight of rohu larvae at the 15th day post hatch. Increased AA might have played an important role in advancing morphological transformation of the digestive tract, protecting gastric mucosa and accelerating growth by the process of tissue formation, which necessitated the requirement of more nutrient thereby, increasing digestive enzyme activity. The regulatory role of AA in the modulation of different digestive

  8. Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display.

    PubMed

    Soares, Tatiane Sanches; Soares Torquato, Ricardo Jose; Alves Lemos, Francisco Jose; Tanaka, Aparecida Sadae

    2013-01-01

    Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1-P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC(50) of 1.1 nM) and chymotrypsin-like enzymes (IC(50) of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC(50) of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC(50) of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.

  9. 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.

  10. 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.

  11. Prenatal diagnosis of megacystis-microcolon-intestinal hypoperistalsis syndrome: contribution of amniotic fluid digestive enzyme assay and fetal urinalysis.

    PubMed

    Muller, Françoise; Dreux, Sophie; Vaast, Pascal; Dumez, Yves; Nisand, Israel; Ville, Yves; Boulot, Pierre; Guibourdenche, Jean; Althusser, Marc; Blin, Gérard; Gautier, Evelyne; Lespinard, Catherine; Perrotin, Franck; Poulain, Patrice; Sarramon, Marie-France

    2005-03-01

    Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a usually lethal disease during the first year of life. There is no specific ultrasound prenatal diagnosis and no identified genetic locus. The value of amniotic fluid digestive enzyme assay and fetal urine biochemistry in the prediction of MMIHS was analysed. Retrospective study of 14 MMIHS cases. Amniotic fluid digestive enzymes and fetal urine biochemistry were compared in MMIHS and megabladder (63 and 264 cases respectively). Abnormal amniotic fluid digestive enzyme profile (vomiting of bile or digestive secretion leakage) was observed in 8/10 MMIHS cases. These profiles were observed in 7/63 controls; 80% sensitivity (95%CI = 55%-100%); 89% specificity (95%CI = 81%-96%). Fetal urinalysis was normal in 12/12 MMIHS cases except high calcium (>0.6 mmol/l). This profile was observed in 33/264 megabladder control cases; 100% sensitivity; 98.7% specificity (95%CI = 83.5%-91.5%). For the first time, we propose a prenatal diagnosis of MMIHS based on amniotic fluid digestive enzyme assay and on fetal urinalysis. Copyright 2005 John Wiley & Sons, Ltd.

  12. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

    PubMed

    Stygar, Dominika; Dolezych, Bogdan; Nakonieczny, Mirosław; Migula, Pawel; Michalczyk, Katarzyna; Zaak, Maria

    2010-10-01

    This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

  13. Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets.

    PubMed

    Silva, Fátima C B L; Alcazar, Alonso; Macedo, Leonardo L P; Oliveira, Adeliana S; Macedo, Francisco P; Abreu, Luiz R D; Santos, Elizeu A; Sales, Mauricio P

    2006-07-01

    The digestive system of Ceratitis capitata was characterized during its larval development and in the insect stage. Disaccharidases against maltose and sucrose were more evident in the 2nd and 3rd day of larval development and in the adult stage, respectively. Glycosil-hydrolyses with higher specific alpha-galactosidasic and beta-galactosidasic activities were detected in the 2nd and 3rd day of the larval stage, respectively. Specific proteolytic activities against azocasein showed an increase in the 4th and 5th day of the larval stage and in the adult stage. Specific hemoglobin activities were constant between 2nd and 6th day of the larval stage. The larvae used mainly serine proteinases, such as trypsin/chymotrypsin, and the adult insects only chymotrypsin-like enzymes in their digestive process. Two serine proteinases were separated from zymogram between the 4th and 5th day of larval development and in the adult stage. Effect of soybean trypsin inhibitor (SBTI, a serine proteinase inhibitor) on development of C. capitata was examined by bioassay. C. capitata was susceptible to SBTI which affected larval mass at ED50 3.01%.

  14. 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. © 2015 Wiley Periodicals, Inc.

  15. 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

  16. Effect of enzymes on anaerobic digestion of primary sludge and septic tank performance.

    PubMed

    Diak, James; Örmeci, Banu; Kennedy, Kevin J

    2012-11-01

    Enzyme additives are believed to improve septic tank performance by increasing the hydrolysis and digestion rates and maintaining a healthy microbial population. Previous studies reported mixed results on the effectiveness of enzymes on mesophilic and thermophilic digestion, and it is not clear whether enzymes would be effective under septic tank conditions where there is no heating or mixing, quantities of enzymes added are small, and they can be washed out quickly. In this study, batch reactors and continuous-flow reactors designed and operated as septic tanks were used to evaluate whether enzymatic treatment would increase the hydrolysis and digestion rates in primary sludge. Total solids, volatile solids, total suspended solids, total and soluble chemical oxygen demand, concentrations of protein, carbohydrate, ammonia and volatile acids in sludge and effluent samples were measured to determine the differences in digestion rates in the presence and absence of enzymes. Overall, no significant improvement was observed in enzyme-treated reactors compared with the control reactors.

  17. 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.

  18. Combination of online enzyme digestion with stable isotope labeling for high-throughput quantitative proteome analysis.

    PubMed

    Wang, Fangjun; Wei, Xiaoluan; Zhou, Hu; Liu, Jing; Figeys, Daniel; Zou, Hanfa

    2012-11-01

    Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10(5) Hela cells).

  19. Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae.

    PubMed

    Tereshchenkova, Valeriia F; Goptar, Irina A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

    2017-08-01

    Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects. © 2017 Wiley Periodicals, Inc.

  20. 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.

  1. Production of digestive enzymes along the gut of the giant keyhole limpet Megathura crenulata (Mollusca: Vetigastropoda).

    PubMed

    Martin, Gary G; Martin, Alanna; Tsai, Whitney; Hafner, John C

    2011-11-01

    The esophagus and intestine form the longest regions of the digestive tract in the giant keyhole limpet and are lined by epithelial cells sharing a common morphology and releasing materials into the gut lumen by apocrine secretion. The purpose of this study was to determine if these morphologically similar regions release similar digestive enzymes and compare their contributions to digestive enzymes released from other regions of the gut. Principal component analysis of enzymes detected by the API ZYM system for 19 enzymes plus EnzChek assays for protease, α-amylase, lipase, cellulase, and lysozyme identify four distinct regions of the gut: 1) crystalline style and style sac, 2) digestive gland, 3) salivary glands, and 4) esophagus and intestine. Heterogeneity in enzymatic activity was observed in regions of the gut with similar cell morphology (middle and posterior esophagus and intestine) as well as regions with different cell morphology (salivary glands, digestive gland and crystalline style). Enzyme activity in each of these regions is compared to other gastropods, in particular the abalone. Although much of the length of the digestive tract is lined by a morphologically similar epithelium, different regions of the alimentary tract produce a different suite of enzymes which may contribute to the digestive process. These data will help enhance our limited understanding of the digestive physiology of Megathura crenulata and lead to improvement of its culture for clinical research. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. 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.

  3. 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

  4. Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers.

    PubMed

    Pinheiro, D F; Cruz, V C; Sartori, J R; Vicentini Paulino, M L M

    2004-09-01

    The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

  5. Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus).

    PubMed

    Dai, Wei; Du, Huahua; Fu, Linglin; Jin, Chengguan; Xu, Zirong; Liu, Huitao

    2009-02-01

    With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.

  6. Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera.

    PubMed

    Dias, Renata O; Via, Allegra; Brandão, Marcelo M; Tramontano, Anna; Silva-Filho, Marcio C

    2015-03-01

    Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic L-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

  7. 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.

  8. Preparation and application of immobilized enzymatic reactors for consecutive digestion with two enzymes.

    PubMed

    Wang, Bingbing; Shangguan, Lulu; Wang, Shulei; Zhang, Lingyi; Zhang, Weibing; Liu, Fan

    2016-12-16

    The bottom up strategy has drawn much attention due to the high accuracy, reliability, and reproducibility in protein identification in which proteins are digested into peptides. However, conventional solution-based digestion and enzymatic reactor with one protease immobilized cannot satisfy high throughput proteolysis of complex samples. Application of consecutive hydrolysis by enzymatic reactor can be a new strategy for high throughput proteolysis of complex samples by adjusting immobilization amount of the enzymes, enzyme ratio, as well as hydrolysis order of two enzymes. In this work, we propose immobilized enzymatic reactor for consecutive digestion with two enzymes by combining two enzyme reactors with trypsin and chymotrypsin immobilized, respectively. Each reactor was prepared individually by immobilizing only one protease (trypsin or chymotrypsin) to hybrid monolith with SBA-15 particles embedded. Proteolysis conditions including hydrolysis order and trypsin to chymotrypsin ratio etc. were studied using standard proteins. Best digestion performance was obtained when the proteins were digested by trypsin first with trypsin to chymotrypsin ratio of 1:1. When applying them to digestion of rat liver proteins, total 1651 proteins and 11011 peptides were identified by combining four enzymolysis strategies with two enzymes including proteolytic digestion in two consecutive enzymatic reactors, synergy enzymolysis with two enzymes in one immobilized enzymatic reactor and consecutive hydrolysis with two enzymes in-solution digestion respectively, in which consecutive enzymolysis in enzymatic reactors gave the best results with 1091 proteins and 5071 peptides identified. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weights, and will play an important role in high efficient and high throughput proteomics research.

  9. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism.

    PubMed

    Sano, Kaori; Kawaguchi, Mari; Yoshikawa, Masayuki; Kaneko, Toyoji; Tanaka, Toshiomi; Iuchi, Ichiro; Yasumasu, Shigeki

    2011-10-01

    We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei. © 2011 The Authors Journal compilation © 2011 FEBS.

  11. Probing the intestinal α-glucosidase enzyme specificities of starch-digesting maltase-glucoamylase and sucrase-isomaltase: synthesis and inhibitory properties of 3'- and 5'-maltose-extended de-O-sulfonated ponkoranol.

    PubMed

    Eskandari, Razieh; Jones, Kyra; Reddy, Kongara Ravinder; Jayakanthan, Kumarasamy; Chaudet, Marcia; Rose, David R; Pinto, B Mario

    2011-12-23

    The synthesis and glucosidase inhibitory activities of two C-3'- and C-5'-β-maltose-extended analogues of the naturally occurring sulfonium-ion inhibitor, de-O-sulfonated ponkoranol, are described. The compounds are designed to test the specificity towards four intestinal glycoside hydrolase family 31 (GH31) enzyme activities, responsible for the hydrolysis of terminal starch products and sugars into glucose, in humans. The target sulfonium-ion compounds were synthesized by means of nucleophilic attack of benzyl protected 1,4-anhydro-4-thio-D-arabinitol at the C-6 position of 6-O-trifluoromethanesulfonyl trisaccharides as alkylating agents. The alkylating agents were synthesized from D-glucose by glycosylation at C-4 or C-2 with maltosyl trichloroacetimidate. Deprotection of the coupled products by using a two-step sequence, followed by reduction afforded the final compounds. Evaluation of the target compounds for inhibition of the four glucosidase activities indicated that selective inhibition of one enzyme over the others is possible. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Functional changes in digestive enzyme activities of meagre (Argyrosomus regius; Asso, 1801) during early ontogeny.

    PubMed

    Suzer, Cüneyt; Kamacı, H Okan; Çoban, Deniz; Yıldırım, Şükrü; Fırat, Kürşat; Saka, Şahin

    2013-08-01

    The ontogenesis of main pancreatic and intestinal enzymes was investigated in the recent promising Mediterranean candidate species of meagre, Argyrosomus regius, during larval development until 40 days after hatching (DAH). The green-water technique was carried out for larval rearing. Whole-body homogenates were used for enzymatic analysis in larvae younger than 15 DAH; after this date, older larvae were dissected into two segments as pancreatic and intestinal segment. Trypsin was detected as early as hatching and sharply increased concurrently with age and exogenous feeding 15 DAH, but constant decline was observed until the end of experiment. Amylase was determined at 2 DAH and sharply increased 10 DAH. Then, slight decreases were found between 10 and 15 DAH, and then slow alterations were continued until the end of the experiment. Lipase was firstly measured on day 3; then, sudden decline was observed between 20 and 25 DAH. After this date, slow fluctuations were maintained until the end of the experiment. Pepsin was firstly assayed 15 DAH related to gastric gland secretion and sharply increased 30 DAH. Then, it slowly varied 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 15 and fluctuated until 20 DAH, aminopeptidase N activity slowly increased 20 DAH. Then, activity of alkaline phosphatase and aminopeptidase N constantly increased 30 DAH, indicating maturation of the intestinal digestive process, and also, these activities continued to slowly increase until the end of the experiment. The specific activity of cytosolic peptidase, leucine-alanine peptidase, smoothly increased on day 8, then fluctuated until 15 DAH. After this date, in contrast to enzymes of brush border membranes, it sharply decreased 25 DAH and continued to gradually decline until the end

  13. The effectiveness of dietary sunflower meal and exogenous enzyme on growth, digestive enzymes, carcass traits, and blood chemistry of broilers.

    PubMed

    Alagawany, Mahmoud; Attia, Adel I; Ibrahim, Zenat A; Mahmoud, Reda A; El-Sayed, Sabry A

    2017-03-29

    High costs of conventional protein feed sources including soybean meal (SBM) generated the need for finding other alternatives. Thus, the present study was designed to evaluate the impact of graded replacements of SBM by sunflower seed meal (SFM) with or without enzyme supplementation on growth performance, digestive enzymes, carcass traits, and blood profile of broiler chickens. A total of 240 unsexed 1-week-old broiler chicks (Hubbard) were randomly divided into eight treatment groups of 30 chicks each in five replicates each of six chicks in a factorial design (4 × 2) arrangement, including four levels of SFM (0, 25, 50, and 75% replacing SBM) and two levels of enzyme (0- or 0.1-g/kg diet) supplementation. Performance traits including feed conversion ratio, body weight, and weight gain were significantly (P < 0.01) improved with increasing SFM up to 50% substitution for SBM or with enzyme supplementation in broiler diet during the experiment. However, feed intake of broiler chicks was decreased with enzyme supplementation (P < 0.05). The activities of digestive enzymes (protease and amylase) were significantly (P < 0.05) influenced and enhanced by SFM and enzyme inclusion in diets, respectively. The activities of protease and amylase were improved with SFM diet supplemented with 0.1 g/kg enzyme in comparison with those with the un-supplemented diet. The evaluated carcass traits were not statistically (P > 0.05) influenced by feeding SFM meal or enzyme addition. Biochemical blood parameters were significantly (P < 0.01) affected by SFM, enzyme, or their interaction in broiler diets, except for globulin that was not affected by dietary enzyme. It is concluded that increasing SFM level in the diet up to 50% replacing SBM with the supplementation of enzyme improved the growth performance and enhanced positively carcass traits as well as the activity of digestive enzymes in broiler chickens.

  14. Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

    PubMed

    Charron, L; Geffard, O; Chaumot, A; Coulaud, R; Queau, H; Geffard, A; Dedourge-Geffard, O

    2013-12-01

    The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms' digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms' health and the chemical quality of aquatic ecosystems.

  15. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

    PubMed

    Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew; van Veelen, Peter; Koning, Frits

    2015-01-01

    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). 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. 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. Currently available digestive enzyme supplements are ineffective in degrading

  16. 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

  17. Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

    PubMed

    Nichols, Buford L; Avery, Stephen E; Quezada-Calvillo, Roberto; Kilani, Shadi B; Lin, Amy Hui-Mei; Burrin, Douglas G; Hodges, Benjamin E; Chacko, Shaji K; Opekun, Antone R; Hindawy, Marwa El; Hamaker, Bruce R; Oda, Sen-Ichi

    2017-08-01

    Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

  18. Motif-directed redesign of enzyme specificity.

    PubMed

    Borgo, Benjamin; Havranek, James J

    2014-03-01

    Computational protein design relies on several approximations, including the use of fixed backbones and rotamers, to reduce protein design to a computationally tractable problem. However, allowing backbone and off-rotamer flexibility leads to more accurate designs and greater conformational diversity. Exhaustive sampling of this additional conformational space is challenging, and often impossible. Here, we report a computational method that utilizes a preselected library of native interactions to direct backbone flexibility to accommodate placement of these functional contacts. Using these native interaction modules, termed motifs, improves the likelihood that the interaction can be realized, provided that suitable backbone perturbations can be identified. Furthermore, it allows a directed search of the conformational space, reducing the sampling needed to find low energy conformations. We implemented the motif-based design algorithm in Rosetta, and tested the efficacy of this method by redesigning the substrate specificity of methionine aminopeptidase. In summary, native enzymes have evolved to catalyze a wide range of chemical reactions with extraordinary specificity. Computational enzyme design seeks to generate novel chemical activities by altering the target substrates of these existing enzymes. We have implemented a novel approach to redesign the specificity of an enzyme and demonstrated its effectiveness on a model system.

  19. [Exo- and endosecretive digestive glands of enzymes as modulators of secretion].

    PubMed

    Korot'ko, G F

    2010-01-01

    Enzymes, exosecreted by the digestive glands plays not only a role of the hydrolases, but also an informational and modulating role in the urgent adaptation of the enzyme secretion to the structure and properties of the luminal content of the gastrointestinal tract. Endosecreted enzymes in the blood not only inform about enzymatic condition of the hydrolase-producing glands and duct system, but also plays an informational and modulating role by the inhibition of the secretion of the same enzymes, and by the stimulation of the secretion of the heteronymic enzyme, defines a parity of their secretion and recretion, integrates enzyme secretion of the pancreas and gastric glands.

  20. Controlling reaction specificity in pyridoxal phosphate enzymes

    PubMed Central

    Toney, Michael D.

    2012-01-01

    Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at Cα of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. PMID:21664990

  1. 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

  2. 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.

  3. 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

  4. Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms.

    PubMed

    Moraes, C S; Lucena, S A; Moreira, B H S; Brazil, R P; Gontijo, N F; Genta, F A

    2012-08-01

    The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source.

  5. Molecular characterization of genes encoding trypsin-like enzymes from Aedes aegypti larvae and identification of digestive enzymes.

    PubMed

    Soares, Tatiane S; Watanabe, Renata M O; Lemos, Francisco J A; Tanaka, Aparecida S

    2011-12-10

    Trypsin-like enzymes play an important role in the Aedes aegypti digestive process. The trypsin-like enzymes present in adults were characterized previously, but little is known about trypsins in larvae. In the present work, we identified one of the trypsin enzymes from Ae. aegypti larval midgut using a library of trypsin gene fragments, which was the sequence known as AAEL005607 from the Ae. aegypti genome. Quantitative PCR analysis showed that AAEL005607 was transcribed in all larval instars, but it was not present in adult midgut. In order to confirm transcription data, the trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified and sequenced. Purified trypsin showed identity with the amino-terminal sequence of AAEL005607, AAEL005609 and AAEL005614. These three trypsins have high amino acids identity, and could all be used as a template for the design of inhibitors. In conclusion, for the first time, digestive enzymes of 4th larval instar of Ae. aegypti were purified and characterized. The knowledge of digestive enzymes present in Ae. aegypti larvae may be helpful in the development of a larvicide. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae).

    PubMed

    Fialho, Maria do Carmo Q; Terra, Walter R; Moreira, Nathália R; Zanuncio, José C; Serrão, Jose Eduardo

    2013-07-01

    The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    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.

    2016-01-01

    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

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

    PubMed

    Goptar, I A; Semashko, T A; Danilenko, S A; Lysogorskaya, E N; Oksenoit, E S; Zhuzhikov, D P; Belozersky, M A; Dunaevsky, Y E; Oppert, B; Filippova, I Yu; Elpidina, E N

    2012-02-01

    The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine. Published by Elsevier Inc.

  9. PANCREATIC DIGESTIVE ENZYME BLOCKADE IN THE SMALL INTESTINE PREVENTS INSULIN RESISTANCE IN HEMORRHAGIC SHOCK

    PubMed Central

    DeLano, Frank A.; Schmid-Schönbein, Geert W.

    2013-01-01

    Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 hours) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution) and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared to control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine lead to a lesser decrease in insulin receptor density compared to controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels two hours after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock, but is restored in when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in

  10. Pancreatic digestive enzyme blockade in the small intestine prevents insulin resistance in hemorrhagic shock.

    PubMed

    DeLano, Frank A; Schmid-Schönbein, Geert W

    2014-01-01

    Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance, but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 h) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution), and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared with control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine led to a lesser decrease in insulin receptor density compared with controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels 2 h after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock but is restored when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in

  11. [Activity of digestive enzymes during intraperitoneal intake of metal compounds].

    PubMed

    Zdol'nik, T D

    2001-01-01

    Digestive function was studied when three compounds from Group VIB of the Mendeleev periodic system of elements were intraperitoneally administered during 100 days. Potassium bichromate, ammonium molybdate in a dose of 0.2 mg/kg and sodium tungstate in a dose of 5.0 mg/kg (in terms of metal) were found to have a resorptive effect on pancreatic function and a local effect on the small intestinal mucosa.

  12. Modelling enzyme reaction mechanisms, specificity and catalysis.

    PubMed

    Mulholland, Adrian J

    2005-10-15

    Modern modelling methods can now give uniquely detailed understanding of enzyme-catalyzed reactions, including the analysis of mechanisms and the identification of determinants of specificity and catalytic efficiency. A new field of computational enzymology has emerged that has the potential to contribute significantly to structure-based design and to develop predictive models of drug metabolism and, for example, of the effects of genetic polymorphisms. This review outlines important techniques in this area, including quantum-chemical model studies and combined quantum-mechanics and molecular-mechanics (QM/MM) methods. Some recent applications to enzymes of pharmacological interest are also covered, showing the types of problems that can be tackled and the insight they can give.

  13. 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.

  14. Superantigenic activity of toxic shock syndrome toxin-1 is resistant to heating and digestive enzymes.

    PubMed

    Li, S-J; Hu, D-L; Maina, E K; Shinagawa, K; Omoe, K; Nakane, A

    2011-03-01

    To elucidate the stability of superantigenic activity and pathogenesis of toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxin A (SEA) against heating and digestive enzymes. Purified TSST-1 and SEA were treated with heating, pepsin and trypsin that are related to food cooking, stomach and intestine conditions. The integrity, superantigenic activity and toxicity of treated TSST-1 and SEA were analysed by Western blotting, spleen cell culture, cytokine assay and toxic shock models. Both TSST-1 and SEA showed strong resistance to heating, pepsin and trypsin digestion. Furthermore, the treated TSST-1 showed significant higher induction of interferon-γ and toxic shock compared with that of SEA. Pepsin- or trypsin-digested TSST-1 fragments still showed significant superantigenic and lethal shock toxicities. The superantigenic activity of TSST-1 was stable to heating and digestive enzymes. Pepsin- and trypsin-digested TSST-1 fragments still showed superantigenic and lethal shock activities, indicating that digested TSST-1 could cross epithelial cells and induce systemic toxicity. This study found, for the first time, that pepsin- or trypsin-digested smaller TSST-1 retained significant superantigenic and lethal shock activities. The different resistance of TSST-1 and SEA participates in the different pathogenic activities during food poisoning and toxic shock syndrome. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

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

    PubMed

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

    2014-03-07

    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.

  16. Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin.

    PubMed

    Rivera-Burgos, Dinelia; Regnier, Fred E

    2013-02-01

    Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin-IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin- IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.

  17. Digestive enzyme activities and gastrointestinal fermentation in wood-eating catfishes.

    PubMed

    German, Donovan P; Bittong, Rosalie A

    2009-11-01

    To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus "Marañon", Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (-600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes' GI tracts. Cellulase and xylanase activities were low (<0.03 U g(-1)), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis-Menten constants (K(m)) of the fishes' beta-glucosidase and N-acetyl-beta-D-glucosaminidase enzymes were significantly lower than the K(m) values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly "xylivorous" taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides.

  18. In planta expression of hyperthermophilic enzymes as a strategy for accelerated lignocellulosic digestion.

    PubMed

    Mir, Bilal Ahmad; Myburg, Alexander A; Mizrachi, Eshchar; Cowan, Don A

    2017-09-13

    Conversion of lignocellulosic biomass to biofuels and biomaterials suffers from high production costs associated with biomass pretreatment and enzymatic hydrolysis. In-planta expression of lignocellulose-digesting enzymes is a promising approach to reduce these cost elements. However, this approach faces a number of challenges, including auto-hydrolysis of developing cell walls, plant growth and yield penalties, low expression levels and the limited stability of expressed enzymes at the high temperatures generally used for biomass processing to release fermentable sugars. To overcome these challenges we expressed codon-optimized recombinant hyperthermophilic endoglucanase (EG) and xylanase (Xyn) genes in A. thaliana. Transgenic Arabidopsis lines expressing EG and Xyn enzymes at high levels without any obvious plant growth or yield penalties were selected for further analysis. The highest enzyme activities were observed in the dry stems of transgenic lines, indicating that the enzymes were not degraded during stem senescence and storage. Biomass from transgenic lines exhibited improved saccharification efficiency relative to WT control plants. We conclude that the expression of hyperthermophilic enzymes in plants is a promising approach for combining pretreatment and enzymatic hydrolysis processes in lignocellulosic digestion. This study provides a valid foundation for further studies involving in planta co-expression of core and accessory lignocellulose-digesting enzymes.

  19. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Effect of formulated diet on digestive enzymes of Labeo rohita (Ham.).

    PubMed

    Sethuramalingam, T A; Haniffa, M A

    2002-01-01

    Six sets of feeding experiments were carried out using formulated diets containing prawn head waste (PW), chicken intestine waste (CW), banana flower (BF), cauliflower waste (CAU) Dolicos lab lab (DLL) and groundnut leaf (GNL) in four levels of inclusion (15, 30, 45 and 60%) to assess the pattern of distribution and activities of digestive enzymes like cellulase, amylase, maltase, invertase, protease and lipase in the digestive tracts of Labeo rohita fingerlings. A control group of fish was fed with diets containing antibiotics to destroy the digestive tract microflora which may induce digestive functions. In general, the activity of digestive enzymes depended on the amount and type of the ingredients present in the diets ingested by the fish. Test animals showed both endogenous and bacterial cellulase activities which suggests the necessity for including cellulose (plant protein source) as dietary ingredient. Occurrence of higher amount of cellulase in the foregut and amylase in the fore and midgut influenced by DNL and GNL diets revealed the possibility of including less than 40% of the respective ingredients in the diet of rohu. Maltase and invertase were highly influenced by GNL, DLL and BF diets than PW and CW diets. More than 40% inclusion of PW and CW was found to increase protease and lipase secretion in the midgut and hindgut regions. The higher secretion of lipase in the midgut suggested the physiological versatility for lipid digestion in rohu fingerlings.

  1. 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.

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

    PubMed Central

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

    2014-01-01

    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

  3. 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

  4. Effect of structural features on enzyme digestibility of corn stover.

    PubMed

    Kim, Sehoon; Holtzapple, Mark T

    2006-03-01

    Corn stover was pretreated with excess calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The enzymatic digestibility of lime-treated corn stover was affected by the change of structural features (acetylation, lignification, and crystallization) resulting from the treatment. Extensive delignification required oxidative treatment and additional consumption of lime (up to 0.17 g Ca(OH)2/g biomass). Deacetylation reached a plateau within 1 week and there were no significant differences between non-oxidative and oxidative conditions at 55 degrees C; both conditions removed approximately 90% of the acetyl groups in 1 week at all temperatures studied. Delignification highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed (or solubilized), but cellulose was not affected by lime pretreatment in mild temperatures (25-55 degrees C), even though corn stover was contacted with alkali for a long time, 16 weeks. The degree of crystallinity slightly increased from 43% to 60% with delignification because amorphous components (lignin, hemicellulose) were removed. However, the increased crystallinity did not negatively affect the 3-d sugar yield of enzymatic hydrolysis. Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity. The non-linear models for 3-d hydrolysis yields of glucan (Y(g)), xylan (Y(x)), and holocellulose (Y(gx)) were empirically established as a function of the residual lignin (L) for the corn stover pretreated with lime and air.

  5. Developmental changes in digestive enzyme activity in American shad, Alosa sapidissima, during early ontogeny.

    PubMed

    Gao, Xiao-Qiang; Liu, Zhi-Feng; Guan, Chang-Tao; Huang, Bin; Lei, Ji-Lin; Li, Juan; Guo, Zheng-Long; Wang, Yao-Hui; Hong, Lei

    2016-12-09

    In order to assess the digestive physiological capacity of the American shad Alosa sapidissima and to establish feeding protocols that match larval nutritional requirements, we investigated the ontogenesis of digestive enzymes (trypsin, amylase, lipase, pepsin, alkaline phosphatase, and leucine aminopeptidase) in larvae, from hatching to 45 days after hatching (DAH). We found that all of the target enzymes were present at hatching, except pepsin, which indicated an initial ability to digest nutrients and precocious digestive system development. Trypsin rapidly increased to a maximum at 14 DAH. Amylase sharply increased until 10 DAH and exhibited a second increase at 33 DAH, which coincided with the introduction of microdiet at 30 DAH, thereby suggesting that the increase was associated with the microdiet carbohydrate content. Lipase increased until 14 DAH, decreased until 27 DAH, and then increased until 45 DAH. Pepsin was first detected at 27 DAH and then sharply increased until 45 DAH, which suggested the formation of a functional stomach. Both alkaline phosphatase and leucine aminopeptidase markedly increased until 18 DAH, which indicated intestinal maturation. According to our results, we conclude that American shad larvae possess the functional digestive system before mouth opening, and the significant increases in lipase, amylase, pepsin, and intestinal enzyme activities between 27 and 33 DAH suggest that larvae can be successfully weaned onto microdiets around this age.

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

    PubMed Central

    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. Images PMID:3009528

  7. 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

    2015-03-06

    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.

  8. 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.

  9. Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.

    PubMed Central

    Saluja, A; Saluja, M; Villa, A; Leli, U; Rutledge, P; Meldolesi, J; Steer, M

    1989-01-01

    The pancreatic duct of anesthetized rabbits was cannulated and, in some animals, flow of pancreatic exocrine secretions was blocked by raising the cannula to a vertical position. Blockage for 3-7 h caused a rapid and significant rise in serum amylase activity and an increase in amylase activity within the pancreas. The concentration of lysosomal enzymes in the pancreas was not altered but they became redistributed among subcellular fractions and, as a result, an increased amount was recovered in the 1,000-g, 15-min pellet, which was enriched in zymogen granules. Immunofluorescence studies indicated that lysosomal enzymes become localized within organelles which, in size and distribution, resemble zymogen granules. They also contain digestive enzyme zymogens. Blockage of pancreatic secretions also caused lysosomal enzyme-containing organelles to become more fragile and subject to in vitro rupture. These changes noted after short-term pancreatic duct obstruction are remarkably similar to those previously noted to occur during the early stages of diet and secretagogue-induced experimental pancreatitis, observations that have suggested that colocalization of digestive enzyme zymogens and lysosomal hydrolases might result in intracellular digestive enzyme activation and be an important early event in the evolution of those forms of experimental acute pancreatitis. Images PMID:2477393

  10. Bacterial enzymes effectively digest Alzheimer's β-amyloid peptide.

    PubMed

    Danilova, Yuliya Vasilyevna; Shagimardanova, Elena Ilyasovna; Margulis, Anna Borisovna; Toymentseva, Anna Aleksandrovna; Balaban, Nelly Pavlovna; Rudakova, Nataliya Leonidovna; Rizvanov, Albert Anatolyevich; Sharipova, Margarita Rashidovna; Palotás, András

    2014-09-01

    Aggregated β-amyloid peptides play key roles in the development of Alzheimer's disease, and recent evidence suggests that microbial particles, among others, can facilitate their polymerization. Bacterial enzymes, however, have been proved to be beneficial in degrading pathological fibrillar structures in clinical settings, such as strepto-kinases in resolving blood-clots. The purpose of this study was to investigate the ability of bacterial substances to effectively hydrolyze β-amyloid peptides. Degrading products of several proteinases from Bacillus pumilus were evaluated using MALDI-TOF mass-spectrometry, and their toxicity was assessed in vitro using cell-culture assays and morphological studies. These enzymes have proved to be non-toxic and were demonstrated to cleave through the functional domains of β-amyloid peptide. By yielding inactive fragments, proteinases of Bacillus pumilus may be used as candidate anti-amyloid agents.

  11. 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.

  12. High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor.

    PubMed

    Wu, Shuaibin; Sun, Liangliang; Ma, Junfeng; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2011-02-15

    A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K=2.65×10(-13) m(2)). With BSA as the model protein, efficient digestion was completed within 20s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.

  13. Enrichment and specific quantification of Methanocalculus in anaerobic digestion.

    PubMed

    Chen, Si; He, Qiang

    2015-12-01

    Members of the genus Methanocalculus are characterized as hydrogenotrophic methanogens and present in diverse natural and engineered environments. Methanocalculus populations were enriched from anaerobic digesters treating dairy waste using formate as the substrate. Methanocalculus sequences retrieved from the enrichment cultures were subsequently used to develop a Methanocalculus-specific TaqMan qPCR assay to determine the abundance of Methanocalculus populations in the environment, representing the first quantitative tool specifically targeting Methanocalculus. The Methanocalculus-specific primer/probe set was shown to have high coverage with perfect match to >80% of all Methanocalculus 16S rRNA gene sequences in the Ribosomal Database Project (RDP). High specificity of the qPCR assay was also validated by both in silico and experimental analyses. Amplification efficiency of the qPCR assay was determined to be 91.9%, which is satisfactory for quantitative applications. Results from the Methanocalculus-specific qPCR analysis of formate-enriched methanogenic cultures were consistent with those from clone library analysis of the same cultures, validating the accuracy of the qPCR assay. Subsequent field application of the qPCR assay found low relative abundance of Methanocalculus in anaerobic digesters treating dairy waste, accounting for 0.01% of the archaeal populations. The qPCR results were consistent with the lack of detection of Methanocalculus in previous studies of the same anaerobic digesters with clone library analyses, which are less sensitive than qPCR. Thus, the Methanocalculus-specific qPCR assay developed in this study is a highly sensitive tool for the rapid and efficient quantification of Methanocalculus populations in methanogenic environments and understanding of the ecological functions of these methanogens.

  14. 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

    2015-11-05

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. Influence of molting and starvation on digestive enzyme activities and energy storage in Gammarus fossarum.

    PubMed

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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. 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…

  19. 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…

  20. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. Microwave-assisted specific chemical digestion for rapid protein identification.

    PubMed

    Hua, Lin; Low, Teck Yew; Sze, Siu Kwan

    2006-01-01

    We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.

  3. Histochemical analyses of digestive enzymes in the intestine of adult large-scaled gurnard (lepidotrigla cavillone, lacepède, 1801).

    PubMed

    Kozarić, Z; Petrinec, Z; Kužir, S; Gjurčević, E; Baždarić, B

    2011-08-01

    Localization and activity levels of the following digestive enzymes in the intestine of free-living large-scaled gurnard were determined: non-specific esterase, alkaline and acid phosphatase as well as aminopeptidase. Enzymatic activity of the four enzymes was confirmed in all intestine parts but with different distribution through the enterocytes and varying in diverse intensity according to intestine part. This research is part of a broader research project on the biology of economically important fish from the Adriatic Sea. Our study reveals that in the large-scaled gurnard, the middle and posterior intestinal segments play the major role in digestion and absorption of proteins, whereas all parts of the intestine participate in lipid absorption and intracellular digestion. The high protein and lipid content in the diet of the large-scaled gurnard is most likely responsible for high activities of esterase, alkaline phosphatase and aminopeptidase, as they are involved in digestion and absorption of proteins and lipids.

  4. Development of digestive enzyme activity in larvae of spotted sand bass Paralabrax maculatofasciatus. 1. Biochemical analysis.

    PubMed

    Alvarez-González, C A; Moyano-López, F J; Civera-Cerecedo, R; Carrasco-Chávez, V; Ortiz-Galindo, J L; Dumas, S

    2008-12-01

    Spotted sand bass Paralabrax maculatofasciatus is a potential aquaculture species in Northwest Mexico. In the last few years it has been possible to close its life cycle and to develop larviculture technology at on pilot scale using live food, however survival values are low (11%) and improvements in growth and survival requires the study of the morpho-physiological development during the initial ontogeny. In this research digestive activity of several enzymes were evaluated in larvae, from hatching to 30 days after hatching (dah), and in live prey (rotifers and Artemia), by use of biochemical and electrophoretic techniques. This paper, is the first of two parts, and covers only the biochemical analysis. All digestive enzyme activities were detected from mouth opening; however the, maximum activities varied among different digestive enzymes. For alkaline protease and trypsin the maximum activities were detected from 12 to 18 dah. Acid protease activity was observed from day 12 onwards. The other digestive enzymes appear between days 4 and 18 after hatching, with marked fluctuations. These activities indicate the beginning of the juvenile stage and the maturation of the digestive system, in agreement with changes that occur during morpho-physiological development and food changes from rotifers to Artemia. All enzymatic activities were detected in rotifers and Artemia, and their contribution to enhancement the digestion capacity of the larvae appears to be low, but cannot be minimised. We concluded that the enzymatic equipment of P. maculatofasciatus larvae is similar to that of other marine fish species, that it becomes complete between days 12 and 18 after hatching, and that it is totally efficient up to 25 dah.

  5. Activities of Digestive Enzymes in the Omnivorous Pest Apolygus lucorum (Hemiptera: Miridae).

    PubMed

    Li, Wenjing; Zhao, Xincheng; Yuan, Wei; Wu, Kongming

    2017-02-01

    The mirid bug Apolygus lucorum Meyer-Dür, 1843, an omnivorous species that feeds on plants and animals, has become a major pest in China as production of Bt-cotton has grown to such a large scale. Its omnivory is likely to be critical for its success, but the digestive mechanism(s) underlying processing and adsorption of such diverse foods is relatively unknown. Here, we examined the activities of digestive enzymes of A. lucorum in the salivary gland complex and midgut and the effect of sex, age, and food source on these activities. Amylase and protease were present in the salivary gland complex and the midgut, but were higher in the salivary gland complex. Trypsin-like enzyme was also present in both organs, but chymotrypsin-like enzyme was present only in the midgut. Sex, age, and food source affected the activities of these digestive enzymes. In general, the activities of these enzymes peaked at 10 d after emergence, and amylase and protease activities were higher in female adults than in males. Of the food sources tested, green bean pods (Gb) induced the highest amylase activity, whereas Helicoverpa armigera Hübner, 1809 eggs (He) and a mixture of Gb and He induced higher activities of the trypsin-like and chymotrypsin-like enzymes. The results from food switching experiments confirmed that amylase activity could be induced by plant sources, and animal sources induced protease activity. Thus, the types and activities of digestive enzymes in A. lucorum provide the physiological basis of the pest's omnivory. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.

  6. Effects of dietary supplementation of multi-enzyme on growth performance, nutrient digestibility, small intestinal digestive enzyme activities, and large intestinal selected microbiota in weanling pigs.

    PubMed

    Zhang, G G; Yang, Z B; Wang, Y; Yang, W R; Zhou, H J

    2014-05-01

    Two experiments were conducted to assess the effects of dietary supplementation of an exogenous multi-enzyme (EME) preparation to 35- to 65-d-old piglets on apparent total tract digestibility (ATTD), growth performance, digestive enzyme activities, and selected microbial populations in feces. In Exp.1, twenty eight 35-d-old piglets were randomly assigned to 7 dietary treatments (corn-soybean based diet supplemented with 0, 100, 150, 200, 250, 300, or 350 mg EME/kg) in a 14-d digestibility study. Piglets fed the diets supplemented with EME had greater ATTD of DM, CP, and GE (P = 0.001, 0.005, and 0.009, respectively) than those fed the diet without EME supplementation, and those ATTD values increased linearly and quadratically (P < 0.001) as the levels of supplemented EME increased. In Exp. 2, two hundred 35-d-old weanling piglets were randomly allocated to 20 pens. The pens were then randomly assigned to 5 dietary treatments (corn-soybean based diet supplemented with 0, 100, 150, 250, or 350 mg EME/kg) with 4 pens per treatment in a 30-d feeding experiment. Piglets has ad libitum access to diets and water, and they were weighed at the beginning (35-d-old), middle (50-d-old), and end (65-d-old) of the experiment. Fecal samples were grabbed directly from the rectum and digesta samples from duodenum, jejunum, and ileum were taken at the end of the experiment for the analysis of selected bacteria populations and digestive-enzyme activities. The ADG and ADFI tended to be greater with the increasing levels of supplemented EME in both periods, whereas G:F was improved (P = 0.012 and 0.017) by EME in the period of 35 to 50 d of age and during the overall experimental period. Furthermore, inclusion of EME in diet increased the counts of Lactobacilli spp. and Bacillus subtilis spp., but reduced the populations of Salmonella spp. and Escherichia coli spp. in the feces. The EME supplementation also enhanced (P < 0.05) the activities of amylase, lipase, and protease in the

  7. Pancreatic Digestive Enzyme Blockade in the Intestine Increases Survival After Experimental Shock

    PubMed Central

    DeLano, Frank A.; Hoyt, David B.; Schmid-Schönbein, Geert W.

    2015-01-01

    Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors—6-amidino-2-naphtyl p-guanidinobenzoate di-methanesulfate, tranexamic acid, or aprotinin—into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multi-organ failure, which can be treated with protease inhibitors that are currently available for use in the clinic. PMID:23345609

  8. Pancreatic digestive enzyme blockade in the intestine increases survival after experimental shock.

    PubMed

    DeLano, Frank A; Hoyt, David B; Schmid-Schönbein, Geert W

    2013-01-23

    Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors--6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate, tranexamic acid, or aprotinin--into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multiorgan failure, which can be treated with protease inhibitors that are currently available for use in the clinic.

  9. In vitro influence of spices and spice-active principles on digestive enzymes of rat pancreas and small intestine.

    PubMed

    Ramakrishna Rao, R; Platel, Kalpana; Srinivasan, K

    2003-12-01

    In vitro influence of 14 individual spices (curcumin, capsaicin, piperine, garlic, onion, ginger, mint, coriander, cumin, ajowan, fennel, fenugreek, mustard, and asafoetida) on the activities of digestive enzymes of rat pancreas and small intestine was examined by including them in the reaction mixture at two different concentrations. A majority of spices enhanced the activity of pancreatic lipase and amylase when they are directly in contact with the enzyme. It is inferred that this positive influence on the activity of enzymes may have a supplementary role in the overall digestive stimulant action of spices, besides causing an enhancement of the titres of digestive enzymes in pancreatic tissue.

  10. Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae.

    PubMed

    Murashita, Koji; Matsunari, Hiroyuki; Kumon, Kazunori; Tanaka, Yosuke; Shiozawa, Satoshi; Furuita, Hirofumi; Oku, Hiromi; Yamamoto, Takeshi

    2014-12-01

    The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6-11, 8-11 and 6-9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40-50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception-increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10-12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.

  11. Inhibition of digestive enzyme activities by copper in the guts of various marine benthic invertebrates.

    PubMed

    Chen, Zhen; Mayer, Lawrence M; Weston, Donald P; Bock, Michael J; Jumars, Peter A

    2002-06-01

    Digestive systems of deposit and suspension feeders can be exposed to high concentrations of copper (Cu) by ingestion of contaminated sediments. We assessed a potential impact of this Cu exposure on digestive enzyme activities in a wide range of benthic organisms by monitoring enzyme activities in their gut fluids during in vitro titrations with dissolved Cu, which mimics Cu solubilization from sediments. Increasing Cu inhibited digestive protease activities at threshold values, which varied widely among organisms, from 8 microM for an echinoderm to 0.4 M for an echiuran. More Cu was required to inhibit proteases in guts containing higher amino acid concentrations because strong Cu-binding sites on amino acids prevent Cu interaction with the enzymatically active sites. Threshold Cu concentrations were similar for proteases, esterases, lipases, and alpha- and beta-glucosidases, suggesting the same inhibition mechanism. Copper was less effective at inhibiting enzymes at lower pH, suggesting that protons can compete with Cu ion for binding to enzymatically active sites or that enzyme conformation is less vulnerable to Cu inhibition at lower pH. These results lead to the counterintuitive conclusion that deposit feeders with low enzyme activity, low amino acid concentration, and high pH values are most vulnerable to harm from sedimentary Cu by this mechanism, although they solubilize less sedimentary Cu than their counterparts with high enzyme activity, high amino acid concentrations, and low gut pH. In general, digestive systems of echinoderms may therefore be more susceptible to Cu contamination than those of polychaetes, with various other phyla showing intermediate susceptibilities. If threshold Cu values are converted to solid-phase sedimentary Cu concentrations, the thresholds are at least consistent with Cu loadings that have been observed to lead to biological impacts in the field.

  12. Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes.

    PubMed

    Kawaguchi, Mari; Yasumasu, Shigeki; Shimizu, Akio; Sano, Kaori; Iuchi, Ichiro; Nishida, Mutsumi

    2010-12-01

    We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

  13. Relationship between protein deficiency in the ration of rats during early ontogeny and function of enzyme systems of digestive and non-digestive organs in adult life.

    PubMed

    Timofeeva, N M; Nikitina, A A; Egorova, V V; Gordova, L A

    2004-07-01

    Low protein content in the ration of rat pups during transfer from mixed to definitive nutrition (days 21-30 of life) has a negative impact on digestive function of the small intestine and trophic and barrier functions of the large intestine, liver, and kidneys and increases (sucrase, glycyl-L-leucin dipeptidase) or decreases (alkaline phosphatase, aminopeptidase M, glycyl-L-leucine dipeptidase) enzyme activities in these organs in 6-month-old rats. Protein deficiency during the early ontogeny modulates functioning of the enzyme systems in digestive and non-digestive organs in adult life, which can lead to the development of not only gastrointestinal, but other visceral diseases.

  14. 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

  15. Monobody-mediated alteration of enzyme specificity.

    PubMed

    Tanaka, Shun-Ichi; Takahashi, Tetsuya; Koide, Akiko; Ishihara, Satoru; Koikeda, Satoshi; Koide, Shohei

    2015-10-01

    Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a β-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics.

  16. Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion.

    PubMed

    Mir, Bilal Ahmad; Mewalal, Ritesh; Mizrachi, Eshchar; Myburg, Alexander A; Cowan, Don A

    2014-05-01

    Plant biomass, as an abundant renewable carbon source, is a promising alternative to fossil fuels. However, the enzymes most commonly used for depolymerization of lignocellulosic biomass are expensive, and the development of cost-effective alternative conversion technologies would be desirable. One possible option is the heterologous expression of genes encoding lignocellulose-digesting enzymes in plant tissues. To overcome simultaneously issues of toxicity and incompatibility with high-temperature steam explosion processes, the use of heterologous genes encoding hyperthermophilic enzymes may be an attractive alternative. This approach could reduce the need for exogenous enzyme additions prior to fermentation, reducing the cost of the complete processing operation. This review highlights recent advances and future prospects for using hyperthermophilic enzymes in the biofuels industry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. The Effects of Enzyme Complex on Performance, Intestinal Health and Nutrient Digestibility of Weaned Pigs

    PubMed Central

    Yi, J. Q.; Piao, X. S.; Li, Z. C.; Zhang, H. Y.; Chen, Y.; Li, Q. Y.; Liu, J. D.; Zhang, Q.; Ru, Y. J.; Dong, B.

    2013-01-01

    Two experiments were conducted to evaluate the effect of supplementing a corn-soybean meal-based diet with an enzyme complex containing amylase, protease and xylanase on the performance, intestinal health, apparent ileal digestibility of amino acids and nutrient digestibility of weaned pigs. In Exp. 1, 108 piglets weaned at 28 d of age were fed one of three diets containing 0 (control), 100, or 150 ppm enzyme complex for 4 wks, based on a two-phase feeding program namely 1 to 7 d (phase 1) and 8 to 28 d (phase 2). At the end of the experiment, six pigs from the control group and the group supplemented with 150 ppm enzyme complex were chosen to collect digesta samples from intestine to measure viscosity and pH in the stomach, ileum, and cecum, as well as volatile fatty acid concentrations and composition of the microflora in the cecum and colon. There were linear increases (p<0.01) in weight gain, gain: feed ratio and digestibility of gross energy with the increasing dose rate of enzyme supplementation during the whole experiment. Supplementation with enzyme complex increased the digesta viscosity in the stomach (p<0.05) and significantly increased (p<0.01) the concentrations of acetic, propionic and butyric acid in the cecum and colon. Enzyme supplementation also significantly increased the population of Lactobacilli (p<0.01) in the cecum and decreased the population of E. coli (p<0.05) in the colon. In Exp. 2, six crossbred barrows (initial body weight: 18.26±1.21 kg), fitted with a simple T-cannula at the distal ileum, were assigned to three dietary treatments according to a replicated 3×3 Latin Square design. The experimental diets were the same as the diets used in phase 2 in Exp. 1. Apparent ileal digestibility of isoleucine (p<0.01), valine (p<0.05) and aspartic acid (p<0.05) linearly increased with the increasing dose rate of enzyme supplementation. In conclusion, supplementation of the diet with an enzyme complex containing amylase, protease and xylanase

  18. 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

  19. Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes.

    PubMed Central

    Apte, M V; Wilson, J S; Korsten, M A; McCaughan, G W; Haber, P S; Pirola, R C

    1995-01-01

    The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant protein deficiency (a known association of alcoholism and pancreatic injury) on these effects. A rat model of chronic ethanol administration was used in which rats were fed in groups of four, and for four weeks, protein sufficient and protein deficient diets with or without ethanol. Ethanol increased the pancreatic content of lipase but did not influence chymotrypsinogen or trypsinogen values. mRNA levels for lipase, trypsinogen, and chymotrypsinogen were raised in rats fed ethanol. Protein deficiency resulted in reduced tissue levels of lipase, chymotrypsinogen, and amylase but did not influence trypsinogen values. mRNA levels for proteases were increased in protein deficient rats, while those for lipase remained unaltered. Both ethanol and protein deficiency increased mRNA levels for cathepsin B. It is concluded that chronic ethanol consumption, in both protein sufficient and protein deficient states, increases the capacity of the pancreatic acinar cell to synthesise digestive and lysosomal enzymes. Images Figure 1 Figure 2 PMID:7533742

  20. Stunting syndrome in broilers: effect of age and exogenous amylase and protease on performance, development of the digestive tract, digestive enzyme activity, and apparent digestibility.

    PubMed

    Shapiro, F; Nir, I

    1995-12-01

    Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with stunting syndrome (SS). Naive controls were kept in a parallel room. The chicks were fed a commercial starter diet supplemented with two levels of enzyme preparations to 14 d of age. The experiment was continued to the age of 6 wk in order to estimate compensatory feed intake and growth. In a parallel study, digestibility of the feed was determined from 1 to 3 wk of age with control or inoculated chicks. The enzymes amylase and proteases were produced by Bacillus subtilis and Penicillium emersonii. Enzyme supplementation had no effect on feed intake, growth, or feed utilization, or on digestibility of fat, starch, protein, or energy. Because enzyme supplementation did not consistently affect performance of chicks and no interactions were observed between enzyme supplementation and infection status, data are presented for effects of infection only. Inoculation of SS-infective material reduced performance to 4 wk. Compensatory growth and feed intake were observed from the age of 4 wk onward. At the age of 6 wk the slight retardation of the inoculated chicks was not significant. On Week 1, retention of fat, starch, protein, and energy was significantly depressed in the inoculated chicks. At the age of 2 wk, retention of starch was not depressed, and at the age of 3 wk, the only consistent depression was that observed for fat. The proventriculus weight and content were consistently higher in inoculated chicks, as were the small intestine and intestinal content. The pH of the gizzard content was higher, and that of the small intestine content was lower, in the inoculated birds than in their control counterparts. Stunting syndrome infection was accompanied by a significant depression of trypsin activity in the pancreas at the age of 1 and 2 wk. At these periods, amylase and chymotrypsin were not affected. At

  1. 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

  2. Development and optimization of a standard method for extraction of microplastics in mussels by enzyme digestion of soft tissues.

    PubMed

    Catarino, Ana I; Thompson, Richard; Sanderson, William; Henry, Theodore B

    2017-04-01

    The authors compared procedures for digestion of mussel soft tissues and extraction of microplastics. Complete tissue digestion was achieved with 1M NaOH, 35% HNO3 , and protease at 9.6 UHb/mL (unit hemoglobin per mL); but use of HNO3 caused unacceptable destruction of some microplastics. Recovery of microplastics spiked into mussels was similar (93 ± 10%) for NaOH and enzyme digestions. The authors recommend use of industrial enzymes based on digestion efficiency, microplastic recovery, and avoidance of caustic chemicals. Environ Toxicol Chem 2017;36:947-951. © 2016 SETAC.

  3. Characterization of peptides generated in proteolytic digest of steamed rice grains by sake koji enzymes.

    PubMed

    Hashizume, Katsumi; Okuda, Masaki; Numata, Mineyo; Zhou, Yan; Koseki, Takuya

    2007-10-01

    High-molecular-weight peptides (approximately 10-30 kDa) generated in a digest of steamed rice grains by sake koji enzymes were characterized. Among 13 major spots resolved by 2-D gel electrophoresis, 12 contained peptides having N-termini of rice glutelin as determined by mass fingerprinting analysis and/or MS/MS. The source of these peptides was presumed to be the acidic subunit of rice glutelin. An addition of up to 25% glucose in the digestion of an isolated rice protein body induced the accumulation of these peptides. The level of accumulation of these peptides in the digest of 70% polished rice samples correlated well with the crude protein content of the rice grains. The degree of accumulation of these peptides in Yamadanishiki and low-polish-rate rice was low, whereas that observed in 90% polished rice samples was extremely low.

  4. 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

  5. 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.

  6. [Analysis of hydrolytic enzyme activities on sludge aerobic/anoxic digestion after ultrasonic pretreatment].

    PubMed

    Ye, Yun-di; Sun, Shui-yu; Zheng, Li; Liu, Bao-jian; Xu, Yan-bin; Zhan, Xing-xing; Liu, Jing-yong

    2012-08-01

    In order to evaluate the function of sludge aerobic/anoxic digestibility by ultrasonic pretreatment. The SS, VSS and hydrolytic enzyme activities (amylase, glucosidase, protease, phosphatase) were measured before and after ultrasonic pretreatment (28 kHz, 0.15 kW x L(-1), 10 min). The results showed that the performances of aerobic/anoxic were greatly improved after ultrasonic pretreatment, the removal efficiency of VSS went to 44.3%, 7.8% better than of traditional aerobic/anoxic digestion. The variational trend of sludge hydrolytic enzyme activities increased firstly and then fell off during 13d digestion, the maximum of amylase activity and glucosidase activity in ultrasonic sludge, appeared in the 5 d, amylase activity was 0.104 micromol x g(-1) and glucosidase activity was 0.637 (micromol x g(-1). The maximum of intracellular protease activity and extracellular proteases activity in ultrasonic sludge, appeared in the 7 d, intracellular protease activity was 23.68 micromol x g(-1), higher than extracellular proteases activity, and it was playing a leading role in sludge digestion. The acid phosphatase activity of ultrasonic sludge was higher than the control sludge, and the alkaline phosphatase was sensitive to environment. So the alkaline phosphatase activity reduced when the internal properties of sludge was changed.

  7. Effects of preparations of Chinese medicinal prescriptions on digestive enzymes in vitro and in vivo.

    PubMed

    Yamasaki, K; Kajimura, K; Nakano, M; Yokoyama, H; Yoneda, K; Umezawa, C

    1998-02-01

    The effects of preparations of Chinese medicinal prescriptions on the activities of digestive enzymes were investigated. The starch dextrinizing activity of pancreatin was inhibited by Keishi-bukuryo-gan, Sho-seiryu-to, Hachimi-jio-gan, Unsei-in and Keishi-ka-shakuyaku-to to below 40% of the control activity. Hachimi-jio-gan and Sho-seiryu-to, in particular, lowered the activity to 4% and 24% of the control activity, respectively. The protein digestive activity of pancreatin was lowered by Keishi-bukuryo-gan, Oren-gedoku-to, Ryutan-shakan-to, Tokaku-joki-to, Yokuinin-to and Hange-shashin-to, to 56 to 70% of the control activity. To investigate the effects of the preparations of Chinese medicinal prescriptions on digestive enzymes in vivo, 600 mg (185 kBq) of 125I-egg white albumin were administered orally to rats 30 min before the administration of 100 mg of Tokaku-joki-to or Oren-gedoku-to. The radioactivity which transferred to the blood was less in the animals pre-fed these prescriptions than in the control animals, indicating that the digestion of egg white albumin was delayed in the presence of the prescriptions.

  8. Induced and constitutive responses of digestive enzymes to plant toxins in an herbivorous mammal.

    PubMed

    Kohl, Kevin D; Dearing, M Denise

    2011-12-15

    Many plants produce plant secondary compounds (PSCs) that bind and inhibit the digestive enzymes of herbivores, thus limiting digestibility for the herbivore. Herbivorous insects employ several physiological responses to overcome the anti-nutritive effects of PSCs. However, studies in vertebrates have not shown such responses, perhaps stemming from the fact that previously studied vertebrates were not herbivorous. The responses of the digestive system to dietary PSCs in populations of Bryant's woodrat (Neotoma bryanti) that vary in their ecological and evolutionary experience with the PSCs in creosote bush (Larrea tridentata) were compared. Individuals from naïve and experienced populations were fed diets with and without added creosote resin. Animals fed diets with creosote resin had higher activities of pancreatic amylase, as well as luminal amylase and chymotrypsin, regardless of prior experience with creosote. The experienced population showed constitutively higher activities of intestinal maltase and sucrase. Additionally, the naïve population produced an aminopeptidase-N enzyme that was less inhibited by creosote resin when feeding on the creosote resin diet, whereas the experienced population constitutively expressed this form of aminopeptidase-N. Thus, the digestive system of an herbivorous vertebrate responds significantly to dietary PSCs, which may be important for allowing herbivorous vertebrates to feed on PSC-rich diets.

  9. 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.

  10. Development of digestive enzyme activity in larvae of spotted sand bass Paralabrax maculatofasciatus II: Electrophoretic analysis.

    PubMed

    Alvarez-González, C A; Moyano-López, F J; Civera-Cerecedo, R; Carrasco-Chávez, V; Ortíz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Dumas, S

    2010-03-01

    The activities of several digestive enzymes during larval development of the spotted sand bass (Paralabrax maculatofasciatus) were evaluated using electrophoretic techniques. The results show the presence of three isoforms of alkaline protease from day 2 after hatching (ah) and the early appearance of one pepsin-like band from day 12 ah onwards. In addition, two lipase bands first appeared on day 2 ah, and there was a change in the molecular weight of one band from day 15 ah onwards. Several alpha-amylase isoforms were observed from hatching up to day 5 ah. These results indicate that the important digestive enzymes develop rapidly in these larvae, supporting the possibility of early weaning at day 12 ah using artificial diets.

  11. 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

  12. 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.

  13. Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island, Indian Ocean.

    PubMed

    Linton, Stuart M; Saborowski, Reinhard; Shirley, Alicia J; Penny, Jake A

    2014-05-01

    The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-β-D-glucosaminidase, the cellulases, endo-β-1,4-glucanase and β-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-β-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-β-1,4-glucanase, β-glucohydrolase and N-acetyl-β-D-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-β-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.

  14. 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.

  15. Changes in digestive enzyme activities of red porgy Pagrus pagrus during a fasting-refeeding experiment.

    PubMed

    Caruso, G; Denaro, M G; Caruso, R; De Pasquale, F; Genovese, L; Maricchiolo, G

    2014-10-01

    An experiment was carried out in red porgy, Pagrus pagrus (Teleostei, Sparidae), to assess the effects of a 14-day fasting period, followed by refeeding to apparent satiation, on the contents of digestive enzymes (total proteases, and particularly pepsin, trypsin, chymotrypsin, carboxypeptidases A and B; amylase and lipase). Two fish groups were considered: one (indicated as fasted/refed group) was fasted for 14 days and then refed during further 7 and 15 days, and the other was fed throughout the study and was taken as a control group. The measured enzymatic values showed that fasting resulted in a generalized, not significant decrease, of the activity of digestive enzymes. Refeeding caused a significant increase for most of the assayed enzymes: total proteases both in the middle and distal intestine, pepsin in the stomach, trypsin in the middle intestine, and amylase and lipase in the proximal intestine. Nevertheless, the detection in the fasted/refed fish of enzymatic values still lower than those measured in the control fish suggested that fish experiencing short-term fasting were partially impaired in their digestive capacity.

  16. Enzyme use in kibble diets formulated with wheat bran for dogs: effects on processing and digestibility.

    PubMed

    Sá, F C; Vasconcellos, R S; Brunetto, M A; Filho, F O R; Gomes, M O S; Carciofi, A C

    2013-05-01

    Recently, there is an interest in technologies that favour the use of coproducts for animal nutrition. The effect of adding two enzyme mixtures in diets for dogs formulated with wheat bran (WB) was evaluated. Two foods with similar compositions were formulated: negative control (NC; without WB) and test diet (25% of WB). The test diet was divided into four treatments: without enzyme (positive control), enzyme mixture 1 (ENZ1; added before extrusion β-glucanase, xylanase, cellulase, glucoamylase, phytase); enzyme mixture 2 (ENZ2; added before extrusion the ENZ1 more α-amylase); enzyme mixture 2 added after the extrusion (ENZ2ex). ENZ1 and ENZ2 were used to evaluate the enzyme effect on extruder pre-conditioner (processing additive) and ENZ2ex to evaluate the effect of enzyme supplementation for the animal. Digestibility was measured through total collection of faeces and urine. The experiment followed a randomized block design with five treatments (diets) and six dogs per diet, totalling 30 dogs (7.0 ± 1.2 years old and 11.0 ± 2.2 kg of body weight). Data were submitted to analysis of variance and means compared by Tukey's test and orthogonal contrasts (p < 0.05). Reducing sugars showed an important reduction after extrusion, suggesting the formation of carbohydrate complexes. The apparent total tract digestibility (ATTD) of dry matter, organic matter, crude protein, acid-hydrolysed fat and energy was higher in NC than in diets with WB (p < 0.001), without effects of enzyme additions. WB diets resulted in higher faecal production and concentration of short-chain fatty acids (SCFA) and reduced pH and ammonia concentration (p < 0.01), with no effect of enzyme addition. The enzyme addition did not result in improved digestibility of a diet high in non-starch polysaccharides; however, only ATTD was measured and nutrient fermentation in the large intestine may have interfered with the results obtained. WB modified fermentation product formation in the colon of dogs

  17. Role of protein, amino acids, and enzyme activity on odor production from anaerobically digested and dewatered biosolids.

    PubMed

    Higgins, Matthew J; Adams, Gregory; Chen, Yen-Chih; Erdal, Zeynep; Forbes, Robert H; Glindemann, Dietmar; Hargreaves, J Ronald; McEwen, David; Murthy, Sudhir N; Novak, John T; Witherspoon, Jay

    2008-02-01

    The main objective of this research was to test the hypothesis that bioavailable protein and, more specifically, the sulfur-containing amino acids within the protein, can be degraded by proteolytic enzymes to produce odor-causing compounds--mainly volatile sulfur compounds (VSCs)--during biosolids storage. To achieve these objectives, samples of digester effluent and cake solids were collected at 11 different wastewater treatment plants in North America, and the samples were analyzed for protein and amino acid content and general protein-degrading enzyme activity. At the same time, cake samples were stored using headspace bottles, the concentration of VSCs were measured using gas chromatography, and olfactometry measurements were made by a trained odor panel. The results showed that the bound cake protein content and methionine content was well-correlated with VSC production and the detection threshold measured by the odor panel.

  18. 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.

  19. Nucleic acids digestion by enzymes in the stomach of snakehead (Channa argus) and banded grouper (Epinephelus awoara).

    PubMed

    Liu, Yu; Zhang, Yanfang; Jiang, Wei; Wang, Jing; Pan, Xiaoming; Wu, Wei; Cao, Minjie; Dong, Ping; Liang, Xingguo

    2017-02-01

    Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.

  20. Effects of cysteamine on growth performance, digestive enzyme activities, and metabolic hormones in broilers.

    PubMed

    Yang, C M; Chen, A G; Hong, Q H; Liu, J X; Liu, J S

    2006-11-01

    A total of 600 avian male broilers at the age of 1 d were used to investigate the effects of cysteamine (CSH) on growth performance, digestive enzyme activities, and concentrations of serum hormones. The broilers received the same basal diets, with CSH added at 0 (control), 60, 90, 120, or 150 mg/kg. The feeding program consisted of a starter diet until 21 d and a grower diet until 42 d. The broilers with addition of CSH at 60 or 90 mg/kg had significantly higher growth rates during d 1 to 21 or d 21 to 42 compared with the control, respectively. However, adding 150 mg of CSH/kg significantly suppressed the growth of broilers. Adding 60 mg of CSH/kg significantly increased the activities of protease, amylase, and lipase in the pancreas and small intestinal contents during d 1 to 21, and the activities of protease and amylase in the small intestinal contents during d 21 to 42. Adding 90 mg of CSH/kg significantly increased the activities of lipase during d 1 to 21 and protease, amylase, and lipase during d 21 to 42 in small intestines. The activities of digestive enzymes during the whole period were suppressed by adding 150 mg of CSH/kg. The concentration of serum thyroxine was higher in the CSH-added birds during the whole period, whereas serum triiodothyronine was higher only during d 1 to 21 compared with the control. These findings indicate that low doses of dietary CSH may improve the growth performance and the activities of the digestive enzyme, but high doses of CSH appear to be detrimental to growth and digestion.

  1. Digestive enzyme activities of turbot (Scophthalmus maximus L.) during early developmental stages under culture condition.

    PubMed

    Tong, X H; Xu, S H; Liu, Q H; Li, J; Xiao, Z Z; Ma, D Y

    2012-06-01

    Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.

  2. Effect of Myracrodruon urundeuva leaf lectin on survival and digestive enzymes of Aedes aegypti larvae.

    PubMed

    Napoleão, Thiago Henrique; Pontual, Emmanuel Viana; de Albuquerque Lima, Thâmarah; de Lima Santos, Nataly Diniz; Sá, Roberto Araújo; Coelho, Luana Cassandra Breitenbach Barroso; do Amaral Ferraz Navarro, Daniela Maria; Paiva, Patrícia Maria Guedes

    2012-02-01

    Aedes aegypti transmits the viruses that cause yellow and dengue fevers. Vector control is essential, since a vaccine for dengue has not as yet been made available. This work reports on the larvicidal activity of Myracrodruon urundeuva leaf lectin (MuLL) against A. aegypti fourth-stage larvae (L(4)). Also, the resistance of MuLL to digestion by L(4) gut proteases and the effects of MuLL on protease, trypsin-like and α-amylase activities from L(4) gut were evaluated to determine if lectin remains active in A. aegypti gut and if insect enzyme activities can be modulated by MuLL. MuLL promoted mortality of L(4) with LC(50) of 0.202 mg/ml. Haemagglutinating activity of MuLL was detected even after incubation for 96 h with L(4) gut preparation containing protease activity. MuLL affected the activity of gut enzymes, inhibiting protease and trypsin activities and stimulating α-amylase activity. The results suggest that MuLL may become a new biodegradable larvicidal agent for dengue control. Larvicidal activity of MuLL may be linked to its resistance to proteolysis by larval enzymes and interference in the activity of digestive larval enzymes.

  3. Evolution of Digestive Enzymes and RNASE1 Provides Insights into Dietary Switch of Cetaceans

    PubMed Central

    Wang, Zhengfei; Xu, Shixia; Du, Kexing; Huang, Fang; Chen, Zhuo; Zhou, Kaiya; Ren, Wenhua; Yang, Guang

    2016-01-01

    Although cetaceans (whales, porpoises, and dolphins) have multi-chambered stomachs, feeding habits of modern cetaceans have dramatically changed from herbivorous to carnivorous. However, the genetic basis underlying this dietary switch remains unexplored. Here, we present the first systematic investigation of 10 digestive enzymes genes (i.e., CYP7A1, CTRC, LIPC, LIPF, PNLIP, PGC, PRSS1, SI, SLC5A1, and TMPRSS15) of representative cetaceans, and the evolutionary trajectory of RNASE1 in cetartiodactylans. Positive selections were detected with proteinases (i.e., CTRC, PRSS1, and TMPRSS15) and lipases (i.e., CYP7A1, LIPF, and PNLIP) suggesting that cetaceans have evolved an enhanced digestion capacity for proteins and lipids, the major nutritional components of their prey (fishes and invertebrates). In addition, it was found that RNASE1 gene duplicated after the cetartiodactylan speciation and two independent gene duplication events took place in Camelidae and Ruminantia. Positive selection was detected with RNASE1 of Camelidae and Bovidae, suggesting enhanced digestive efficiency in the ruminants. Remarkably, even though the ancestors of cetaceans were terrestrial artiodactyls that are herbivorous, modern cetaceans lost the pancreatic RNASE1 copy with digestive function, which is in accordance with the dietary change from herbivorous to carnivorous. In sum, this is the first study that provides new insights into the evolutionary mechanism of dietary switch in cetaceans. PMID:27651393

  4. Evolution of Digestive Enzymes and RNASE1 Provides Insights into Dietary Switch of Cetaceans.

    PubMed

    Wang, Zhengfei; Xu, Shixia; Du, Kexing; Huang, Fang; Chen, Zhuo; Zhou, Kaiya; Ren, Wenhua; Yang, Guang

    2016-12-01

    Although cetaceans (whales, porpoises, and dolphins) have multi-chambered stomachs, feeding habits of modern cetaceans have dramatically changed from herbivorous to carnivorous. However, the genetic basis underlying this dietary switch remains unexplored. Here, we present the first systematic investigation of 10 digestive enzymes genes (i.e., CYP7A1, CTRC, LIPC, LIPF, PNLIP, PGC, PRSS1, SI, SLC5A1, and TMPRSS15) of representative cetaceans, and the evolutionary trajectory of RNASE1 in cetartiodactylans. Positive selections were detected with proteinases (i.e., CTRC, PRSS1, and TMPRSS15) and lipases (i.e., CYP7A1, LIPF, and PNLIP) suggesting that cetaceans have evolved an enhanced digestion capacity for proteins and lipids, the major nutritional components of their prey (fishes and invertebrates). In addition, it was found that RNASE1 gene duplicated after the cetartiodactylan speciation and two independent gene duplication events took place in Camelidae and Ruminantia. Positive selection was detected with RNASE1 of Camelidae and Bovidae, suggesting enhanced digestive efficiency in the ruminants. Remarkably, even though the ancestors of cetaceans were terrestrial artiodactyls that are herbivorous, modern cetaceans lost the pancreatic RNASE1 copy with digestive function, which is in accordance with the dietary change from herbivorous to carnivorous. In sum, this is the first study that provides new insights into the evolutionary mechanism of dietary switch in cetaceans. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study.

    PubMed

    López-Rodríguez, M J; Trenzado, C E; Tierno de Figueroa, J M; Sanz, A

    2012-05-01

    Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. 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. © 2015 Japanese Society of Animal Science.

  8. 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.

  9. 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 ...

  10. 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

  11. Effect of exogenous fibrolytic enzymes on ruminal fermentation and nutrient digestion in dairy cows.

    PubMed

    Peters, Anja; Lebzien, Peter; Meyer, Ulrich; Borchert, Ulrike; Bulang, Michael; Flachowsky, Gerhard

    2010-06-01

    The aim of the present study was to examine the effects of an exogenous fibrolytic enzyme product applied to a total mixed ration (TMR) prior to feeding on ruminal fermentation, microbial protein synthesis, nutrient digestion, and milk yield and composition. Six multiparous lactating Holstein cows (598 +/- 29 kg initial live weight and 98 +/- 30 days in milk) fitted with rumen and duodenal cannulae were allocated to two treatments in a crossover design over three consecutive 28-d periods. The TMR containing 50% concentrates, 30% corn silage and 20% grass silage on dry matter (DM) basis, was mixed once daily and fed twice a day. Treatments were TMR alone (Control) or TMR with an enzyme product containing primarily cellulase and xylanase activities (9000 U endo-1,4-beta glucanase, 24000 U endo-1,3(4)-beta glucanase and 40000 U 1,4-beta xylanase per ml). The enzyme product was applied at a rate of 6.2 ml/kg TMR (DM basis). It was diluted at a rate of 1:5 with water and applied daily to the TMR. During the control period the cows received a TMR supplemented with 36 ml water/kg TMR on DM basis. Duodenal digesta flow was measured using Cr2O3 as flow marker and microbial protein in the duodenal digesta was estimated by near-infrared spectroscopy (NIRS). There were no significant differences in ruminal pH-values, NH3-N concentrations, total SCFA concentrations and molar proportions of SCFA. No treatment effects on microbial N flow to the duodenum and efficiency of microbial protein synthesis were observed. The apparent ruminal digestibilities of DM, organic matter, NDF and ADF, milk yield and composition were also not affected by the enzyme supplementation. In this study the application of exogenous fibrolytic enzymes fed to dairy cows did not show a significant effect on any parameter tested.

  12. 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.

  13. 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

  14. 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.

  15. Digestive System

    MedlinePlus

    ... other substances that aid in digestion. Examples include: Cystic fibrosis is a chronic, inherited illness that not only ... To help manage their digestive problems, people with cystic fibrosis can take digestive enzymes and nutritional supplements. Hepatitis , ...

  16. Simulated digestion of proanthocyanidins in grape skin and seed extracts and the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitory activity.

    PubMed

    Fernández, Katherina; Labra, Javiera

    2013-08-15

    This study investigated the effect of in vitro gastrointestinal digestion on the stability and composition of flavan-3-ols from red grape skin and seed extracts (raw and purified, which are high in proanthocyanidins (PAs)). In addition, the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitory activities of these extracts were evaluated. The extracts were digested with a mixture of pepsin-HCl for 2 h, followed by a 2 h incubation with pancreatin and bile salts including a cellulose dialysis tubing (molecular weight cut-off 12 kDa) at 37°C with shaking in the dark and under N2. Under gastric conditions, the mean degree of polymerisation (mDP) of seed extracts, raw (mDP≈6, p<0.05), and purified (mDP≈10, p<0.05) was stable. The mDP of the raw skin extracts increased from 19 to 25 towards the end of the digestion. The PAs were significantly degraded (up to 80%) during the pancreatic digestion, yielding low-molecular-weight compounds that diffused into the serum-available fraction (mDP≈2). The overall mass transfer coefficient (K) of the seed extracts was 10(-7) m(2)/s. After simulated gastrointestinal digestion, over 80% of ACE inhibition by raw seed and skin extracts was preserved. However, the purified seed and skin extracts lost their ability to inhibit ACE after intestinal digestion.

  17. Influence of canola meal-based diets supplemented with exogenous enzyme and digestible lysine on performance, digestibility, carcass, and immunity responses of broiler chickens.

    PubMed

    Mushtaq, T; Sarwar, M; Ahmad, G; Mirza, M A; Nawaz, H; Mushtaq, M M Haroon; Noreen, U

    2007-10-01

    The response of broiler chickens to 2 levels of endo-1,4-beta xylanase and endo-1,3-beta glucanase combination (with and without), 3 levels of digestible Lys (0.8, 0.9, and 1.0%), and 2 levels of canola meal (CM; 20 and 30%) were evaluated in 2 x 3 x 2 factorial arrangement. A total of 2,448 male Hubbard broiler chicks were fed on practical mash diets having 2,750 kcal of ME.kg(-1) and 19.6% CP from 1 to 42 d of age. The BW gain was significantly reduced when 30% CM was added in the diets during 1 to 21 d. Feed:gain and mortality were also observed to be high. No significant effect of enzyme addition or Lys level was observed on feed intake, BW gain, feed:gain, and mortality during the starter phase. When the data were pooled for 42 d, BW gain and feed:gain were unaffected by enzyme addition or Lys levels. A depression in breast weight was observed due to 30% CM or 0.8 and 0.9% digestible Lys at 43 d. Leg weights were significantly depressed by enzyme addition or increasing digestible Lys to 1.0% of the diets. The AME, nitrogen digestibility, and antibody titers against Newcastle and infectious bursal diseases were also unaffected by the dietary treatments. In conclusion, the 30% CM is not recommended in broiler diets especially during starter phase (1 to 21 d). However, the CM may be used up to 30% of the diets during finishing phase. The digestible Lys can be lowered to 0.8% when amino acids in proportion to digestible Lys follow the ideal AA ratio. The glucanase and xylanase cocktail have no pronounced effect on the growth performance, nutrient digestibility, and carcass characteristics.

  18. Ontogeny changes and weaning effects in gene expression patterns of digestive enzymes and regulatory digestive factors in spotted rose snapper (Lutjanus guttatus) larvae.

    PubMed

    Moguel-Hernández, I; Peña, R; Andree, K B; Tovar-Ramirez, D; Bonacic, K; Dumas, S; Gisbert, E

    2016-10-01

    The study of digestive physiology is an important issue in species that have been introduced in aquaculture like the spotted rose snapper (Lutjanus guttatus). The aims of this study were to describe the expression of digestive enzymes (trypsinogen, chymotrypsinogen, α-amylase, lipoprotein lipase, phospholipase A and pepsinogen) and their relation with orexigenic (neuropeptide Y, NPY) and anorexigenic (cholecystokinin, CCK) factors during the larval development and to evaluate the effect of weaning in their expression. The results showed that the transcripts of all the assayed digestive enzymes, with the exception of pepsinogen, and NPY and CCK were already present in L. guttatus from the hatching stage. The expression of all the enzymes was low during the yolk-sac stage (0-2 days after hatching, DAH), whereas after the onset of exogenous feeding at 2 DAH, their expression increased and fluctuated throughout larval development, which followed a similar pattern as in other marine fish species and reflected changes in different types of food items and the progressive maturation of the digestive system. On the other hand, weaning of L. guttatus larvae from live prey onto a microdiet between 25 and 35 DAH significantly affected the relative expression of most pancreatic digestive enzymes during the first weaning days, whereas chymotrypsinogen 2 and lipoprotein lipase remained stable during this period. At the end of co-feeding, larvae showed similar levels of gene expression regardless of the diet (live prey vs. microdiet), which indicated that larvae of L. guttatus were able to adapt their digestive capacities to the microdiet. In contrast, feeding L. guttatus larvae with live feed or microdiet did not affect the expression of CCK and NPY. The relevance of these findings with regard to current larval rearing procedures of L. guttatus is discussed.

  19. Interaction of digestive enzymes with tunable light emitting quantum dots: a thorough Spectroscopic investigation.

    PubMed

    Ellappan, Vaishnavi; Kesavan, Manibalan; Ramalingam, Parameshwari; Kulandaivel, Jeganathan; Rajalingam, Renganathan

    2015-11-01

    In this article, we have examined the direct spectroscopic and microscopic evidence of efficient quantum dots-α-chymotrypsin (ChT) interaction. The intrinsic fluorescence of digestive enzyme is reduced in the presence of quantum dots through ground-state complex formation. Based on the fluorescence data, quenching rate constant, binding constant, and number of binding sites are calculated under optimized experimental conditions. Interestingly, fluorescence quenching method clearly illustrated the size dependent interaction of MPA-CdTe quantum dots. Conformational change of ChT was traced using synchronous fluorescence measurements, circular dichroism and FTIR spectroscopic methods. Furthermore, the AFM results revealed that the individual enzyme molecule dimensions were changed after interacting with quantum dot. Consequently, this result could be helpful for constructing safe and effective utilisation of QDs in biological applications. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Digestive enzyme activity of two stonefly species (Insecta, Plecoptera) and their feeding habits.

    PubMed

    de Figueroa, J M Tierno; Trenzado, C E; López-Rodríguez, M J; Sanz, A

    2011-11-01

    The digestive enzymes of two stoneflies species, Hemimelaena flaviventris and Isoperla morenica, were studied for the first time. These species are temporary water inhabitants and exhibit great feeding plasticity. Although they are traditionally referred to as predators, a previous study revealed that H. flaviventris incorporates some diatoms into its diet in addition to feeding usually on several prey, and I. morenica (in that study under the name of I. curtata) only feeds on animals occasionally. The enzymatic activities of digestive amylase, lipase, protease, trypsin and chymotrypsin were determined for each species at the same developmental stage. The results show that H. flaviventris has a greater digestive enzymatic pool and higher relative and absolute protease, lipase and trypsin activities than I. morenica. The latter has a relative higher amylase activity. As higher amylase activity is typical of phytophagous species and higher protease activity typical of carnivorous species; these results reveal that H. flaviventris is a more efficient zoophagous species than I. morenica. The ecological implications of these findings, including the higher secondary production of H. flaviventris in its habitat, are discussed.

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

    PubMed

    Gibson, Donna M; King, Brian C; Hayes, Marshall L; Bergstrom, Gary C

    2011-06-01

    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 noted that plant pathogenic fungi are highly competent producers of lignocellulolytic enzymes, and their enzyme activity patterns reflect host specificity. We propose that plant pathogens may contribute to biofuel production as diverse sources of accessory enzymes for more efficient conversion of lignocellulose into fermentable sugars.

  2. 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.

  3. Specific effects of fiber size and fiber swelling on biomass substrate surface area and enzymatic digestibility.

    PubMed

    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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. IMPAIRED SMALL BOWEL BARRIER INTEGRITY IN THE PRESENCE OF LUMENAL PANCREATIC DIGESTIVE ENZYMES LEADS TO CIRCULATORY SHOCK

    PubMed Central

    Kistler, Erik B.; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W.

    2012-01-01

    In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small bowel lumen. It is unresolved, however, whether ischemically-mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access, and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of non-ischemic rats was perfused for two hours with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase and lipase. Control (n=6) and experimental animals perfused with pancreatic enzymes only (n=6) or single enzymes (n=3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n=6) developed mild hypotension (p<0.001 compared to groups perfused with pancreatic enzymes only after 90 minutes) and increased intestinal permeability to intralumenally perfused FITC-dextrans-20kD (p<0.05) compared to control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n=6) developed hypotension and increased intestinal permeability (p<0.001 after 90 minutes). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These

  5. Impaired small-bowel barrier integrity in the presence of lumenal pancreatic digestive enzymes leads to circulatory shock.

    PubMed

    Kistler, Erik B; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W

    2012-08-01

    In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P < 0.001 compared with groups perfused with pancreatic enzymes only after 90 min) and increased intestinal permeability to intralumenally perfused fluorescein isothiocyanate-dextran 20 kd (P < 0.05) compared with control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n = 6) developed hypotension and increased intestinal permeability (P < 0.001 after 90 min). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress

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

    PubMed

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

    2014-05-01

    Avian coccidiosis is a disease caused by intestinal protozoa in the genus Eimeria. Clinical signs of coccidiosis include intestinal lesions and reduced feed efficiency and BW gain. This 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 examine the differential expression of digestive enzymes, transporters of amino acids, peptides, sugars, and minerals, and an antimicrobial peptide in the small intestine of Eimeria acervulina-infected broilers and layers. Uninfected broilers and layers, in general, expressed these genes at comparable levels. Some differences included 3-fold and 2-fold greater expression of the peptide transporter PepT1 and the antimicrobial peptide LEAP2 (liver expressed antimicrobial peptide 2), respectively, in the jejunum of layers compared with broilers and 17-fold greater expression of LEAP2 in the duodenum of broilers compared with layers. In the duodenum of Eimeria-infected broilers and layers, there was downregulation of aminopeptidase N; sucrase-isomaltase; the neutral, cationic, and anionic amino acid transporters b(o,+)AT/rBAT, B(o)AT, CAT2, and EAAT3; the sugar transporter GLUT2; the zinc transporter ZnT1; and LEAP2. In the jejunum of infected layers there was downregulation of many of the same genes as in the duodenum plus downregulation of PepT1, b(o,+)AT/rBAT, and the y(+) L system amino acid transporters y(+) LAT1 and y(+) LAT2. In the ileum of infected layers there was downregulation of CAT2, y(+)LAT1, the L type amino acid transporter LAT1, and the sugar transporter GLUT1, and upregulation of APN, PepT1, the sodium glucose transporter SGLT4, and LEAP2. In E. acervulina-infected broilers, there were no gene expression changes in the jejunum and ileum. These changes in intestinal digestive enzyme and nutrient transporter gene expression may result in a decrease in the efficiency of protein digestion, uptake of important amino acids

  7. Digestive enzyme activity in the intestine of Nile tilapia (Oreochromis niloticus L.) under pond and cage farming systems.

    PubMed

    Santos, Juliana Ferreira; Soares, Karollina Lopes Siqueira; Assis, Caio Rodrigo Dias; Guerra, Carlos Augusto Martins; Lemos, Daniel; Carvalho, Luiz Bezerra; Bezerra, Ranilson Souza

    2016-10-01

    The effect of different farming systems (cage, pond) upon digestive enzyme activities of Nile tilapia was evaluated. Juvenile Nile tilapia (87.61 ± 1.52 g) were simultaneously cultured in pond and cage systems during 90 days. Cages used nutritional biphasic plan (35 and 32 % crude protein-CP feeds) and ponds used nutritional triphasic plan (35, 32 and 28 % CP feeds). Biometric measurements were monthly performed for adjustments in feeding regimes and removal of intestine tissues to evaluate the performance of enzyme activities. Total proteolytic, amylase and lipase activities were not statistically different between the treatments throughout the periods analyzed (31, 63 and 94 days of culture). However, trypsin and chymotrypsin activities were higher with 31 and 63 days of culture in fish from pond system, suggesting that natural food may have influenced these activities. A positive correlation was observed between the recommended concentration of essential amino acids for Nile tilapia and specific aminopeptidases activity in fish cage system. Substrate-SDS-PAGE revealed 12 active proteolytic bands in both systems. However, integrated density (ID) values were higher in the bands of ponds. Specimens of either cage or pond exhibited five bands of amylolytic activity. Fish from cage and pond systems showed the highest values of ID within 31 days of cultivation. In this study, the complexity of digestive functions could be verified for animals maintained under commercial conditions. Some of the assessed enzymes may show adaptations of their activities and/or expression that allow the fish to achieve a more efficient nutrient assimilation.

  8. 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

  9. 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

  10. Comparative efficiency of microbial enzyme preparations versus pancreatin for in vitro alimentary protein digestion.

    PubMed

    Andriamihaja, Mireille; Guillot, Alain; Svendsen, Allan; Hagedorn, Joerg; Rakotondratohanina, Sahondra; Tomé, Daniel; Blachier, François

    2013-02-01

    Utilisation of microbial enzymes may represent an alternative strategy to the use of conventional pancreatin obtained from pig pancreas for the treatment of severe pancreatic insufficiency. In this study, we focused on the capacity of two microbial preparations for their capacity to digest alimentary proteins (caseins and soya proteins) in comparison with pancreatin. These microbial enzymatic preparations were found to be able to generate small, medium-size and larger polypeptides from caseins and soya proteins but were inactivated at pH 3.0. As determined by Liquid Chromatography-Mass Spectrometry analysis, microbial enzymes generated very different peptides from caseins when compared with peptides generated through pancreatin action. These microbial preparations were characterised by relatively low trypsin- and low carboxypeptidase-like activities but high chymotrypsin-like activities and strong capacity for cleavage of caseins at the methionine sites. Although the efficiency of these microbial preparations to increase the rate of absorption of nitrogen-containing compounds in severe pancreatic insufficiency remains to be tested in vivo, our in vitro data indicate proteolytic capacities of such preparations for alimentary protein digestion.

  11. In vitro digestibility of individual amino acids in rumen-undegraded protein: the modified three-step procedure and the immobilized digestive enzyme assay.

    PubMed

    Boucher, S E; Calsamiglia, S; Parsons, C M; Stern, M D; Moreno, M Ruiz; Vázquez-Añón, M; Schwab, C G

    2009-08-01

    Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy(II) incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA

  12. Purification and characterization of cellulase from North Pacific krill (Euphausia pacifica). Analysis of cleavage specificity of the enzyme.

    PubMed

    Tsuji, Akihiko; Sato, Shiori; Kondo, Ayumi; Tominaga, Keiko; Yuasa, Keizo

    2012-01-01

    Krill are filter feeders that consume algae, plankton and detritus, indicating that krill possess an adequate cellulose digesting system. However, less is known about the enzymatic properties of crustacean cellulases compared to termite cellulases. In the present study, 48 kDa-cellulase was highly purified from krill (Euphausia pacifica) in an effort to determine the cleavage specificity of the enzyme. The most notable characteristic of the enzyme was its high activity against both lichenan and carboxymethyl cellulose. The enzyme hydrolyzed internal β-1,4 glycosidic bonds within lichenan as well as carboxymethyl cellulose to release oligosaccharides and glucose. The effects of pH and temperature on the activity and stability of both enzyme activities were almost identical. Cello-oligosaccharides with a degree of polymerization of 4-6 were hydrolyzed by the enzyme, and the same endo-products, cellotriose, cellobiose and glucose, were produced from these oligosaccharides. Neither cellotriose nor cellobiose was hydrolyzed by the enzyme. The enzyme digested filter paper and sea lettuce to produce cellobiose, cellotriose and glucose as major products. Although amino acid sequence homology of the enzyme with termite cellulases and the presence of oligosaccharides in the enzyme suggested that the enzyme is produced by krill itself, further analysis is necessary.

  13. Influence of enzymes on performance and digestive parameters of broilers fed rye-based diets.

    PubMed

    Lázaro, R; García, M; Medel, P; Mateos, G G

    2003-01-01

    An experiment was conducted to study the influence of enzyme supplementation (ES) to rye-based diets on rate of food passage through the digestive tract, viscosity of jejunum content, volatile fatty acid concentration in ceca, and performance of broilers. There were seven treatments; six diets arranged factorially with three varieties of rye (Petkus, Prima, and Saratov V) and two levels of ES (0 or 500 ppm of an enzyme complex containing 858 IU of beta-glucanase and 864 IU of xylanase/g) and an additional control diet based on corn. Each treatment was replicated seven times (12 chicks caged together), and the trial lasted 25 d. Rye feeding increased intestinal viscosity and impaired bird performance at 25 d (P < 0.001). Among rye diets the greatest feed intake and weight gain were obtained with Petkus variety, which also produced the lowest intestinal viscosity. Enzyme addition reduced the time needed to recover 1% (0.78 vs. 0.98 h; P < 0.05) and 50% (4.2 vs. 6.5 h; P < 0.01) of the marker in feces and reduced the mean retention time of marker in the gastrointestinal tract (17.1 vs. 18.8 h; P < 0.05). Also, ES reduced intestinal viscosity (P < 0.001) and improved feed intake, daily gain, and feed conversion of birds from 4 to 25 d (P < 0.01) but did not modify volatile fatty acid concentration in ceca. We concluded that ES added to rye diets decreased intestinal viscosity and accelerated digestive transit, improving productive performance of broilers.

  14. Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes.

    PubMed

    Ahn, J-E; Lovingshimer, M R; Salzman, R A; Presnail, J K; Lu, A L; Koiwa, H; Zhu-Salzman, K

    2007-06-01

    Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

  15. Effect of adding cofactors to exogenous fibrolytic enzymes on preingestive hydrolysis, in vitro digestibility, and fermentation of bermudagrass haylage.

    PubMed

    Romero, J J; Ma, Z X; Gonzalez, C F; Adesogan, A T

    2015-07-01

    Our objectives were to examine if adding metal ion cofactors (COF) to exogenous fibrolytic enzymes (EFE) would increase the beneficial effects of the EFE on the preingestive hydrolysis and in vitro digestibility and fermentation of bermudagrass haylage. In experiment 1, 5 COF (Mn(2+), Co(2+), Fe(2+), Ca(2+), and Mg(2+)) were screened to select the best candidates for synergistically enhancing release of water-soluble carbohydrates (WSC) from bermudagrass haylage by 5 EFE. The 5 EFE (1A, 2A, 11C, 13D, and 15D) were sourced from Trichoderma reesei and Aspergillus oryzae and they were the most effective of 12 EFE at increasing the neutral detergent fiber digestibility of bermudagrass haylage in a previous trial. Adding 1mM of each of the COF to EFE 2A or 11C synergistically increased release of WSC from bermudagrass haylage, as did adding (1mM) Fe(2+) to 1A, Mn(2+), Co(2+), or Fe(2+) to 13D, or Co(2+)or Fe(2+) to 15D. The greatest release of WSC responses were obtained by adding Mn(2+) to 11C (38%) or by adding Fe(2+) to 2A or 13D (10 and 21.9%, respectively). In experiment 2, the effect of increasing the COF dose on in vitro digestibility and fermentation of bermudagrass haylage was examined using the best EFE-COF combinations from experiment 1. Effects of adding increasing doses of these COF on EFE-mediated changes in vitro digestibility depended on the COF-EFE combination. Adding 10mM Mn(2+) alone to bermudagrass haylage increased DMD and NDFD by 2.7 and 6.3% and adding 11C alone increased these measures by 6.6 and 15.5%, respectively. However, adding 10mM Mn(2+) with 11C resulted in 3.5 and 8.1% increases in DMD and NDFD, respectively, beyond the increases caused by adding 11C alone. Adding Fe(2+) to 2A had no effects on EFE-mediated digestibility responses, but 2A prevented adverse effects of adding Fe(2+) alone on DMD and NDFD. In contrast, adding Fe(2+) to 13D reduced the increases in DMD and NDFD caused by adding the EFE alone. This study shows that adding COF

  16. Examination of digestive enzyme distribution in gut tract and functions of intestinal caecum, in megascolecid earthworms (Oligochaeta: Megascolecidae) in Japan.

    PubMed

    Nozaki, Mana; Ito, Katsutoshi; Miura, Chiemi; Miura, Takeshi

    2013-09-01

    Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.

  17. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

    PubMed

    Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

    Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  18. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

    PubMed Central

    Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

    Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  19. 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

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

    PubMed

    Zou, Jialing; Zheng, Ping; Zhang, Keying; Ding, Xuemei; Bai, Shiping

    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.

  1. Effects of Geroprotectors on Age-Related Changes in Proteolytic Digestive Enzyme Activities at Different Lighting Conditions.

    PubMed

    Morozov, A V; Khizhkin, E A; Svechkina, E B; Vinogradova, I A; Ilyukha, V A; Anisimov, V N; Khavinson, V Kh

    2015-10-01

    We studied the effect of melatonin and epithalon on age-related changes in proteolytic digestive enzyme activity in the pancreas and gastric mucosa of rats kept under different lighting conditions. In rats kept under standard illumination, pepsin activity and the total proteolytic activity in the stomach and pancreas increased by the age of 12 months, but then decreased. Constant and natural lighting disturbed the age dynamics of proteolytic digestive enzyme activity. Administration of melatonin and epithalon to animals exposed to constant lighting restored age dynamics of pepsin activity and little affected total proteolytic activity.

  2. Digestive enzyme activities during early ontogeny in Common snook (Centropomus undecimalis).

    PubMed

    Jimenez-Martinez, L D; Alvarez-González, C A; Tovar-Ramírez, D; Gaxiola, G; Sanchez-Zamora, A; Moyano, F J; Alarcón, F J; Márquez-Couturier, G; Gisbert, E; Contreras-Sánchez, W M; Perales-García, N; Arias-Rodríguez, L; Indy, J R; Páramo-Delgadillo, S; Palomino-Albarrán, I G

    2012-04-01

    Common snook (Centropomus undecimalis) is one of the most important marine species under commercial exploitation in the Gulf of Mexico; for this reason, interest in developing its culture is a priority. However, larviculture remains as the main bottleneck for massive production. In this sense, our objective was to determine the changes of digestive enzymes activities using biochemical and electrophoretic techniques during 36 days of Common snook larviculture fed with live preys (microalgae, rotifers, and Artemia). During larviculture, all digestive enzymatic activities were detected with low values since yolk absorption, 2 days after hatching (dah) onwards. However, the maximum values for alkaline protease (6,500 U mg protein(-1)), trypsin (0.053 mU × 10(-3) mg protein(-1)), and Leucine aminopeptidase (1.4 × 10(-3) mU mg protein(-1)) were detected at 12 dah; for chymotrypsin at 25 dah (3.8 × 10(-3) mU mg protein(-1)), for carboxypeptidase A (280 mU mg protein(-1)) and lipase at 36 dah (480 U mg protein(-1)), for α-amylase at 7 dah (1.5 U mg protein(-1)), for acid phosphatases at 34 dah (5.5 U mg protein(-1)), and finally for alkaline phosphatase at 25 dah (70 U mg protein(-1)). The alkaline protease zymogram showed two active bands, the first (26.3 kDa) at 25 dah onwards, and the second (51.6 kDa) at 36 dah. The acid protease zymogram showed two bands (RF = 0.32 and 0.51, respectively) at 34 dah. The digestive enzymatic ontogeny of C. undecimalis is very similar to other strictly marine carnivorous fish, and we suggest that weaning process should be started at 34 dah.

  3. The effect of dietary wheat middlings and enzyme supplementation II: apparent nutrient digestibility, digestive tract size, gut viscosity, and gut morphology in two strains of leghorn hens.

    PubMed

    Jaroni, D; Scheideler, S E; Beck, M M; Wyatt, C

    1999-12-01

    An experiment was conducted with two strains of Leghorn hens, DeKalb Delta (D) and Hisex White (H), to investigate the effect of a commercial poultry enzyme preparation (EZ; xylanase plus protease) on the digestibility of protein, fat, Ca, and P and to determine any changes in the relative size of the digestive tract, gut morphology, and gut viscosity (GV) of birds fed wheat middlings (WM) over an 18-wk period. Three hundred birds (150 birds per strain) were randomly assigned to six diets: Diet 1, control (corn-soybean); Diets 2 and 3, 8% and 16% WM, respectively; Diet 4, 8% WM and 0.1% enzyme (EZ); and Diets 5 and 6, 16% WM and 0.1% and 0.2% EZ, respectively. There were five replicates per diet per strain. At 50 wk, protein digestibility increased significantly with supplementation of EZ, but, at 60 wk, all responses were similar. Protein digestibility was greater in DeKalb Deltas for WM with EZ compared with Hisex on the same treatment. Fat digestibility was greater for Diet 1 than the other diets at 50 wk but showed a similar response at 60 wk. The H strain showed a reduction in fat digestibility with WM diets with EZ. The control diet showed greater Ca digestibility than the other diets at 50 wk but did not differ at 60 wk. Phosphorus digestibility increased significantly for WM diets with or without EZ at 60 wk. Intestinal weight was significantly higher for WM with or without EZ at 50 wk, but was equal to the control diet at 60 wk. At 60 wk, gizzard weights (GW) were also lower in birds fed WM and WM with EZ compared with birds fed the control, but GV was not affected by dietary treatments. Histological observations on jejunum of birds fed WM without EZ showed shortening, thickening, and atrophy of the villi, all of which improved when EZ was included in the diet. Availability of some nutrients in WM diets was improved with supplementation of enzyme. Gastrointestinal (GI) tract and organ size were increased, and gut morphology appeared to be improved.

  4. 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.

  5. Effect of soaking, germination, and enzyme treatment of whole barley on nutritional value and digestive tract parameters of broiler chickens.

    PubMed

    Svihus, B; Newman, R K; Newman, C W

    1997-09-01

    1. An experiment was carried out to determine the effect of soaking at 0 degrees C, soaking at room temperature, germination, or enzyme treatment of whole barley on feeding value and digestive tract parameters of 2- to 4-week old broiler chickens given diets with 700g/kg whole barley. 2. Soaking or germination decreased the soluble and total beta-glucan content (P < 0.05) and, except for soaking at 0 degrees C, the acid extract viscosity of the grain also decreased (P < 0.05). Germination and soaking in the presence of enzymes produced the lowest beta-glucan content and viscosity. 3. Except for soaking in cold water, the soaking, germination and enzyme treatments increased weight gain and decreased food:gain ratio (P < 0.05). Correspondingly, the digestibility of protein, fat, and ash, and the digestible energy content, increased (P < 0.05) after enzyme treatment or germination. 4. Chickens fed on enzyme-treated or germinated barley diets had intestinal contents with a greater proportion of dry matter and lower viscosity than chickens fed on untreated barley (P < 0.05). Consequently, the cages and chickens were cleaner (P < 0.05) and the weight of digestive organs as proportion of live weight was lower. 5. Particle size analysis of excreta revealed that whole barley was efficiently ground by the gizzards of 16-d-old chickens, and very few whole kernels were found.

  6. 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.

  7. 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

    2016-06-05

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Enzymic dephosphorylation of bovine casein to improve acid clotting properties and digestibility for infant formula.

    PubMed

    Li-Chan, E; Nakai, S

    1989-01-01

    To improve acid clotting properties, enzymic dephosphorylation of caseins with calf intestinal alkaline phosphatase (CAP) or potato acid phosphatase (PAP) was investigated. Greater dephosphorylation was achieved using alpha s1- or beta-casein as substrates, compared to whole casein or skim milk. Electrophoresis of PAP-modified caseins revealed bands with lower mobility and a multibanded pattern in the beta-casein region which was similar to that of human beta-casein. On the other hand, CAP modification produced electrophoretic bands having lower mobility of the beta-casein component, but with higher mobility in the alpha s1-casein component as well as increased net negative charge in the CAP-casein. PAP-casein formed a fine dispersion upon acidification to pH 4, with a microstructure similar to that of acidified human casein. Greater initial rates of hydrolysis by pepsin at pH 4 were observed for both CAP- and PAP-modified caseins, compared to bovine and human caseins. The rate and extent of hydrolysis remained high for CAP-casein but tended to level off with PAP-casein during sequential digestion with pepsin and pancreatin. There may be advantages in the use of partial dephosphorylation to improve acid clotting and digestibility properties of bovine casein for infant feeding.

  9. Changes on digestive enzymes during initial ontogeny in the three-spot cichlid Cichlasoma trimaculatum.

    PubMed

    Toledo-Solís, F J; Uscanga-Martínez, A; Guerrero-Zárate, R; Márquez-Couturier, G; Martínez-García, R; Camarillo-Coop, S; Perales-García, N; Rodríguez-Valencia, W; Gómez-Gómez, M A; Álvarez-González, C A

    2015-02-01

    A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.

  10. Differential regulation of pancreatic digestive enzymes during chronic high-fat diet-induced obesity in C57BL/6J mice.

    PubMed

    Birk, Ruth Z; Rubio-Aliaga, Isabel; Boekschoten, Mark V; Danino, Hila; Müller, Michael; Daniel, Hannelore

    2014-07-28

    Exocrine pancreatic digestive enzymes are essential for the digestion of dietary components and are regulated by them. Chronic excess dietary high fat (HF) consumption is a contributing factor of diet-induced obesity (DIO) and associated chronic diseases and requires adaptation by the pancreas. The aim of the present study was to investigate the effects of chronic HF diet feeding on exocrine pancreatic digestive enzyme transcript levels in DIO C57BL/6J mice. C57BL/6J mice were fed diets containing either 10 or 45% energy (E%) derived from fat for 12 weeks (n 10 mice per diet group). Pancreatic tissue and blood samples were collected at 0, 4 and 12 weeks. The expression of a panel of exocrine pancreatic digestive enzymes was analysed using quantitative RT-PCR and Western blot analysis. The HF (45 E%) diet-fed C57BL/6J mice developed obesity, hyperleptinaemia, hyperglycaemia and hyperinsulinaemia. The transcript levels of pancreatic lipase (PL), pancreatic lipase-related protein 2 (PLRP2) and pancreatic phospholipase A2 (PLA2) were initially elevated; however, they were down-regulated to basal control levels at week 12. The transcript levels of colipase were significantly affected by diet and time. The protein levels of PL and PLRP2 responded to HF diet feeding. The transcript levels of amylase and proteases were not significantly affected by diet and time. The transcript levels of specific lipases in hyperinsulinaemic, hyperleptinaemic and hyperglycaemic DIO C57BL/6J mice are down-regulated. However, these mice compensate for this by the post-transcriptional regulation of the levels of proteins that respond to dietary fat. This suggests a complex regulatory mechanism involved in the modulation of fat digestion.

  11. 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.

  12. 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.

  13. Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers.

    PubMed

    Xu, Z R; Hu, C H; Xia, M S; Zhan, X A; Wang, M Q

    2003-06-01

    Two hundred forty male Avian Farms broiler chicks, 1 d of age, were randomly allocated to four treatments, each of which had five pens of 12 chicks per pen. The chicks were used to investigate the effects of fructooligosaccharide (FOS) on digestive enzyme activities and intestinal microflora and morphology. The chicks received the same basal diet based on corn-soybean meal, and FOS was added to the basal diet at 0, 2.0, 4.0, and 8.0 g/kg diet at the expense of corn. Addition of 4.0 g/kg FOS to the basal diet significantly increased average daily gain of broilers. The feed-to-gain ratios were significantly decreased for the birds fed diets with 2.0 and 4.0 g/kg FOS versus the control. Addition of 4.0 g/kg FOS enhanced the growth of Bifidobacterium and Lactobacillus, but inhibited Escherichia coli in the small intestinal and cecal digesta. Supplementation of 2.0 or 4.0 g/kg FOS to chicks significantly improved the activities of amylase compared to the control (12.80 or 14.75 vs. 8.42 Somogyi units). A significant increase in the activities of total protease was observed in 4.0 g/kg FOS-treated birds versus controls (83.91 vs. 65.97 units). Morphology data for the duodenum, jejunum, and ileum showed no significant differences for villus height, crypt depth, or microvillus height at the duodenum. By contrast, addition of 4.0 g/kg FOS significantly increased ileal villus height, jejunal and ileal microvillus height, and villus-height-to-crypt-depth ratios at the jejunum and ileum and decreased crypt depth at the jejunum and ileum. However, addition of 8.0 g/kg FOS had no significant effect on growth performance, digestive enzyme activities, intestinal microflora, or morphology.

  14. Consequences of lower food intake on the digestive enzymes activities, the energy reserves and the reproductive outcome in Gammarus fossarum.

    PubMed

    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.

  15. 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

  16. Production of lignocellulolytic enzymes and enhancement of in vitro digestibility during solid state fermentation of wheat straw by Phlebia floridensis.

    PubMed

    Sharma, Rakesh Kumar; Arora, Daljit Singh

    2010-12-01

    Degradation by white rot fungi has the potential to increase digestibility of wheat straw and thus improve its value as animal feed. To optimize conditions for production of lignocellulolytic enzymes by Phlebia floridensis during solid state fermentation of wheat straw along with enhancement of in vitro digestibility, a response surface methodology (RSM) based experiment was designed. Effect of moisture content, inorganic nitrogen source (NH(4)Cl) and malt extract on lignocellulolytic enzymes, changes in chemical constituents and digestibility of wheat straw was evaluated. With increase in moisture content, laccase production increased up to 34-fold, while Manganese peroxidase was optimally produced in the presence of almost equal amount (50-55 mg/g of WS) of NH(4)Cl and malt extract. These supplements also significantly (p<0.05) enhanced the production of CMCase and xylanase. In vitro digestibility was increased by almost 50% with a loss of 27.6% and 14.6% in lignin and total organic matter, respectively. The present findings revealed P. floridensis to be an efficient organism for lignocellulolytic enzymes production and simultaneous enhancement in in vitro digestibility of wheat straw. 2010 Elsevier Ltd. All rights reserved.

  17. Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

    PubMed Central

    Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

    2013-01-01

    Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

  18. [Effect of light intensity on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas under two kinds of culture methods].

    PubMed

    Wei, Zi-Zhong; Zhao, Wen

    2014-01-01

    The effects of light intensity (0, 1000, 2000 and 3000 1x) on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicus under two kinds of culture methods (compound Chinese medicine preparation and microbial preparation) were studied. Results showed that the relative mass gain rate (WGR) and the specific growth rate (SGR) of juvenile sea cucumber were significantly affected by light intensity (P < 0.05) , and the orders of WGR and SGR (form high to low) of juvenile sea cucumber under different light intensities were 2000 1x > 1000 1x > 3000 1x > 0 1x. Under the same light intensity, the growth of juvenile sea cucumber under the two kinds of culture methods were significantly different (P < 0.05), with the WGR and SGR of the Chinese medicine treatment being greater than those of the microbial treatment. The light intensity also significantly affected the digestive enzyme activity of juvenile sea cucumber. The order of amylase and lipase activity was 2000 1x > 1000 1x > 3000 1x > 0 1x, while that of protease activity was 1000 1x > 2000 1x > 0 1x > 3000 1x. Under the same light intensity, the digestive enzyme activities of the Chinese medicine treatment were generally higher than those of the microbial treatment.

  19. 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

  20. Molecular motors that digest their track to rectify Brownian motion: processive movement of exonuclease enzymes.

    PubMed

    Xie, Ping

    2009-09-16

    A general model is presented for the processive movement of molecular motors such as λ-exonuclease, RecJ and exonuclease I that use digestion of a DNA track to rectify Brownian motion along this track. Using this model, the translocation dynamics of these molecular motors is studied. The sequence-dependent pausing of λ-exonuclease, which results from a site-specific high affinity DNA interaction, is also studied. The theoretical results are consistent with available experimental data. Moreover, the model is used to predict the lifetime distribution and force dependence of these paused states.

  1. Techniques for quantitative LC-MS/MS analysis of protein therapeutics: advances in enzyme digestion and immunocapture.

    PubMed

    Fung, Eliza N; Bryan, Peter; Kozhich, Alexander

    2016-04-01

    LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. Digestive organ sizes and enzyme activities of refueling western sandpipers (Calidris mauri): contrasting effects of season and age.

    PubMed

    Stein, R Will; Place, Allen R; Lacourse, Terri; Guglielmo, Christopher G; Williams, Tony D

    2005-01-01

    We examined seasonal and age-related variation in digestive organ sizes and enzyme activities in female western sandpipers (Calidris mauri) refueling at a coastal stopover site in southern British Columbia. Adult sandpipers exhibited seasonal variation in pancreatic and intestinal enzyme activities but not in digestive system or organ sizes. Spring migrants had 22% higher total and 67% higher standardized pancreatic lipase activities but 37% lower total pancreatic amylase activity than fall migrants, which suggests that the spring diet was enriched with lipids but low in glycogen. Spring migrants also had 47% higher total intestinal maltase activity as well as 56% higher standardized maltase and 13% higher standardized aminopeptidase-N activities. Spring migrants had higher total enzymic capacity than fall migrants, due primarily to higher total lipase and maltase activities. During fall migration, the juvenile's digestive system was 10% larger than the adult's, and it was composed differently: juveniles had a 16% larger small intestine but a 27% smaller proventriculus. The juvenile's larger digestive system was associated with lower total enzymic capacity than the adult's due to 20% lower total chitinase and 23% lower total lipase activities. These results suggest that juvenile western sandpipers may process food differently from adults and/or have a lower-quality diet.

  4. Release of EPA and DHA from salmon oil - a comparison of in vitro digestion with human and porcine gastrointestinal enzymes.

    PubMed

    Aarak, K E; Kirkhus, B; Holm, H; Vogt, G; Jacobsen, M; Vegarud, G E

    2013-10-01

    In the present study, we hypothesised whether in vitro digestion of salmon oil would release different amounts of PUFA depending on the origin of the lipolytic enzymes used. For this purpose, in vitro digestion of salmon oil (SO) was performed using human duodenal juice (HDJ) or a commercial enzyme preparation consisting of porcine pancreatin and bile (PB). The lipolytic effect was determined by measuring the release of fatty acids (FA) using solid-phase extraction and GC-flame ionisation detection, withdrawing samples every 20 min during digestion. The amount of FA released indicated that a plateau was reached after 80 min with approximately similar amounts of FA detected using both HDJ and PB (379 (sd 18) and 352 (sd 23) mg/g SO, respectively). However, the release of 18 : 2, EPA (20 : 5) and DHA (22 : 6) was significantly different during in vitro digestion. At 80 min, HDJ and PB released 43 and 33% of 18 : 2, 14 and 9% of EPA and 11 and 9% of DHA, respectively. Both enzyme preparations released approximately the same amounts of the other FA analysed. The effect of the addition of bile salts (BS) was significantly different in the two enzyme systems, where porcine pancreatin highly responded to the increase in BS concentration, in contrast to HDJ.

  5. Effects of treating sorghum wet distillers grains with solubles with fibrolytic enzymes on nutrient digestibility and performance in finishing beef steers

    USDA-ARS?s Scientific Manuscript database

    Two experiments were conducted to determine the effects of treating sorghum WDG with solubles (SWDG) with an enzyme, or enzyme-buffer combination on diet digestibility and feedlot performance. Experimental treatments are; 1) untreated SWDG (control), 2) addition of an enzyme complex to SWDG (enzyme...

  6. 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.

  7. Angiotensin I-converting enzyme inhibitory peptides generated from in vitro gastrointestinal digestion of pork meat.

    PubMed

    Escudero, Elizabeth; Sentandreu, Miguel Angel; Arihara, Keizo; Toldrá, Fidel

    2010-03-10

    The main purpose of this work was to study the generation of Angiotensin I-converting enzyme inhibitory (ACEI) peptides after gastrointestinal digestion of pork meat by the action of pepsin and pancreatin at simulated gut conditions. The hydrolysate was further subjected to reverse phase chromatography in order to separate the fractions with ACEI activity. Using MALDI-TOF/TOF mass spectrometry, 12 peptides were identified in these fractions. It is worth highlighting the novel peptides ER, KLP, and RPR with IC(50) values of 667 microM, 500 microM, and 382 microM, respectively. Results obtained by MALDI-TOF/TOF mass spectrometry were complemented by a second approach consisting of the analysis of the hydrolysate directly by nanoLC-ESI-MS/MS followed by a study of the obtained sequences and comparison with known ACEI peptide sequences. By using these two approaches, a total of 22 peptides were selected for its synthesis and further in vitro assay of ACEI activity. The strongest ACE inhibition was observed for peptide KAPVA (IC(50) = 46.56 microM) followed by the sequence PTPVP (IC(50) = 256.41 microM). Sequence similarity searches revealed that these two peptides derive from muscle titin, constituting the first identified ACEI peptides coming from this protein. This is also the first time that ACEI sequences MYPGIA and VIPEL have been reported. Other identified and synthesized sequences showed less ACEI activity. The obtained results evidence the potential of pork meat proteins as a source of antihypertensive peptides after gastrointestinal digestion.

  8. Kernel composition, starch structure, and enzyme digestibility of opaque-2 maize and quality protein maize.

    PubMed

    Hasjim, Jovin; Srichuwong, Sathaporn; Scott, M Paul; Jane, Jay-Lin

    2009-03-11

    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 (12.7-13.3%). Kernels of a severe o2 mutant B46o2 also contained less starch (66.9%) than those of B46wt (73.0%). B46o2 and QPM starches contained less amylose (28.0 and 26.0%, respectively) than others (31.9-33.7%). The B46o2 starch also consisted of amylopectin with the fewest branch chains of DP 13-24. Thus, the B46o2 starch was the most susceptible to porcine pancreatic alpha-amylase (PPA) hydrolysis. Starches of the dry-ground o2 maize and QPM were hydrolyzed faster than that of the dry-ground WT maize, resulting from the reduced protein content of the o2-maize kernels and the reduced amylose content of the B46o2 and QPM starch. Starch in the dry-ground maize sample was hydrolyzed faster by PPA (85-91%) than was the isolated starch (62-71%), which could be attributed to the presence of mechanically damaged starch granules and endogenous amylases in the dry-ground maize samples. These results showed that o2 maize and QPM had highly digestible starch and could be desirable for feed and ethanol production.

  9. Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes.

    PubMed

    Speda, Jutta; Johansson, Mikaela A; Odnell, Anna; Karlsson, Martin

    2017-01-01

    Enzymatic treatment of lignocellulosic material for increased biogas production has so far focused on pretreatment methods. However, often combinations of enzymes and different physicochemical treatments are necessary to achieve a desired effect. This need for additional energy and chemicals compromises the rationale of using enzymes for low energy treatment to promote biogas production. Therefore, simpler and less energy intensive in situ anaerobic digester treatment with enzymes is desirable. However, investigations in which exogenous enzymes are added to treat the material in situ have shown mixed success, possibly because the enzymes used originated from organisms not evolutionarily adapted to the environment of anaerobic digesters. In this study, to examine the effect of enzymes endogenous to methanogenic microbial communities, cellulolytic enzymes were instead overproduced and collected from a dedicated methanogenic microbial community. By this approach, a solution with very high endogenous microbial cellulolytic activity was produced and tested for the effect on biogas production from lignocellulose by in situ anaerobic digester treatment. Addition of enzymes, endogenous to the environment of a mixed methanogenic microbial community, to the anaerobic digestion of ensiled forage ley resulted in significantly increased rate and yield of biomethane production. The enzyme solution had an instant effect on more readily available cellulosic material. More importantly, the induced enzyme solution also affected the biogas production rate from less accessible cellulosic material in a second slower phase of lignocellulose digestion. Notably, this effect was maintained throughout the experiment to completely digested lignocellulosic substrate. The induced enzyme solution collected from a microbial methanogenic community contained enzymes that were apparently active and stable in the environment of anaerobic digestion. The enzymatic activity had a profound effect on the

  10. Digestive enzyme as benchmark for insecticide resistance development in Culex pipiens larvae to chemical and bacteriologic insecticides.

    PubMed

    Kamel, Nashwa H; Bahgat, Iman M; El Kady, Gamal A

    2013-04-01

    This work monitored changes in some digestive enzymes (trypsin and aminopeptidase) associated with the building up of resistance in Cx. pipiens larvae to two chemical insecticides (methomyl and/or malathion) and one biological insecticide (Bacillus thuringiensis-H14 or B.t H 14). The LC50 value of methomyl for both field- and the 12th generation (F12) of the selected strain was 1.789 ppm and 8.925 ppm respectively. The LC50 value of malathion for both field and the F12 of the selected strain was 0.082 ppm and 0.156 ppm respectively, and those of B.t H14 of field strain and the F12 was 2.550ppm & 2.395ppm respectively. The specific activity of trypsin enzyme in control susceptible colony was 20.806 +/- 0.452micromol/min/mg protein; but at F4 and F8 for malathion and methomyl treated larvae were 10.810 +/- 0.860 & 15.616+/-0.408 micromol/min/mg protein, respectively. Trypsin activity of F12 in treated larvae with B.t.H14 was 2.097 +/- 0.587 microiol/min/mg protein. Aminopeptidase specific activity for susceptible control larvae was 173.05 +/- 1.3111 micromol/min/mg protein. This activity decreased to 145.15 +/- 4.12, 152.497 +/- 6.775 & 102.04 +/- 3.58a micromol/min/mg protein after larval (F 12) treatment with methomyl, malathion and B.t H 14 respectively.

  11. A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots.

    PubMed

    Wouters, Bert; Dapic, Irena; Valkenburg, Thalassa S E; Wouters, Sam; Niezen, Leon; Eeltink, Sebastiaan; Corthals, Garry L; Schoenmakers, Peter J

    2017-03-31

    A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Site-specific DNA transesterification catalyzed by a restriction enzyme.

    PubMed

    Sasnauskas, Giedrius; Connolly, Bernard A; Halford, Stephen E; Siksnys, Virginijus

    2007-02-13

    Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5' terminus of the cleaved DNA. Under certain conditions, the terminal 3'-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that BfiI first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively.

  13. 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

  14. Diverse enzymatic specificities of digestive proteases, 'intestains', enable Colorado potato beetle larvae to counteract the potato defence mechanism.

    PubMed

    Gruden, Kristina; Popovic, Tatjana; Cimerman, Nina; Krizaj, Igor; Strukelj, Borut

    2003-02-01

    In response to insect attack, high levels of proteinase inhibitors are synthesised in potato leaves. This can cause inefficient protein digestion in insects, leading to reduced growth, delayed development and lower fecundity. It has been suggested that Colorado potato beetle overcomes this defence mechanism by inducing the production of a set of cysteine proteases that are resistant to potato proteinase inhibitors. Experiments with gut extracts showed that these proteases have unusual inhibition profiles as they are not inhibited by most of the cystatins but are strongly inhibited by thyropins. In this study we have isolated three cysteine proteases from adapted guts of Colorado potato beetle larvae, named intestains 1, 2 and 3, the first cysteine proteases known to be involved in extracellular protein digestion. The N-terminal sequences suggest their classification into the papain family. Intestains differ in substrate specificities and inhibitory profiles. Their substrate specificities suggest that intestains 1 and 2 are general digestive enzymes, while intestain 3 has a more specific function. The inhibitory profile of intestain 1 is similar to that of proteases of the papain family. However, the Ki values for the interaction of intestain 2 with the same set of inhibitors are several hundred fold higher, which would enable the enzyme to circumvent the potato defence mechanism characterised by high concentrations of protease inhibitors in attacked potato leaves. A further, different strategy of the Colorado potato beetle to avoid potato defence is exhibited by intestain 3, which is able to cleave off the N-terminus of model cystatin and thus inactivate the inhibitor. These results suggest that the Colorado potato beetle combines different strategies to counteract plant defence mechanisms.

  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. Pretreatment of rice straw by proton beam irradiation for efficient enzyme digestibility.

    PubMed

    Kim, Sung Bong; Kim, Jun Seok; Lee, Jong Ho; Kang, Seong Woo; Park, Chulhwan; Kim, Seung Wook

    2011-08-01

    Biomass was pretreated with proton beam irradiation (PBI) in order to enhance enzyme digestibility. Rice straw and soaking in aqueous ammonia (SAA)-treated rice straw were treated with 1-25 kGy doses of PBI at a beam energy of 45 MeV. The optimal doses of PBI for efficient sugar recovery were 15 and 3 kGy for rice straw and SAA-treated rice straw, respectively. When PBI was applied to rice straw at 15 kGy, the glucose conversion reached 68% of the theoretical maximum at 72 h. When 3 kGy of PBI was applied to SAA-treated rice straw, approximately 90% of the theoretical glucose conversion was obtained at 12 h compared to a 89% conversion at 48 h. After 2 h of enzymatic saccharification, the initial reaction rates of raw rice straw pretreated with 15 kGy of PBI and SAA-treated rice straw pretreated with 3 kGy of PBI were 1.4 × 10⁻⁴ and 9.7 × 10⁻⁴ g L⁻¹ s⁻¹, respectively. Further, the results of X-ray diffractometry support the effect of PBI on sugar recovery, whereas scanning electron microscopy images revealed a more rugged rice straw surface.

  17. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Control of the release of digestive enzymes in the larvae of the fall armyworm, Spodoptera frugiperda.

    PubMed

    Lwalaba, Digali; Hoffmann, Klaus H; Woodring, Joseph

    2010-01-01

    There is a basal level of enzyme activity for trypsin, aminopeptidase, amylase, and lipase in the gut of unfed larval (L6) Spodoptera frugiperda. Trypsin activity does not decrease with non-feeding, possibly because of the low protein levels in plants along with high amino acid requirements for growth and storage (for later reproduction in adults). Therefore, trypsin must always be present so that only a minimal protein loss via egestion occurs. Larvae, however, adjust amylase activity to carbohydrate ingestion, and indeed amylase activity is five-fold higher in fed larvae compared to unfed larvae. Gut lipase activity is low, typical of insects with a high carbohydrate diet. A flat-sheet preparation of the ventriculus was used to measure the release of enzymes in response to specific nutrients and known brain/gut hormones in S. frugiperda. Sugars greatly increase (>300%) amylase release, but starch has no effect. Proteins and amino acids have little or no effect on trypsin or aminopeptidase release. The control of enzyme release in response to food is likely mediated through neurohormones. Indeed, an allatostatin (Spofr-AS A5) inhibits amylase and trypsin, and allatotropin (Manse- AT) stimulates amylase and trypsin release. Spofr-AS A5 also inhibits ileum myoactivity and Manse-AT stimulates myoactivity. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter-current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. (c) 2009 Wiley Periodicals, Inc.

  19. Specificity of infant digestive conditions: some clues for developing relevant in vitro models.

    PubMed

    Bourlieu, Claire; Ménard, Olivia; Bouzerzour, Karima; Mandalari, Giuseppina; Macierzanka, Adam; Mackie, Alan R; Dupont, Didier

    2014-01-01

    Digestion of nutrients is an essential function of the newborn infant gut to allow growth and development and understanding infant digestive function is essential to optimize nutrition and oral drug delivery. Ethical considerations prohibit invasive in vivo trials and as a consequence in vitro assays are often conducted. However, the choice of in vitro model parameters are not supported by an exhaustive analysis of the literature and do not mimic precisely the digestive conditions of the infant. This review contains a compilation of the studies which characterized the gastroduodenal conditions in full-term or preterm infants of variable postnatal age from birth up to six months. Important data about healthy full-term infants are reported. The enzymatic (type of enzymes and level of activity) and nonenzymatic (milk-based diet, frequency of feeding, bile salt concentrations) conditions of digestion in infants are shown to differ significantly from those in adults. In addition, the interindividual and developmental variability of the digestive conditions in infants is also highlighted.

  20. 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

    2016-02-05

    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.

  1. 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

  2. Pectinases From Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): Putative Accessory Digestive Enzymes

    PubMed Central

    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

  3. O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity, and enzyme regulation.

    PubMed

    Vocadlo, David J

    2012-12-01

    The addition of N-acetylglucosamine (GlcNAc) O-linked to serine and threonine residues of proteins is known as O-GlcNAc. This post-translational modification is found within multicellular eukaryotes on hundreds of nuclear and cytoplasmic proteins. O-GlcNAc transferase (OGT) installs O-GlcNAc onto target proteins and O-GlcNAcase (OGA) removes O-GlcNAc. Their combined action makes O-GlcNAc reversible and serves to regulate cellular O-GlcNAc levels. Here I review select recent literature on the catalytic mechanism of these enzymes and studies on the molecular basis by which these enzymes identify and process their substrates. Molecular level understanding of how these enzymes work, and the basis for their specificity, should aid understanding how O-GlcNAc contributes to diverse cellular processes ranging from cellular signaling through to transcriptional regulation.

  4. [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.

  5. [Effect of different environmental factors on the activities of digestive enzymes and alkaline phosphatase of Macrobrochium nipponense].

    PubMed

    Wang, Weina; Sun, Ruyong; Wang, Anli; Bao, Lei; Wang, Peng

    2002-09-01

    The activities of digestive enzymes and alkaline phosphatase from the hepatopancreas of Macrobrochium nipponense were determined under different environmental factors (calcium concentrations 20 mg.L-1, 35 mg.L-1, 60 mg.L-1, 80 mg.L-1, 150 mg.L-1; salinity 7@1000, 14@1000, pH 7.6, 8.8, 9.8). The results showed that higher Ca2+ concentration could enhance the pepsin activity, but inhibit the trysin-like activity in hepatopancreas of M. nipponense. The activities of pepsin, trysin-like, alkaline phosphatase in hepatopancreas of M. nipponense were higher under salinity of 14@1000 than under salinity of 7@1000 and 20@1000. It showed that the activities of digestive enzymes and alkaline phosphatase of shrimp increased gradually with increasing pH value from 7.6 to 9.8.

  6. Effect of Flavonoid-Rich Extract of Glycyrrhiza glabra on Gut-Friendly Microorganisms, Commercial Probiotic Preparations, and Digestive Enzymes.

    PubMed

    Asha, Mannanthendil Kumaran; Debraj, Debnath; Dethe, Shekhar; Bhaskar, Anirban; Muruganantham, Nithyanantham; Deepak, Mundkinajeddu

    2017-05-04

    Flavonoid-rich extract prepared from Glycyrrhiza glabra has been found to be beneficial in patients with functional dyspepsia and was reported to possess some gut health-promoting properties such as antioxidant, anti-inflammatory and anti-Helicobacter pylori activities. In the present study, the flavonoid-rich extract of Glycyrrhiza glabra was evaluated for its compatibility with probiotic strains (Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, and Streptococcus thermophilus), commercial probiotic drinks, and digestive enzymes (pancreatic α-amylase, α-glucosidase, phytase, xylanase, and pancreatic lipase). Results of this study indicated that the flavonoid-rich extract of Glycyrrhiza glabra is compatible with the tested probiotic strains, probiotic drinks and digestive enzymes.

  7. Different digestion enzymes used for human pancreatic islet isolation: a mixed treatment comparison (MTC) meta-analysis.

    PubMed

    Rheinheimer, Jakeline; Ziegelmann, Patrícia Klarmann; Carlessi, Rodrigo; Reck, Luciana Ross; Bauer, Andrea Carla; Leitão, Cristiane Bauermann; Crispim, Daisy

    2014-01-01

    Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval - CrI) = -2.19 (-4.25 to -0.21) and -2.28 (-4.49 to -0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = -1.69 (-2.87 to -0.51) and -1.07 (-1.79 to -0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF.

  8. 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.

  9. Extraction and analysis of chlorpromazine and its major metabolites in post mortem material by enzymic digestion and HPLC.

    PubMed

    Allender, W J; Archer, A W; Dawson, A G

    1983-01-01

    A method is described for the determination of chlorpromazine and some of its major metabolites in post mortem specimens by enzymic digestion of the tissues with ethyl acetate using a simple, single micro-extraction method, followed by HPLC of the extracts using a 10 micron silica column packing and a mobile phase consisting of ethanolamine:methanol:water. Separation and quantitation of 7-hydroxy-chlorpromazine, chlorpromazine, chlorpromazine sulfoxide, norchlorpromazine and norchlorpromazine sulfoxide was achieved employing mesoridazine as an internal standard.

  10. Substrate specificity and enzyme recycling using chitosan immobilized laccase.

    PubMed

    Skoronski, Everton; Fernandes, Mylena; Magalhães, Maria de Lourdes Borba; da Silva, Gustavo Felippe; João, Jair Juarez; Soares, Carlos Henrique Lemos; Júnior, Agenor Fúrigo

    2014-10-17

    The immobilization of laccase (Aspergillus sp.) on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method as compared to immobilization via physical adsorption. To assess the influence of different substrate functional groups on the enzyme's catalytic efficiency, substrate specificity was investigated using chitosan-immobilized laccase and eighteen different phenol derivatives. It was observed that 4-nitrophenol was not oxidized, while 2,5-xylenol, 2,6-xylenol, 2,3,5-trimethylphenol, syringaldazine, 2,6-dimetoxyphenol and ethylphenol showed reaction yields up 90% at 40 °C. The kinetic of process, enzyme recyclability and operational stability were studied. In batch reactors, it was not possible to reuse the enzyme when it was applied to syringaldazne bioconversion. However, when the enzyme was applied to bioconversion of 2,6-DMP, the activity was stable for eight reaction batches.

  11. Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.

    PubMed

    Rago, Florencia; Saltzberg, Daniel; Allen, Karen N; Tolan, Dean R

    2015-11-04

    Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.

  12. 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. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Site-specific DNA transesterification catalyzed by a restriction enzyme

    PubMed Central

    Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

    2007-01-01

    Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5′ terminus of the cleaved DNA. Under certain conditions, the terminal 3′-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that BfiI first makes a covalent enzyme–DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively. PMID:17267608

  14. 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.

  15. LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function.

    PubMed

    Holmstrom, Sam R; Deering, Tye; Swift, Galvin H; Poelwijk, Frank J; Mangelsdorf, David J; Kliewer, Steven A; MacDonald, Raymond J

    2011-08-15

    We have determined the cistrome and transcriptome for the nuclear receptor liver receptor homolog-1 (LRH-1) in exocrine pancreas. Chromatin immunoprecipitation (ChIP)-seq and RNA-seq analyses reveal that LRH-1 directly induces expression of genes encoding digestive enzymes and secretory and mitochondrial proteins. LRH-1 cooperates with the pancreas transcription factor 1-L complex (PTF1-L) in regulating exocrine pancreas-specific gene expression. Elimination of LRH-1 in adult mice reduced the concentration of several lipases and proteases in pancreatic fluid and impaired pancreatic fluid secretion in response to cholecystokinin. Thus, LRH-1 is a key regulator of the exocrine pancreas-specific transcriptional network required for the production and secretion of pancreatic fluid.

  16. [Digestion of fat and protein and its relation to the activity of faecal enzymes in mucoviscidosis infants under substitution therapy with pancreatic enzymes (author's transl)].

    PubMed

    Tolckmitt, W; Lohmann, A; Kutschera, J

    1981-09-01

    The article reports on a study to determine the influence of different dosage schedules of a pancreas enzyme preparation on the digestion of fat and protein and on the lipolytic, tryptic and chymotryptic activity of the faeces in children suffering from mucoviscidose. After administration of the pancreatin preparation at the low dose level, the quantity of faeces, the excretion of fatty acids and of nitrogen are reduced and the absorption coefficient is improved. The faecal tryptic and chymotryptic activity increases, whereas the lipase activity in the faeces merely shows a tendency to rise. Increasing the pancreatin dose up to a still tolerable value by doubling the dosage does not result in further reduction of the excretion of fat and nitrogen although it enhances the proteolytic activity of the faeces while the lipase activity remains unaffected. Determination of the lipolytic, tryptic or chymotryptic activity is, therefore, unsuitable for assessing the fat and protein digestion and for estimating the required dose of pancreatic enzyme. No increase in uric acid excretion was seen in the urine collected for 24 hours after administration of both enzyme doses.

  17. 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

  18. 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.

  19. Synthesis of. beta. -D-glucan in vitro: HPLC separation of oligosaccharides from enzymic digests of glucans synthesized by Golgi apparatus and UDP-glucose

    SciTech Connect

    Gibeaut, D.M.; Carpita, N.C. )

    1990-05-01

    Anion exchange HPLC (Dionex) resolves {beta}-D-glucose oligosaccharides which vary in (1{yields}3) and (1{yields}4) linkage structure. The products from incubation of UDPG and cellular membranes from maize enriched for Golgi apparatus, ER, and plasma membrane are digested with glucan hydrolases specific for either (1{yields}3) or (1{yields}4) linkages and one which requires a specific (1{yields}3){beta}-D-glc-(1{yields}4){beta}-D-glc sequence. Two oligomers released by the latter enzyme are diagnostic of cereal grass mixed-linkage {beta}-D-glucan and are used as standards to examine reaction conditions for synthesis of this polymer with radioactive substrates. Our preliminary data indicate that formation of specific sequences of {beta}-D-glucan is increased by high concentrations of UDPG. Other radioactive oligomers are present in greater quantity than those from authentic mixed-linkage glucan and their linkage structure is now under investigation.

  20. Stability of Rosmarinic Acid in Aqueous Extracts from Different Lamiaceae Species after in vitro Digestion with Human Gastrointestinal Enzymes

    PubMed Central

    Zorić, Zoran; Markić, Joško; Pedisić, Sandra; Bučević-Popović, Viljemka; Generalić-Mekinić, Ivana; Grebenar, Katarina

    2016-01-01

    Summary The present study compares the gastrointestinal stability of rosmarinic acid in aqueous extracts of thyme, winter savory and lemon balm with the stability of pure rosmarinic acid. The stability of rosmarinic acid was detected after two-phase in vitro digestion process (gastric and duodenal) with human gastrointestinal enzymes. The concentration of rosmarinic acid in undigested and digested samples was detected using HPLC-DAD. Results showed that gastrointestinal stability of pure rosmarinic acid was significantly higher than that of rosmarinic acid from plant extracts after both gastric and intestinal phases of digestion. Among plant extracts, rosmarinic acid was the most stable in lemon balm after gastric (14.10%) and intestinal digestion phases (6.5%). The temperature (37 °C) and slightly alkaline medium (pH=7.5) did not affect the stability of rosmarinic acid, while acid medium (pH=2.5) significantly decreased its stability (≥50%). In addition, the stability rate of rosmarinic acid is influenced by the concentration of human gastrointestinal juices. PMID:27904398

  1. Stability of Rosmarinic Acid in Aqueous Extracts from Different Lamiaceae Species after in vitro Digestion with Human Gastrointestinal Enzymes.

    PubMed

    Zorić, Zoran; Markić, Joško; Pedisić, Sandra; Bučević-Popović, Viljemka; Generalić-Mekinić, Ivana; Grebenar, Katarina; Kulišić-Bilušić, Tea

    2016-03-01

    The present study compares the gastrointestinal stability of rosmarinic acid in aqueous extracts of thyme, winter savory and lemon balm with the stability of pure rosmarinic acid. The stability of rosmarinic acid was detected after two-phase in vitro digestion process (gastric and duodenal) with human gastrointestinal enzymes. The concentration of rosmarinic acid in undigested and digested samples was detected using HPLC-DAD. Results showed that gastrointestinal stability of pure rosmarinic acid was significantly higher than that of rosmarinic acid from plant extracts after both gastric and intestinal phases of digestion. Among plant extracts, rosmarinic acid was the most stable in lemon balm after gastric (14.10%) and intestinal digestion phases (6.5%). The temperature (37 °C) and slightly alkaline medium (pH=7.5) did not affect the stability of rosmarinic acid, while acid medium (pH=2.5) significantly decreased its stability (≥50%). In addition, the stability rate of rosmarinic acid is influenced by the concentration of human gastrointestinal juices.

  2. 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.

  3. 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

  4. Effect of dietary or abomasal supplementation of exogenous polysaccharide-degrading enzymes on rumen fermentation and nutrient digestibility.

    PubMed

    Hristov, A N; McAllister, T A; Cheng, K J

    1998-12-01

    The effect of site of supplementation of a mixture of two crude preparations (Enzyme C and Enzyme X) of exogenous polysaccharide-degrading enzymes (EPDE) was studied in vivo using four ruminally and duodenally cannulated heifers (Exp. 1). The treatments were as follows: control (no EPDE), EPDE supplied through the diet (EF, 47.0 g/d), and EPDE infused continuously into the abomasum (EA, 41.6 g/d). Enzyme treatment increased the concentration of soluble reducing sugars (P < .05) and decreased NDF content (P < .05) in the treated feed, but this did not increase the rate or extent of in sacco disappearance of DM from the feed. Compared with control, ruminal fermentation was not affected by EF, but abomasal infusion increased (P < .05) rumen ammonia levels and shifted ruminal VFA patterns. Ruminal carboxymethylcellulase (CMCase) and xylanase activities were not affected by treatment. Abomasal infusion increased (P < .05) duodenal xylanase activity as compared with control and EF, but apparent digestion of DM, NDF, and CP were not affected by treatment. Negligible levels of CMCase and amylase reached the duodenum. During an in vitro experiment (Exp. 2), abomasal stability of the two EPDE was studied over a range of pH from 3.39 to .85, with or without pepsin. Carboxymethylcellulase activity (in Enzymes C and X) and beta-glucanase activity (in Enzyme C) were largely unstable against pepsin proteolysis (P < .001) and low pH (P < .001). Xylanase and amylase activities were resistant to pepsin but irreversibly inactivated at low pH. These two experiments showed that abomasal supplementation of EPDE did not successfully supply cellulases and amylases to the intestine, due partially to their limited resistance to low pH and pepsin proteolysis. Although EPDE significantly increased the level of xylanase activity at the duodenum, this did not significantly improve total tract digestion.

  5. Structural studies of the Trypanosoma cruzi Old Yellow Enzyme: insights into enzyme dynamics and specificity.

    PubMed

    Murakami, Mário T; Rodrigues, Nathalia C; Gava, Lisandra M; Honorato, Rodrigo V; Canduri, Fernanda; Barbosa, Leandro R S; Oliva, Glaucius; Borges, Júlio C

    2013-12-31

    The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/β)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of β-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.

  6. Characteristics of digestive enzymes of calanoid copepod species from different latitudes in relation to temperature, pH and food.

    PubMed

    Freese, Daniela; Kreibich, Tobias; Niehoff, Barbara

    2012-08-01

    In calanoid copepods it is poorly understood how enzymatic activities and patterns are affected by abiotic and biotic factors. Such knowledge, however, is crucial to assess metabolic functioning and performance of organisms in different habitats. Therefore, our study focuses on digestive enzyme activities in relation to temperature, pH and food in the Arctic species Calanus glacialis and in Centropages hamatus and Temora longicornis from the North Sea. Enzyme activities were measured over a range from 0 to 70 °C (lipases/esterases, proteinases) and pH 5 to 9 (proteinases). In all species, relative proteinases activity peaked at 40/50 °C and pH 6; relative lipases/esterases activity peaked at 30 °C. Between 0 and 20 °C, lipase activity of C. glacialis was higher (40-70% of maximum) than that of the boreal copepods (25-64%), which suggests thermal adaptation of the lipid metabolism in the polar species. Incubating C. glacialis with the diatom Thalassiosira weissflogii showed (i) that enzyme activities increased especially in the alkaline range and (ii) that enzyme patterns, revealed by gel electrophoresis, differed from that of starving individuals, indicating that feeding induced enzyme expression. Such studies, linking abiotic and biotic conditions to enzyme functioning, can help elucidating the capacity of copepods to respond to environmental changes. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Effect of prebiotic konjac mannanoligosaccharide on growth performances, intestinal microflora, and digestive enzyme activities in yellow catfish, Pelteobagrus fulvidraco.

    PubMed

    Wu, Zhi-xin; Yu, Yan-mei; Chen, Xi; Liu, Hong; Yuan, Juan-fa; Shi, Yan; Chen, Xiao-xuan

    2014-06-01

    In the present study, konjac mannanoligosaccharide (KMOS) was evaluated as a prebiotic in yellow catfish. The fish were fed with diets containing KMOS in four concentrations: 0 g kg(-1) (C), 1.0 g kg(-1) (KM1), 2.0 g kg(-1) (KM2), and 3.0 g kg(-1) (KM3) for 49 days, respectively. Another group fed with diets containing 3.0 g kg(-1) yeast cell wall mannanoligosaccharide (MOS) (M3) was set as positive control. The results indicated that fish receiving the diets supplemented with KMOS or MOS showed higher relative gain rate (RGR), specific growth rate (SGR), and lower feed conversion ratio (FCR) with significantly differences (P < 0.05) than those fed with the basal diets. Moreover, fish receiving the diets with 2.0 g kg(-1) KMOS inclusion showed higher RGR, SGR, and lower FCR (P < 0.05) than that feeding the diets supplemented with 3.0 g kg(-1) MOS. The quantities of Bifidobacterium spp. were significantly increased (P < 0.05). Meanwhile, Escherichia coli and Aeromonas spp. were significantly reduced (P < 0.05) in the fish-feeding diets with 2.0 g kg(-1) KMOS supplement. Compared with the control group, the significantly enhancement of protease and amylase activity (P < 0.05) in intestine and pancreas was observed in fish fed with diets containing KMOS or MOS. Collectively, an optimum level of KMOS inclusion in diets could modulate intestinal microflora, induce digestive enzyme activity, and improve the growth performance of yellow catfish significantly.

  8. Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

    PubMed

    Izagirre, U; Angulo, E; Wade, S C; ap Gwynn, I; Marigómez, I

    2009-02-01

    In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

  9. 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.

  10. 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.

  11. THE SECRETION OF DIGESTIVE ENZYMES AND CAECAL SIZE ARE DETERMINED BY DIETARY PROTEIN IN THE CRICKET Gryllus bimaculatus.

    PubMed

    Woodring, Joseph; Weidlich, Sandy

    2016-11-01

    In Gryllus bimaculatus, the size of the caecum decreases in the latter half of each instar to a stable minimal size with a steady minimal rate of digestive enzyme secretion until feeding resumes after ecdysis. The higher the percent protein in the newly ingested food, the faster and larger the caecum grows, and as a consequent the higher the secretion rate of trypsin and amylase. When hard boiled eggs (40% protein) are eaten the caecum is 2× larger, the trypsin secretion is almost 3× greater, and amylase 2.5× greater then when fed the same amount of apples (1.5% protein). Only dietary protein increases amylase secretion, whereas dietary carbohydrates have no effect on amylase secretion. The minimal caecal size and secretion rate must be supported by utilization of hemolymph amino acids, but the growth of the caecum and increasing enzymes secretions after the molt depend upon an amino acid source in the lumen. This simple regulation of digestive enzyme secretion is ideal for animals that must stop feeding in order to molt. This basic control system does not preclude additional regulation mechanisms, such as prandal, which is also indicated for G. bimaculatus, or even paramonal regulation. © 2016 Wiley Periodicals, Inc.

  12. Processing technologies and cell wall degrading enzymes to improve nutritional value of dried distillers grain with solubles for animal feed: an in vitro digestion study.

    PubMed

    de Vries, Sonja; Pustjens, Annemieke M; Kabel, Mirjam A; Salazar-Villanea, Sergio; Hendriks, Wouter H; Gerrits, Walter J J

    2013-09-18

    Currently, the use of maize dried distillers grain with solubles (DDGS) as protein source in animal feed is limited by the inferior protein quality and high levels of non-starch polysaccharides (NSP). Processing technologies and enzymes that increase NSP degradability might improve digestive utilization of DDGS, enhancing its potential as a source of nutrients for animals. The effects of various combinations of processing technologies and commercial enzyme mixtures on in vitro digestion and subsequent fermentation of DDGS were tested. Wet-milling, extrusion, and mild hydrothermal acid treatment increased in vitro protein digestion but had no effect on NSP. Severe hydrothermal acid treatments, however, effectively solubilized NSP (48-78%). Addition of enzymes did not affect NSP solubilization in unprocessed or processed DDGS. Although the cell wall structure of DDGS seems to be resistant to most milder processing technologies, in vitro digestion of DDGS can be effectively increased by severe hydrothermal acid treatments.

  13. In vitro digestion of purified β-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

    PubMed

    Petrat-Melin, B; Andersen, P; Rasmussen, J T; Poulsen, N A; Larsen, L B; Young, J F

    2015-01-01

    Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory

  14. Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

    PubMed

    Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

    2015-01-01

    The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

  15. 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.

  16. RNA-Seq reveals the dynamic and diverse features of digestive enzymes during early development of Pacific white shrimp Litopenaeus vannamei.

    PubMed

    Wei, Jiankai; Zhang, Xiaojun; Yu, Yang; Li, Fuhua; Xiang, Jianhai

    2014-09-01

    The Pacific white shrimp (Litopenaeus vannamei), with high commercial value, has a typical metamorphosis pattern by going through embryo, nauplius, zoea, mysis and postlarvae during early development. Its diets change continually in this period, and a high mortality of larvae also occurs in this period. Since there is a close relationship between diets and digestive enzymes, a comprehensive investigation about the types and expression patterns of all digestive enzyme genes during early development of L. vannamei is of considerable significance for shrimp diets and larvae culture. Using RNA-Seq data, the types and expression characteristics of the digestive enzyme genes were analyzed during five different development stages (embryo, nauplius, zoea, mysis and postlarvae) in L. vannamei. Among the obtained 66,815 unigenes, 296 were annotated as 16 different digestive enzymes including five types of carbohydrase, seven types of peptidase and four types of lipase. Such a diverse suite of enzymes illustrated the capacity of L. vannamei to exploit varied diets to fit their nutritional requirements. The analysis of their dynamic expression patterns during development also indicated the importance of transcriptional regulation to adapt to the diet transition. Our study revealed the diverse and dynamic features of digestive enzymes during early development of L. vannamei. These results would provide support to better understand the physiological changes during diet transition.

  17. Effect of extracellular enzyme activity on digestion performance of mesophilic UASB reactor treating high-strength municipal wastewater.

    PubMed

    Turkdogan-Aydinol, F Ilter; Yetilmezsoy, Kaan; Comez, Sezen

    2011-05-01

    Effect of extracellular enzyme activity on digestion performance of up-flow anaerobic sludge blanket (UASB) reactor was investigated for enhancement of anaerobic treatability of municipal wastewater. Two identical UASB reactors (9 L), namely Reactor-A (without enzyme addition) and Reactor-B (with enzyme addition),were simultaneously operated at mesophilic conditions (32 ± 2°C) with a hydraulic retention time of 24 h. Preliminary test results showed that the highest total chemical oxygen demand (TCOD) removal were achieved with an extracellular enzyme dosage of 0.2 mL/L. In the activation period of the extracellular enzyme (on days 186-212), while Reactor-A removed up to 69.3% of TCOD and 55.9% of soluble chemical oxygen demand (SCOD), Reactor-B effectively removed up to 81.9% of TCOD and 72.2% of SCOD. The average VFA/alkalinity ratios were determined to be about 0.40 (±0.03) and 0.28 (±0.08) for Reactor-A and Reactor-B, respectively.

  18. 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

  19. 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.

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

    PubMed

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

    2016-02-10

    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.

  1. Stage-specific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata.

    PubMed

    Krishnan, Natraj; Kodrík, Dalibor; Turanli, Ferit; Sehnal, Frantisek

    2007-01-01

    The titers of reactive oxygen species (ROS) represented by superoxide anion and general peroxides, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), are regulated in the midgut of the Colorado potato beetle (CPB) relative to the gut compartment, developmental stage, and food intake. ROS concentration is low in the potato leaves but it is very high in their digest in insect's anterior midgut. It is proposed that intensive ROS production in this gut region is linked to the processing of allelochemicals. SOD and CAT activities, low oxygen tension, and unidentified redox systems that maintain a slightly reducing milieu in the midgut lumen (pe+pH=6.95 declining to 5.36), obviously contribute to the decrease of ROS concentration along the gut length to a minimum in the wall of posterior midgut region. SOD and CAT activities are higher in the potato leaves than in the midgut tissues but the role of plant enzymes in ROS elimination within the gut lumen remains to be shown. A lower level of ROS and a higher antioxidant potential in the adult than in the larval midgut indicate stage specificity in the management of oxidative stress. The antioxidant defense is high in the diapausing adults that contain no detectable superoxide and about ten times less peroxides than the reproducing adults.

  2. 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.

  3. Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV).

    PubMed

    Amorim, Ticiana M L; Macedo, Leonardo L P; Uchoa, Adriana F; Oliveira, Adeliana S; Pitanga, Joelma C M; Macedo, Francisco P; Santos, Elizeu A; de Sales, Mauricio P

    2008-09-10

    The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.

  4. The effect of aerobic exercise training on growth performance, digestive enzyme activities and postprandial metabolic response in juvenile qingbo (Spinibarbus sinensis).

    PubMed

    Li, Xiu-Ming; Yu, Li-Juan; Wang, Chuan; Zeng, Ling-Qing; Cao, Zhen-Dong; Fu, Shi-Jian; Zhang, Yao-Guang

    2013-09-01

    Continual swimming exercise usually promotes growth in fish at a moderate water velocity. We hypothesized that the improvement in growth in exercise-trained fish may be accompanied by increases in digestive enzyme activity, respiratory capacity and, hence, postprandial metabolism. Juvenile qingbo fish (Spinibarbus sinensis) were subjected to aerobic training for 8weeks at a water velocity of control (3cms(-1)), 1, 2 and 4 body length (bl)s(-1) at a constant temperature of 25°C. The feed intake (FI), food conversion rate (FCR), specific growth rate (SGR), whole-body composition, trypsin and lipase activities, maximal oxygen consumption (M˙O2max) and postprandial M˙O2 response were measured at the end of the training period. Aerobic exercise training induced a significant increase in FI compared with the control group, while the FCR of the 4bls(-1) group was significantly lower than for the other three groups (P<0.05). The 1 and 2bls(-1) groups showed a significantly higher SGR over the control group (P<0.05). The whole-body fat and protein contents were significantly altered after aerobic exercise training (P<0.05). Furthermore, aerobic exercise training elevated the activity of both trypsin and lipase in the hepatopancreas and intestinal tract of juvenile S. sinensis. The M˙O2max of the 4bls(-1) training group was significantly higher than for the control group. The resting M˙O2 (M˙O2rest) and peak postprandial M˙O2 (M˙O2peak) in the three training groups were significantly higher than in the control group (P<0.05). Time to M˙O2peak was significantly shorter in the 1, 2 and 4bls(-1) training groups compared with the control group, while exercise training showed no effect on SDA (specific dynamic action) duration, factorial metabolic scope, energy expended on SDA and the SDA coefficient when compared to the control group. These data suggest that (1) the optimum water velocity for the growth of juvenile S. sinensis occurred at approximately 2.4bls(-1); (2

  5. 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…

  6. 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…

  7. Effects of lactic acid fermentation and gamma irradiation of barley on antinutrient contents and nutrient digestibility in mink (Mustela vison) with and without dietary enzyme supplement.

    PubMed

    Skrede, Anders; Sahlstrøm, Stefan; Ahlstrøm, Oystein; Connor, Kirsti Hjelme; Skrede, Grete

    2007-06-01

    The experiment was conducted to study the effects of fermentation of barley, using two different strains of lactic acid bacteria, a Lactobacillus plantarum/pentosus strain isolated from spontaneously fermented rye sourdough (AD2) and a starch-degrading Lactobacillus plantarum (AM4), on contents of mixed-linked (1 --> 3) (1 --> 4)-beta-glucans, alpha-amylase inhibitor activity, inositol phosphates, and apparent digestibility of macronutrients in mink. Effects of fermentation were compared with effects of gamma irradiation (gamma-irradiation: 60Co gamma-rays at 25 kGy). The diets were fed to mink with and without a supplementary enzyme preparation. Both lactic acid fermentation and gamma-irradiation followed by soaking and incubation, reduced concentrations of soluble beta-glucans, phytate and alpha-amylase inhibitor activity. Dietary enzyme supplementation increased significantly digestibility of crude protein, fat, starch and crude carbohydrate (CHO). Fermentation of the barley increased digestibility of starch and CHO. Fermentation with lactic acid bacteria AD2 resulted in higher starch and CHO digestibility than strain AM4, and had greater effect than gamma-irradiation, soaking and incubation. The highest digestibility of starch and CHO was obtained after AD2 fermentation followed by enzyme supplementation. It is concluded that both lactic acid fermentation of barley and enzyme supplementation have positive nutritional implications in the mink by limiting the effects of antinutrients and improving digestibility and energy utilization.

  8. Characterization of the heterochromatin of the darkling beetle Misolampus goudoti: cloning of two satellite DNA families and digestion of chromosomes with restriction enzymes.

    PubMed

    Pons, J; Petitpierre, E; Juan, C

    1993-01-01

    The darkling beetle Misolampus goudoti Er. has 58% of C-banded chromosome material. In this paper we deal with the study of the heterochromatin of this insect both by molecular and cytogenetical methods. Two different satellite DNA families have been characterized in Misolampus goudoti by agarose gel electrophoresis of EcoRI and PstI restriction fragments, respectively. The EcoRI family is composed of a monomeric unit of 196 bp (64.3% A-T rich) DNA sequence, representing about 120,000 copies per haploid genome. The presence of frequent intermediate-size satellite variants and an internal direct repetition of 61 bp in the EcoRI repetitive main monomer suggest that the evolution of this satellite proceeded by unequal crossing-over, occurring both within and between the 196 bp unit. Another highly repetitive sequence, defined by digestion of genomic DNA with PstI, has a more complex unit of 1.2 kb with about 70,000 copies per haploid genome. In situ digestion of M. goudoti chromosomes with restriction enzymes shows a non-specific chromosome DNA extraction from pericentromeric positions with EcoRI and chromosome specific extraction of DNA with PstI and HinfI. This is discussed in relation to the chromosomal location of both satellites.

  9. Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

    ERIC Educational Resources Information Center

    Kin, Ng Hong; Ling, Tan Aik

    2016-01-01

    The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

  10. Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

    ERIC Educational Resources Information Center

    Kin, Ng Hong; Ling, Tan Aik

    2016-01-01

    The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

  11. 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.

  12. Ligation-mediated PCR amplification of specific fragments from a class-II restriction endonuclease total digest.

    PubMed Central

    Guilfoyle, R A; Leeck, C L; Kroening, K D; Smith, L M; Guo, Z

    1997-01-01

    A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5'-end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide 'indexing sequence.' The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual lambda Bcl I fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligation- mediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases. PMID:9108171

  13. Performance, intestinal microflora, and amino acid digestibility altered by exogenous enzymes in broilers fed wheat- or sorghum-based diets.

    PubMed

    Wu, D; Wu, S B; Choct, M; Swick, R A

    2017-02-01

    The objective of the current study was to compare the effects of dietary enzymes and nutrient restriction on performance and bone mineralization in birds fed wheat- or sorghum-based diets. A total of 720 d-old male broiler chicks were randomly allocated to 8 treatments, with 6 replicates per treatment and 15 birds per replicate. Birds were reared in floor pens from 0 to 35 d. The study used a 2 × 4 factorial arrangement with 2 grains (sorghum or wheat) and 4 diets: positive control (no enzyme and ME, digestible Lys, Ca, and P sufficient, negative control (NC; no enzyme and reduced ME [-100 kcal/kg], digestible AA [-2%], Ca [-0.12 percentage points], and available P [-0.18 percentage points in the starter phase and -0.22 percentage points in the grower phase]), NC + nonstarch polysaccharide-degrading enzymes + phytase (500 phytase units [FTU]; NCCP), and NC + phytase (1,000 FTU; NCP). From 0 to 35 d, birds fed wheat-based diets had greater G:F (4.5%), BW gain (9.2%), breast meat yield (6.8%), and tibia ash (2.0%) compared with birds fed sorghum-based diets. Across grain types, the NCCP treatment improved BW gain ( < 0.001), feed intake ( < 0.001), G:F ( < 0.05), and livability ( < 0.001) compared with the NC treatment. Birds fed NCP had greater BW gain ( < 0.001), feed intake ( < 0.001), G:F ( < 0.001), and livability ( < 0.001) compared with birds fed NC. Birds fed the NCP diet had greater BW gain ( < 0.001), toe ash ( < 0.01), and tibia ash ( < 0.001) compared with birds fed the NCCP diet. There was a grain × diet interaction for feed intake ( < 0.01), BW gain ( < 0.001), tibia ash ( < 0.01), and tibia breaking strength ( < 0.05). The influence of enzymes was more pronounced in sorghum-based diets than in wheat-based diets. Birds fed wheat-based diets had greater ileal digestibility of His, Met, Val, Phe, Ile, Leu, Trp, Glu, Pro, Ala, Tyr, and Cys compared with those fed sorghum-based diets ( < 0.05). Across grain types, NCP had greater apparent ileal

  14. Effects of inclusion of poultry by-product meal and enzyme-prebiotic supplementation in grower diets on performance and feed digestibility of broilers.

    PubMed

    Kirkpinar, F; Açikgöz, Z; Bozkurt, M; Ayhan, V

    2004-04-01

    1. Two experiments were conducted to determine the effects of level of inclusion of poultry by-product and enzyme-prebiotic supplementation on grower diet digestibility and the performance of broilers. 2. Six grower diets were formulated to provide a similar nutrient profile with the exception of using three graded levels of poultry by-product, namely 0, 25, 40 g/kg of the diet with and without supplementation of enzyme preparation at the rate of 1 kg per tonne of feed and prebiotic preparation at the rate of 2 kg per tonne of feed. The experimental diets were used from 3 to 6 weeks of age. 3. Body weights, feed intake and feed conversion efficiency were not affected by poultry by-product; however, enzyme-prebiotic had a significant positive effect on feed conversion efficiency at 0 to 6 weeks in experiment 1. 4. Crude protein digestibility was decreased by feeding the diet containing poultry by-product while ether extract digestibility was increased by poultry by-product at the rate of 25 g per kg of feed only. Dry matter retention, crude fibre digestibility and organic matter retention were not affected by poultry by-product. Dry matter and organic matter retentions, crude protein, ether extract and crude fibre digestibilities were not affected by enzyme-prebiotic. 5. Protein efficiency ratio (PER) values were increased by poultry by-product at the rate of 40 g per kg of feed and addition of enzyme-prebiotic.

  15. Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform.

    PubMed

    Deng, Zhenzhen; Mao, Jiawei; Wang, Yan; Zou, Hanfa; Ye, Mingliang

    2017-01-01

    Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 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.

  17. Evidence-based benefits of specific mixtures of non-digestible oligosaccharides on the immune system.

    PubMed

    Nauta, Alma J; Garssen, Johan

    2013-03-01

    Non-digestible carbohydrates (NDC) are natural constituents of many foods. They are mostly referred to as dietary fibre and are associated with many health benefits mostly connected to gut health. NDC have emerged as a promising nutritional concept to modulate immune function as well. In the world of immunology non-digestible carbohydrates are recognized now as key immunomodulating molecules. Both pharma and food industries realize the enormous potency of these immune active components. Although the mechanisms underlying the effects of NDC on the immune system are not totally clear yet many studies have reported beneficial effects on both mucosal and systemic immunity in humans. The aim of this review is to summarize the available evidence on the immune modulatory effects of specific mixtures of oligosaccharides. Both mechanistic in vitro and in vivo studies have been performed and will be discussed. Finally the potential use of these unique structures will be evaluated. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. 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. © 2015 Poultry Science Association Inc.

  19. 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

  20. 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

  1. 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

  2. The inhibitory effects of hull polysaccharides and tannins of field beans (Vicia faba L.) on the digestion of amino acids, starch and lipid and on digestive enzyme activities in young chicks.

    PubMed

    Longstaff, M; McNab, J M

    1991-03-01

    The effects of polysaccharides and tannins present in the hulls of field beans (Vicia faba L.) on the digestion of amino acids, starch and lipid were studied in poultry. A control diet without hulls and the same diet substituted with 400 g hulls/kg diet from three different varieties of beans were fed to 3-week-old chicks for 4 d. Digestibility coefficients for amino acids, starch and lipid were calculated from measurements made of these nutrients in the diets and the freeze-dried excreta with the aid of titanium dioxide as a marker. Activities of trypsin (EC 3.4.21.4), alpha-amylase (EC 3.2.1.1), and lipase (EC 3.1.1.3) in digesta removed from the upper jejunum, sucrase (EC 3.2.1.48) in the gut mucosa from the upper jejunum, and alpha-amylase and lipase in the pancreas were measured. The hulls were analysed for their polysaccharide and tannin contents. Results showed that the hulls were mostly carbohydrate in composition, with cellulose the predominant polysaccharide. Tannins present in the hulls of two coloured-flowering varieties (Brunette and Minica) were of the condensed type. The diet with tannin-free hulls (white-flowering variety Medes) lowered slightly the digestion of amino acids, starch and lipid compared with the control diet. This effect was believed to be due to inhibition of digestive enzymes, possibly through their adsorption onto the hulls. Diets with tannin-rich hulls (varieties Brunette and Minica) caused a large reduction in the digestion of amino acids, starch and lipid compared with the control diet mainly due to inactivation of digestive enzymes by the formation of tannin-enzyme complexes in the digestive tract. Enzyme activities could be partially restored by the addition of polyvinylpyrrolidone to the digesta. Tannins inactivated trypsin the most, alpha-amylase to a lesser extent and lipase the least and as a consequence lowered the digestion of amino acids the most, starch to a lesser extent and lipid the least. Tannins did not induce an

  3. 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.

  4. Substrate specificity and some physicochemical properties of autolytic enzymes of the bacterium Lysobacter sp. XL 1.

    PubMed

    Tsfasman, I M; Sitkin, B V; Lysanskaya, V Ya; Stepnaya, O A; Kulaev, I S

    2007-07-01

    The substrate specificity of autolytic enzymes of the bacterium Lysobacter sp. XL 1 has been established. The periplasmic enzyme A8, the cytosolic enzyme A1, and the enzyme A10 solubilized from the cell walls and membranes with Triton X-100 exhibit glucosaminidase activity; the cytosolic enzyme A4 and the enzyme A9 solubilized from the cell walls and membranes with LiCl exhibit the muramidase activity. The cytosolic enzymes A3 and A6 have N-acetylmuramoyl-L-alanine amidase activity, and the enzyme A5 exhibits the diaminopimelinoyl-alanine endopeptidase activity. Some physicochemical properties of the most active autolytic cytosolic enzymes of Lysobacter sp. XL 1 (endopeptidases A5 and A7 and N-acetylmuramoyl-L-alanine amidase A6) were studied. The enzymes exhibit maximal activity over a wide range of buffer concentrations in weakly alkaline medium and moderate temperatures. The investigated enzymes are comparatively thermolabile proteins.

  5. 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

  6. 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.

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

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-06-14

    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.

  8. Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei.

    PubMed

    Zokaeifar, Hadi; Balcázar, José Luis; Saad, Che Roos; Kamarudin, Mohd Salleh; Sijam, Kamaruzaman; Arshad, Aziz; Nejat, Naghmeh

    2012-10-01

    We studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8) (BM8) CFU g(-1) feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P < 0.05) in the shrimp fed BM5 and BM8 diets compared to the control group. These findings demonstrate that administration of B. subtilis strains, L10 and G1, can improve growth performance and disease resistance through an enhanced immune response in shrimp.

  9. Bacillus Probiotic Enzymes: External Auxiliary Apparatus to Avoid Digestive Deficiencies, Water Pollution, Diseases, and Economic Problems in Marine Cultivated Animals.

    PubMed

    Olmos Soto, Jorge

    2017-01-01

    Exploitation of marine fishes is the main source of several life-supporting feed compounds such as proteins, lipids, and carbohydrates that maintain the production of most trading marine organisms by aquaculture. However, at this rate the marine inventory will go to the end soon, since fishery resources are finite. In this sense, the availability of the principal ingredients obtained from marine fishes is going to decrease considerably, increasing the diet prices and affecting the economy of this activity. Therefore, aquaculture industry needs to find nonexpensive land unconventional resources of protein, carbohydrates, and lipids and use bacterial probiotics to improve digestion-assimilation of these unfamiliar compounds. Bacillus subtilis is a cosmopolitan probiotic bacterium with a great enzymatic profile that could improve nutrient digestion-assimilation, induce healthy growth, and avoid water pollution, decreasing economic problems and increasing yields in the aquaculture industry. In this chapter, we present how Bacillus enzymes can help marine animals to assimilate nutrients from unconventional and economic plant resources.

  10. Growth, metabolic rate, and digestive enzyme activity in the white shrimp Litopenaeus setiferus early postlarvae fed different diets.

    PubMed

    Brito; Chimal; Gaxiola; Rosas

    2000-12-01

    Growth rate, soluble-protein content, oxygen consumption, ammonia excretion, and digestive-enzyme activity were studied in Litopenaeus setiferus early postlarvae under four feeding regimens that included combinations of freshly hatched Artemia nauplii, microparticulate commercial diet, and algae. Growth and of postlarvae fed a mixed diet were significantly higher. Artificial diet used alone caused the lowest growth, lowest soluble-protein content, higher ammonia excretion, lowest O:N ratio, and higher proteolytic and amylase activities. The artificial diet stimulated proteolytic activity and ammonia excretion of postlarvae, apparently in response to some deficiency in protein composition of the diet. Based on results in growth, soluble-protein content, enzymatic activity, and metabolic substrate, we determined that partial substitution of Artemia nauplii by artificial diet, with or without addition of algae when rearing early postlarval stages, will benefit the growth and nutritional state of L. setiferus postlarvae.

  11. The interactive effect of digesting a meal and thermal acclimation on maximal enzyme activities in the gill, kidney, and intestine of goldfish (Carassius auratus).

    PubMed

    Turner, Leah A; Bucking, Carol

    2017-04-05

    Surrounding environmental temperatures affect many aspects of ectotherm physiology. Generally, organisms can compensate at one or more biological levels, or allow temperature to dictate processes such as enzyme activities through kinetic effects on reaction rates. As digestion also alters physiological processes such as enzyme activities, this study determined the interacting effect of thermal acclimation (8 and 20 °C) and digesting a single meal on maximal enzyme activities in three tissues of the goldfish (Carrassius auratus). Acclimation to elevated temperatures decreased branchial Na(+), K(+), ATPase (NKA) activity. In contrast, acclimation to elevated temperatures had no effect on citrate synthase (CS) or pyruvate kinase (PK) activity in any tissue, nor were renal NKA or glutamine synthetase (GS) activities impacted. Warm water-acclimation exaggerated the positive impact of digestion on intestinal and branchial NKA activities and intestinal GS activity only, but digestion had no effect in the kidney. CS and PK did not display intestinal zonation; however, there was a distinct increase towards the distal intestine in NKA and GS activities. Zonation of NKA was more prominent in warm-acclimated animals, while acclimation temperature did not affect intestinal heterogeneity of GS. Finally, the impact of tissue protein content on enzyme activity was discussed. We conclude that the intestine and gill of warm-acclimated goldfish exhibited an augmented capacity for increasing several enzyme activities in response to digestion while the kidney was unaffected by thermal acclimation or digesting a single meal. However, this amplified capacity was ameliorated by alterations in tissue protein content. Amplified increases in NKA activity may ultimately have implications for ATP demand in these tissues, while increased GS activity may beneficially increase ammonia-detoxifying capacity in the intestine.

  12. 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.

  13. 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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  15. Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity.

    PubMed

    Zhao, Qiang; Li, Xing-Fang; Le, X Chris

    2011-12-15

    Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.

  16. Surprising results from abiotic enzyme digestion of dissolved organic matter at the molecular scale

    NASA Astrophysics Data System (ADS)

    Hess, N. J.; Tfaily, M. M.; Heredia-Langnar, A.; Rodriguez, L.; Purvine, E.; Todd-Brown, K. E.

    2016-12-01

    Sometimes even the simplest of experiments leads to unexpected results and new understanding. We extract dissolved organic matter using water from peat soil obtained from the S1 bog at the Marcell Experimental Forest in northern Minnesota. We characterized the dissolved organic matter in the water extract before and after adding glucosidase, peroxidase and β-N-Acetylglucosaminidase enzymes using electrospray Fourier transform ion cyclotron resonance mass spectrometry in negative ion mode. Based on mass measurement accuracy of less than 1 ppm for singly charged ions, we assigned putative chemical formula to greater than 80% of the measured mass spectrometry features. For each enzyme tested we are able to easily distinguish between the types and composition of dissolved organic molecules that are susceptible to enzyme degradation - and those that are not - based on the presence new compounds in reacted extracts and loss of compounds from the initial water extract. Next, we created a consensus molecular network analysis based on the neutral mass loss between the measured compounds for each enzyme. The connectivity within these networks suggested a unique, distinctive chemistry for each enzyme. Some results were expected, like the nondiscriminatory oxidation of organic molecules by peroxidase and preferential loss of lignin and tannin-like molecules by glucosidase. However, surprising results include the apparent reactivity of glucosidase enzymatic products to reassemble, forming larger mass organic molecules. While these experiments were conducted abiotically, these molecular-resolved results suggest that biotic enzymatic processes may result in product compounds with unexpected chemistry and reactivity, implying that our current conceptual model of microbial enzymatic activity may be overly simplistic.

  17. Cellular localization of lead using an antibody-based detection system and enzyme activity changes in the gills and digestive gland of the blue mussel Mytilus edulis.

    PubMed

    Einsporn, Sonja; Bressling, Jana; Koehler, Angela

    2009-02-01

    Marine organisms are continuously exposed to heavy metals in their environment. Bivalve mollusks such as the blue mussel Mytilus edulis accumulate high levels of heavy metals effecting cellular homeostasis and functions. Lead (Pb) exposure (2.5 mg/L of lead (II) nitrate for 10 d) and depuration (10 d in clean seawater) experiments were conducted to study its intracellular fate in the gills and digestive gland of M. edulis. For this purpose, an antibody-based detection method for ultrastructural localization and a subcellular fractionation approach for chemical analysis of Pb were used. In addition, effects of Pb on enzyme activities involved in oxyradical scavenging, such as the conjugative enzyme glutathione-S-transferase and the antioxidative enzyme catalase, were determined. The ultrastructural studies showed that Pb was mainly detected in lysosomes of gill epithelial cells and digestive cells. Lead was also detected in cell nuclei and granular hemocytes. Higher metal concentrations were measured by chemical analysis in subcellular fractions of the gills compared to those of the digestive gland. Increased activities of glutathione-S-transferase were found in gills after exposure and remained elevated during the depuration period, whereas glutathione-S-transferase activity remained unaffected in the digestive gland. Catalase activities showed no changes after Pb exposure, neither in the gills nor in the digestive gland. We conclude that gill cells are major sites of uptake and accumulation for dissolved Pb and are involved in sequestration and detoxification of this metal in M. edulis.

  18. Quantitative classification of DNA damages induced by submicromolar cadmium using oligonucleotide chip coupled with lesion-specific endonuclease digestion.

    PubMed

    Pal, Sukdeb; Kim, Ji Yeon; Park, Sun Hee; Lim, Heung Bin; Lee, Kyeong-Hee; Song, Joon Myong

    2011-05-15

    Implementation of proper analytical tool for systematic investigation and quantitative determination of different classes of cadmium ion-induced DNA damages, especially at low metal ion concentrations, is still lacking. Using lesion-specific enzymes that cleave DNA at specific classes of damage and a fluorometric approach developed for quantifying fluorophore-labeled oligonucleotides bound to chip surfaces, we determined the frequencies of different lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites) induced by submicromolar Cd(2+). Cd(2+)-treated oligonucleotide chips were digested with various endonucleases (Fpg protein, endonuclease III, endonuclease IV), producing a de novo single strand break (SSB) at their substrate modifications. The frequency of SSB and double strand break (DSB) was computed from the difference of pre- and post-Cd(2+)-treatment oligonucleotide coverage on the chip. While the frequency of SSBs and oxidized bases were successfully quantified even at 0.5 μM of Cd(2+), DSB frequency could be easily quantitated at 8.7 μM [Cd(2+)]. The numbers of abasic sites were below the oligonucleotide detection limit (2.4 amole; equivalent to 0.24 fM for a reaction volume of 100 μL). SSBs were found to constitute about 85-90% of single strand damages, while oxidized bases comprise only 4-7% of the total at 0.9 to 8.7 μM [Cd(2+)].

  19. 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

  20. In vitro and in vivo digestibility of corn starch for weaned pigs: Effects of amylose:amylopectin ratio, extrusion, storage duration, and enzyme supplementation.

    PubMed

    Li, Y; Zhang, A R; Luo, H F; Wei, H; Zhou, Z; Peng, J; Ru, Y J

    2015-07-01

    The objective of this study was to investigate the effects of amylose (AM):amylopectin (AP) ratio, extrusion, storage duration, and enzyme supplementation on starch digestibility of corn. Three corn varieties with high (0.60; HA), medium (0.44; MA), and low (0.39; LA) AM:AP ratios, respectively, were selected from 74 corn samples to evaluate the in vitro and in vivo digestibility of starch. In Exp. 1, during wk 4 after extrusion, resistant starch (RS) content of the 3 selected corn varieties (LA, MA, and HA) increased (P < 0.05) each week and starch digestibility in vitro decreased as storage time increased (P < 0.05). The AM:AP ratio affected the formation of RS (P < 0.01). The RS content of the 3 corn varieties was ranked as LA < MA < HA in each week (P < 0.05). Correlation analysis showed that AM:AP ratio and storage duration were both positively correlated with RS content (P < 0.01). Furthermore, a significant quadratic relation was found between storage duration and RS content in each corn variety as well as storage duration and digestibility. Starch digestibility was negatively correlated with RS content (P < 0.001). In Exp. 2, digestion trials were performed on cannulated pigs with BW of 13.20 ± 0.94 kg. Extrusion increased ileal digestibility of GE and starch of either HA or LA compared with the enzyme-supplemented diets (P < 0.001). Enzyme supplementation did not improve ileal energy and starch digestibility. The ileal digestibility of starch and GE of LA varieties was greater than HA samples (P < 0.05). The results implied that AM:AP ratio and storage duration after extrusion may be important determinants of RS formation and digestibility of starch for corn. In addition, RS content could be an important indicator of digestibility of starch in extruded corn. Using a lower AM:AP ratio corn or reducing the storage duration of extruded corn would help to reduce the formation of RS and improve the starch bioavailability of corn for piglets.

  1. 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.

  2. Why can't young fish eat plants? Neither digestive enzymes nor gut development preclude herbivory in the young of a stomachless marine herbivorous fish.

    PubMed

    Day, Ryan D; German, Donovan P; Tibbetts, Ian R

    2011-01-01

    Most young fishes lack the ability to function as herbivores, which has been attributed to two aspects of the digestive system: elevated nitrogen demand and a critical gut capacity. We compared the digestive morphology and biochemistry of two size classes of the marine herbivore Hyporhamphus regularis ardelio, pre-ontogenetic trophic shift (pre-OTS, <100mm) and post-ontogenetic trophic shift (post-OTS, >100mm), to determine what limits the onset of herbivory and how their digestive processes fit with current models of digestion. Two gut-somatic indices comparing gut length to body length (relative gut length) and body mass (Zihler's Index) demonstrated a significant decrease (RGL 0.59→0.49, P<0.01; ZI 3.24→2.44, P<0.01) in gut length relative to body size. There was little difference in enzyme activity between the two classes, with juveniles showing similar levels of carbohydrase and lipase and less protease compared with adults, indicating that juveniles did not preferentially target nitrogen and were as capable of digesting an herbivorous diet. These findings suggest that herbivory in this fish is not limited by the function of the post-oesophageal digestive tract, but rather the ability of the pharyngeal mill to mechanically process plants. Our findings offer partial support for the current model of stomachless digestion, indicating that further refinement may be necessary.

  3. Symbiotic Chlorella variabilis incubated under constant dark conditions for 24 hours loses the ability to avoid digestion by host lysosomal enzymes in digestive vacuoles of host ciliate Paramecium bursaria.

    PubMed

    Kodama, Yuuki; Fujishima, Masahiro

    2014-12-01

    Endosymbiosis between symbiotic Chlorella and alga-free Paramecium bursaria cells can be induced by mixing them. To establish the endosymbiosis, algae must acquire temporary resistance to the host lysosomal enzymes in the digestive vacuoles (DVs). When symbiotic algae isolated from the alga-bearing paramecia are kept under a constant dark conditions for 24 h before mixing with the alga-free paramecia, almost all algae are digested in the host DVs. To examine the cause of algal acquisition to the host lysosomal enzymes, the isolated algae were kept under a constant light conditions with or without a photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea for 24 h, and were mixed with alga-free paramecia. Unexpectedly, most of the algae were not digested in the DVs irrespective of the presence of the inhibitor. Addition of 1 mM maltose, a main photosynthetic product of the symbiotic algae or of a supernatant of the isolated algae kept for 24 h under a constant light conditions, did not rescue the algal digestion in the DVs. These observations reveal that unknown factors induced by light are a prerequisite for algal resistance to the host lysosomal enzymes. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  4. Aqueous extract of Psidium guajava leaves: phenolic compounds and inhibitory potential on digestive enzymes.

    PubMed

    Simão, Anderson A; Marques, Tamara R; Marcussi, Silvana; Corrêa, Angelita D

    2017-06-29

    Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.

  5. The effects of digestive enzymes on characteristics of placental insulin receptor. Comparison of particulate and soluble receptor preparations.

    PubMed Central

    Clark, S; DeLuise, M; Larkins, R G; Melick, R A; Harrison, L C

    1978-01-01

    The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function. PMID:100106

  6. The maturation of mucus-secreting gastric epithelial progenitors into digestive-enzyme secreting zymogenic cells requires Mist1.

    PubMed

    Ramsey, Victoria G; Doherty, Jason M; Chen, Christopher C; Stappenbeck, Thaddeus S; Konieczny, Stephen F; Mills, Jason C

    2007-01-01

    Continuous regeneration of digestive enzyme (zymogen)-secreting chief cells is a normal aspect of stomach function that is disrupted in precancerous lesions (e.g. metaplasias, chronic atrophy). The cellular and genetic pathways that underlie zymogenic cell (ZC) differentiation are poorly understood. Here, we describe a gene expression analysis of laser capture microdissection purified gastric cell populations that identified the bHLH transcription factor Mist1 as a potential ZC regulatory factor. Our molecular and ultrastructural analysis of proliferation, migration and differentiation of the gastric unit in Mist1(-/-) and control mice supports a model whereby wild-type ZC progenitors arise as neck cells in the proliferative (isthmal) zone of the gastric unit and become transitional cells (TCs) with molecular and ultrastructural characteristics of both enzyme-secreting ZCs and mucus-secreting neck cells as they migrate to the neck-base zone interface. Thereafter, they rapidly differentiate into mature ZCs as they enter the base. By contrast, Mist1(-/-) neck cells differentiate normally, but ZCs in the mature, basal portion of the gastric unit uniformly exhibit multiple apical cytoplasmic structural abnormalities. This defect in terminal ZC differentiation is also associated with markedly increased abundance of TCs, especially in late-stage TCs that predominantly have features of immature ZCs. Thus, we present an in vivo system for analysis of ZC differentiation, present molecular evidence that ZCs differentiate from neck cell progenitors and identify Mist1 as the first gene with a role in this clinically important process.

  7. 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

  8. 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).

  9. Comparison of pepsin-digestion and enzyme-linked immunosorbent assay for the diagnosis of trichinosis in swine.

    PubMed Central

    Smith, H J

    1988-01-01

    Comparison of parasitological and serological diagnosis of trichinosis in swine was carried out on 36 pigs given 15,400 infective larvae each by gavage. Circulating eosinophil levels were determined and sera were examined by enzyme-linked immunosorbent assay for anti-Trichinella antibodies. Two pigs were killed per day from days 15 to 29 postinfection. Muscle was examined by pepsin-digestion and comparable tissue was fed to a rat. Eosinophil counts increased at about day 6 and reached peak levels about day 25 postinfection and returned to approximate preinfection levels about two months postinfection in those pigs still in the study. Infective larvae were recovered from all pigs killed at greater than or equal to 18 days postinfection. Using the criterion of 5 x mean optical density readings of negative sera as positive, seroconversion occurred between days 19 and 26 postinfection. Use of a lower criterion of 3 x mean optical density readings of negative sera resulted in only three of 30 pigs killed greater than or equal to 18 days postinfection seroconverting less than or equal to 18 days postinfection, when infective larvae were first recovered in the musculature. In pigs, even in those heavily infected, there is a lag between the period that trichinae in musculature become infective and development of antibodies as detected by enzyme-linked immunosorbent assay which results in false negative reactions in many animals. This study demonstrated that the enzyme-linked immunosorbent assay using an excretory-secretory antigen should not be used to certify pork or pork products free of infective Trichinella larvae or safe for human consumption. PMID:3127035

  10. Impact of levels of total digestible nutrients on microbiome, enzyme profile and degradation of feeds in buffalo rumen

    PubMed Central

    Kala, Anju; Kamra, D. N.; Kumar, Avinash; Agarwal, Neeta; Chaudhary, L. C.; Joshi, C. G.

    2017-01-01

    The present study was aimed at understanding a shift in rumen microbiome of buffaloes fed various levels of total digestible nutrients. To understand the process, the metagenomics of rumen microbes, in vivo and in vitro rumen fermentation studies were carried out. Three rumen fistulated adult male Murrah buffaloes were fed three isonitrogenous diets varying in total digestible nutrients (70, 85 and 100% of TDN requirement) in 3X3 switch over design. On dry matter basis, wheat straw/ roughage content were 81, 63 and 51% and that of maize grain was 8, 16 and 21% in three diets respectively. After 20 d of feeding, rumen liquor and rumen contents were sampled just before (0h) and 4h post feeding. Ruminococcus flavefaciens and R. albus (estimated with real time PCR) were higher in high roughage diets. The predominant phyla in all the three groups were Bacteroidetes, Firmicutes followed by Proteobacteria, Actinobacteria and Fibrobacteres. A core group of more than fifty rumen bacteria was present in all the animals with very little variations due to level of TDN. The most predominant bacterial genera reported in order of decreasing abundance were: Prevotella, Bacteroides, Clostridium, Ruminococcus, Eubacterium, Parabacteroides, Fibrobacter, Butyrivibrio etc. The higher diversity of the enyzmes families GH 23, GH 28, GH 39, GH 97, GH 106, and GH 127 (the enzymes active in fibre and starch degradation) were significantly higher on 100%TDN diet while CE 14 (required for the hydrolysis of bond between carbohydrate and lignin) was higher on low TDN (70%) diet, indicating ester bond cleavage was better in animals fed high roughage (wheat straw) diet. PMID:28207851

  11. 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

    2015-01-07

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Impact of levels of total digestible nutrients on microbiome, enzyme profile and degradation of feeds in buffalo rumen.

    PubMed

    Kala, Anju; Kamra, D N; Kumar, Avinash; Agarwal, Neeta; Chaudhary, L C; Joshi, C G

    2017-01-01

    The present study was aimed at understanding a shift in rumen microbiome of buffaloes fed various levels of total digestible nutrients. To understand the process, the metagenomics of rumen microbes, in vivo and in vitro rumen fermentation studies were carried out. Three rumen fistulated adult male Murrah buffaloes were fed three isonitrogenous diets varying in total digestible nutrients (70, 85 and 100% of TDN requirement) in 3X3 switch over design. On dry matter basis, wheat straw/ roughage content were 81, 63 and 51% and that of maize grain was 8, 16 and 21% in three diets respectively. After 20 d of feeding, rumen liquor and rumen contents were sampled just before (0h) and 4h post feeding. Ruminococcus flavefaciens and R. albus (estimated with real time PCR) were higher in high roughage diets. The predominant phyla in all the three groups were Bacteroidetes, Firmicutes followed by Proteobacteria, Actinobacteria and Fibrobacteres. A core group of more than fifty rumen bacteria was present in all the animals with very little variations due to level of TDN. The most predominant bacterial genera reported in order of decreasing abundance were: Prevotella, Bacteroides, Clostridium, Ruminococcus, Eubacterium, Parabacteroides, Fibrobacter, Butyrivibrio etc. The higher diversity of the enyzmes families GH 23, GH 28, GH 39, GH 97, GH 106, and GH 127 (the enzymes active in fibre and starch degradation) were significantly higher on 100%TDN diet while CE 14 (required for the hydrolysis of bond between carbohydrate and lignin) was higher on low TDN (70%) diet, indicating ester bond cleavage was better in animals fed high roughage (wheat straw) diet.

  13. Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products.

    PubMed

    Beneito-Cambra, M; Herrero-Martínez, J M; Ramis-Ramos, G; Lindner, W; Lämmerhofer, M

    2011-10-14

    Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 μm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 μm or 3 μm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.

  14. A novel angiotensin converting enzyme inhibitory peptide derived from proteolytic digest of Chinese soft-shelled turtle egg white proteins.

    PubMed

    Rawendra, Reynetha D S; Aisha; Chang, Chi-I; Aulanni'am; Chen, Ho-Hsien; Huang, Tzou-Chi; Hsu, Jue-Liang

    2013-12-06

    In this study, soft-shelled turtle (Pelodiscus sinensis) egg white (SSTEW) proteins were digested by thermolysin and the resulting small peptides were further fractionated by reverse phase chromatography. Peptides with angiotensin I-converting enzyme inhibitory (ACEI) activity from these fractions were screened. A lysozyme-derived peptide, IW-11, from the fraction with the most effective ACEI was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its purified form showed effective ACEI activity in vitro (IC50=4.39±0.31μM). The Lineweaver-Burk plots indicated that the inhibition towards ACE caused by this peptide is a competitive inhibition. The molecular docking study further revealed that the ACEI activity of IW-11 is mainly attributed to the formation of hydrogen bonds between the N-terminal residue of IW-11 and the S1 pocket (Ala354 and Tyr523) and the S2' region (His513 and His353) of ACE. Moreover, the digestion parameters were further optimized and the target peptide (82% purity) was readily obtained (15% yield) without any cumbersome purification procedure. Notably, lysozyme C is the most abundant protein in SSTEW, which implies that an efficient production of this ACEI peptide from SSTEW is promising. Inhibition of ACE has proven to be an effective strategy in prevention and treatment of hypertension and related diseases. Unlike typical synthetic ACE inhibitors which exert well described side effects, food-derived peptides with ACE inhibitory activity may be safer alternatives for hypertension treatment. In this study, we comprehensively identified peptides derived from SSTEW digest using a proteomic approach. IW-11, which is derived from lysozyme, the most abundant protein in SSTEW, showed remarkable inhibition towards ACE. This peptide has been demonstrated to have a competitive inhibitory property which is able to bind to ACE active site and found to be a true inhibitor against ACE according to Lineweaver-Burk plots. Using an

  15. Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera).

    PubMed

    Oliveira, Carolina Turatti; Kunz, Daniele; Silva, Carlos Peres; Macedo, Maria Lígia Rodrigues

    2015-10-01

    Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella. Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Fabrication of an on-line enzyme micro-reactor coupled to liquid chromatography-tandem mass spectrometry for the digestion of recombinant human erythropoietin.

    PubMed

    Foo, Hsiao Ching; Smith, Norman W; Stanley, Shawn M R

    2015-04-01

    Our aim was to develop a fast and efficient on-line method using micro-reactors for the digestion and deglycosylation of recombinant human erythropoietin extracted from equine plasma. The trypsin digestion micro reactors were fabricated using fused silica capillaries with either a dextran-modified coating or a porous monolith that was able to immobilise the enzyme. These were both found to be reasonably robust and durable, with the trypsin immobilised on dextran-modified fused silica capillaries offering better reproducibility than the micro-reactor based upon covalent attachment of this enzyme to the polymer. It is also evident that the enzyme attached micro reactors produced some tryptic peptides in a greater yield than in-solution digestion. A peptide-N-glycosidase F reactor was also fabricated and, when coupled with the trypsin reactor, the deaminated peptides T5 DAM and T9 DAM from recombinant human erythropoietin could also be detected by LC-ESI-MS/MS analysis. These results were better than those achieved using off-line digestion plus deglycosylation reactions and the analysis required far less time and effort to complete. The use of this on-line approach improved the sensitivity, efficiency and speed of our confirmation methodology that is based upon detecting the unique peptide segments of recombinant human erythropoietin that has been affinity extracted from positive equine plasma samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. LYSIS-FROM-WITHOUT OF STAPHYLOCOCCUS AUREUS STRAINS BY COMBINATIONS OF SPECIFIC PHAGES AND PHAGE-INDUCED LYTIC ENZYMES

    PubMed Central

    Ralston, Doris J.; McIvor, Mary

    1964-01-01

    Ralston, Doris J. (University of California, Berkeley) and Mary McIvor. Lysis-from-without of Staphylococcus aureus strains by combinations of specific phages and phage-induced lytic enzymes. J. Bacteriol. 88:676–681. 1964—Several typing phages, adsorbed in sufficient concentrations to their homologous propagating strains, altered the cell surface so as to render the cells sensitive to rapid and synergistic lysis by extra-cellular additions of wall lysins. Lysis was effected both by lysins induced by the individual phages and by phage K1 virolysin. Phage K1 also rendered cells sensitive to the lysins of the typing phages. With the exception of lysins from PS 53, 70, and 77, none of the lysins nor purified phages tested separately caused significant lysis of living cells. Lysis-from-without in Staphylococcus aureus appears to be a stepwise process: sensitization by phage followed by digestion of the wall by lysin. PMID:14208506

  19. Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.

    PubMed

    Triantaphyllidou, I E; Sklaviadis, T; Vynios, D H

    2006-12-15

    The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.

  20. Stress-induced changes in accumulation of sorbitol and in activities of concomitant enzymes in digestive gland of freshwater snail.

    PubMed

    Tsvetkov, I L; Konichev, A S

    2009-11-01

    Sorbitol content was determined in the digestive gland of freshwater snail (Viviparus viviparus L.) in different seasons and in a short-term experiment on the water temperature decrease and on intoxication with cadmium chloride. In the model experiments, changes in activities of enzymes involved in sorbitol metabolism (acid phosphatases, sorbitol dehydrogenase, and aldose reductase) were also studied. Sorbitol was accumulated by the snail in response to the temperature decrease (as a cryoprotectant) and under conditions of acute intoxication (as a probable metabolic regulator or a nonspecific protective factor). However, the mechanisms of this accumulation are different: on cold adaptation sorbitol is produced as a result of reduction of glucose under the influence of aldose reductase, and on intoxication sorbitol is mainly produced from fructose under the influence of sorbitol dehydrogenase. Pathways of the sorbitol accumulation and its re-involvement into metabolism are not always the same, and this might be a mechanism for regulation of carbohydrate metabolism (at the initial stage of glycolysis) on adaptation to unfavorable factors of the environment.

  1. [Effect of Hydrochloric acid on invasion of Ascaris suum; enzyme activity in the digestive system and serum of newborn piglets].

    PubMed

    Jabłonowski, Z; Romaniuk, K; Piechocki, D; Zółtowska, K; Lukaszewicz-Babecka, J; Dziekońska-Rynko, I J

    1995-01-01

    The studies were carried out on twenty newborn piglets. They were divided into four groups. The groups no 3 and no 4 were given intragastric 0.18% HCl from the 3th day of experiment. The groups no 2 and no 4 were infected on the 7th day with 10,000 invasive eggs of Ascaris suum. The presence of A. suum larvae in the lungs and liver was examined after one week lasting invasion by Baermann method. The total acidity in the gastric content was measured. The activity of alpha-amylase, lipase and proteases was determined in the extracts from pancreas and in the contents of stomach, duodenum and jejunum. The level of pepsinogens and alpha-amylase in the animals serum was studied. The intensity of Ascaris invasion was slightly higher in the group which was given HCl than in the infected group without HCl. The activity of digestive enzymes in the both groups was similar. Only in the stomach content from the 4th group the activity of pepsin was higher (p < 0.05), and alpha-amylase and lipase were lower (p < 0.01) than in the 2nd and in the 3th group. The level of pepsinogens was always lower and alpha-amylase higher in the serum of infected animals than in uninfected groups.

  2. 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.

  3. Diurnal feeding rhythms in north sea copepods measured by Gut fluorescence, digestive enzyme activity and grazing on labelled food

    NASA Astrophysics Data System (ADS)

    Baars, M. A.; Oosterhuis, S. S.

    Results obtained with three methods for measuring feeding rhythms of copepods were different. Gut fluorescence showed clear day-night variation during 2 out of 3 cruises at the Oyster Ground in the North Sea. The species studied ( Pseudocalanus, Temora, Centropages, Calanus) had highest gut fluorescence during the night in May and September, the larger species demonstrating the largest difference. Gut fluorescence was positively correlated with ambient chlorophyll concentrations. Gut clearance rate was not dependent on temperature but on gut fullness. Gut passage times at high gut fluorescence levels were ˜25 minutes, at low levels 2 hours. In grazing experiments with 14C labelled food, filtering rates declined after 5 to 15 minutes, presumably before the first defecation of radioactive material. Filtering rates in Temora were higher at night than by day during May and July, but not in Pseudocalanus and Calanus during September. No diurnal pattern of amylase and tryptic activity was found, except in July for amylase but then probably due to vertical migration. The activity of these digestive enzymes appeared to be least and gut fluorescence most suitable for the detection of grazing rhythms. The occurrence of high fluorescence levels at night in all species studied suggests that intermittent feeding by copepods on phytoplankton is a general phenomenon from spring to autumn. The increase in foraging activity appeared to start well before complete darkness, during May and July even one hour or more before sunset.

  4. Effects of tonicity, digestive enzymes and bile salts, and nutrient media on the survival of excysted metacercariae of Echinostoma caproni.

    PubMed

    Fried, Bernard; Schneck, Jessica L; Ponder, Elizabeth L

    2004-05-01

    The effects of tonicity, digestive enzymes and bile salts, and various nutrients added to Locke's solution were studied on the chemically excysted metacercariae of Echinostoma caproni. Metacercariae were maintained at 37.5 degrees C in multiwell chambers, ten per 0.5 ml of test solution; each experiment was replicated five times. Most metacercariae maintained in deionized water or Locke's 2x solution were dead within 2 h. About 85% and 55% of the metacercariae were alive at 8 h in Locke's 1x and Locke's 0.5x, respectively. Metacercariae of this species are osmoconformers, as is the case for adult digeneans. All metacercariae were dead in an acid saline or acid pepsin medium by 2 h. About 50% of the metacercariae were alive in an alkaline trypsin-bile salts medium at 4 h. These results suggest that the acidic pepsin environment in the stomach of a definitive host would be detrimental to the survival of excysted metacercariae, but prolonged survival in alkaline trypsin-bile salts would facilitate establishment of this larval stage in the mucosa of the host small intestine. Studies on excysted metacercariae in Locke's 1x supplemented with various nutrients showed that optimal survival occurred in Locke's plus 0.1% glucose and in Locke's 1x plus 1% hen's egg yolk. Significant survival of excysted metacercariae in Locke's 1x supplemented with either 0.1% proline, 0.1% threonine, or 0.1% serine did not occur. Copyright 2004 Springer-Verlag

  5. Preimaginal exposure to azadirachtin affects food selection and digestive enzymes in adults of Drosophila melanogaster (Diptera: Drosophilidae).

    PubMed

    Kilani-Morakchi, Samira; Bezzar-Bendjazia, Radia; Ferdenache, Maroua; Aribi, Nadia

    2017-08-01

    Among the plant derived product, azadirachtin, a neem-based insecticide, is exceptional in having a broad range of bioactivity including toxicity, growth, development and reproduction effects, repellency and antifeedancy. If considerable progress on the physiological and biological activities and agricultural application of azadirachtin has been achieved, its exact mechanism of action remains uncertain. In this study, we aimed at assessing the lethal and sublethal behavioral and physiological effects of azadirachtin on Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae) as biological model. Azadirachtin was applied topically at two doses LD25 (0.28μg) and LD50 (0.67μg) on early third instar larvae. Results showed that flies preferentially ingested control medium rather than azadirachtin-treated medium. Pre-imaginal exposure (L3) to azadirachtin increased aversion to this substance suggesting a memorability of the learned avoidance. In addition, all tested flies revealed a clear preference for solvent odour rather than azadirachtin odour. Moreover, azadirachtin treatment decreased significantly the amount of food intake in the adults of both sexes. Finally, azadirachtin was found to affect digestive enzyme activities in the midgut of flies. Indeed, an inhibition of α-amylase, chitinase, and protease activities and an increase of lipasic activity were noted. These results may reflect interference of azadirachtin with regulation of feeding and metabolism, and provide some evidence of a long term antifeedancy and delayed effects through developmental stage which may reinforce the insecticidal activity of this bioinsecticide. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Effects of periplocoside X on midgut cells and digestive enzymes activity of the soldiers of red imported fire ant.

    PubMed

    Li, Yan; Zeng, Xin-Nian

    2013-07-01

    The pathological effects of ingested periplocoside X, an insecticidal component isolated from the root of Periploca sepium Bunge, on the midgut epithelial cells of the soldiers of red imported fire ant were studied and the symptom was described. The results showed that periplocoside X could induce a severe, time-dependent cytotoxicity in the midgut epithelial cells. An optical microscopy showed that epithelial cells swelled firstly and then lysed. Transmission electron microscopy (TEM) showed that numerous swollen lysosomes were appeared, microvilli were disrupted and sloughed off, and the numbers of the rough endoplasmic reticulum and the mitochondria decreased sharply in earlier stage. Numerous vacuoles were observed in the later stage. Finally, periplocoside X resulted in cell death by cytolysis. Assay of main three digestive enzymes activity indicated that amylase activity was significantly inhibited, but no significant changes were seen for lipase activity and total protease activity. So it is suggested that periplocoside X induced mainly to organic damage of midgut epithelium cells of insect. In all, insect midgut is one of targets for periplocoside X.

  7. The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

    SciTech Connect

    Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.; Nicora, Carrie D.; Webb-Robertson, Bobbie-Jo M.; White, Richard A.; Monroe, Matthew E.; Huang, Eric L.; Smith, Richard D.; Currie, Cameron R.

    2016-10-26

    Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles were significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.

  8. 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

  9. Assessment of enzyme supplementation on growth performance and apparent nutrient digestibility in diets containing undecorticated sunflower seed meal in layer chicks.

    PubMed

    Fafiolu, A O; Oduguwa, O O; Jegede, A V; Tukura, C C; Olarotimi, I D; Teniola, A A; Alabi, J O

    2015-08-01

    Six hundred and forty one-day-old layer chicks were used to investigate the effect of replacing soybean meal with undecorticated sunflower seed meal protein for protein at 0, 25, 50, and 75% levels. Diets were without enzyme supplementation or with enzyme supplementation with four replications of twenty birds. Growth performance and nutrient utilization were determined. Proximate composition of the undecorticated sunflower seed meal used revealed that undecorticated sunflower seed meal contained 925.9, 204.5, 336.2, 215.1, 52.0 and 192.2g/kg dry matter, crude protein, ether extract, crude fibre, ash and soluble carbohydrates, respectively. Results showed that the final weight of 484.4 g/bird was obtained for birds on 75% undecorticated sunflower seed meal diet, while the lowest value of 472.2g/bird was obtained for birds on 25% undecorticated sunflower seed meal diet. Weight gain per bird per day was not significantly (P > 0.05) affected as the level of undecorticated sunflower seed meal increased in the diets. Feed intake per bird per day increased (P < 0.05) across the treatment as a result of increased undecorticated sunflower seed meal inclusion in the diet. However, enzyme supplementation of the diets showed marked (P < 0.05) improvements in feed intake, weight gain, and final weight as well as the feed to gain ratio. Survivability was not affected by the treatments imposed. Dry matter digestibility were significantly (P < 0.05) reduced due to high undecorticated sunflower seed meal inclusion in the diet while crude protein digestibility progressively reduced (P < 0.05) as the level of undecorticated sunflower seed meal increased in the diet. Ash digestibility values were, however, increased (P < 0.05) as the level of undecorticated sunflower seed meal increased in the diets. Birds on enzyme-supplemented diets consistently showed superior (P < 0.05) digestibility values than those on diets without enzyme supplementation. However ether extract digestibility was

  10. Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus.

    PubMed

    Liu, Haitian; Wang, Shifeng; Cai, Yan; Guo, Xiaohui; Cao, Zhenjie; Zhang, Yongzheng; Liu, Shubin; Yuan, Wei; Zhu, Weiwei; Zheng, Yu; Xie, Zhenyu; Guo, Weiliang; Zhou, Yongcan

    2017-01-01

    The probiotic properties of Bacillus subtilis HAINUP40 isolated from the aquatic environment, and the effects of dietary administration of B. subtilis HAINUP40 on the growth performance, intestinal probiotic recovery, digestive enzyme activities, innate immunity and disease resistance of tilapia (Oreochromis niloticus) were evaluated. The probiotic properties investigated include tolerance to simulated gastrointestinal stress, auto-aggregation, cell surface hydrophobicity and extracellular enzyme production. The cell number of B. subtilis changed little after 4 h in simulated gastric fluid at pH = 2.0, 3.0, 4.0 and simulated intestinal fluid at pH = 6.8.B.subtilis HAINUP40 revealed strong auto-aggregation property (34.6-87.0%) after 24 h incubation period. It exhibited significant cell surface hydrophobicity in xylene (28.8%) and chloroform (41.3%) and produced extracellular proteases and amylase. After tilapia (mean weight = 95 ± 8 g) were fed with a diet containing 10(8) cfu/g B. subtilis HAINUP40, their final body weight, percent weight gain (PWG), specific growth rate (SGR), total antioxidant capacity (T-AOC) and serum superoxide dismutase (SOD) increased significantly (p < 0.05) after 8 weeks; feed conversion rate (FCR) is significantly lower (p < 0.05) after 8 weeks; the protease and amylase activity in the digestive tract increased significantly (p < 0.05) after 4 and 8 weeks; and respiratory bursts and serum lysozyme activity increased significantly (p < 0.05) after 2 weeks. Moreover, being challenged with pathogenic Streptococcus agalactiae for 2 weeks, the relative percent survival (RPS%) is 52.94%. The results of this study strongly suggest that dietary supplement of B. subtilis HAINUP40 can effectively enhances the growth performance, immune response, and disease resistance of Nile tilapia.

  11. 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

  12. 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

  13. Characterization and study of the orientation of immobilized enzymes by tryptic digestion and HPLC-MS: design of an efficient catalyst for the synthesis of cephalosporins.

    PubMed

    Temporini, Caterina; Bonomi, Paolo; Serra, Immacolata; Tagliani, Auro; Bavaro, Teodora; Ubiali, Daniela; Massolini, Gabriella; Terreni, Marco

    2010-06-14

    An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117alpha for PGA-Eupergit C; Lys 163alpha and Lys 165alpha for PGA-glyoxyl-agarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins.

  14. Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics.

    PubMed

    Wu, Shuaibin; Zhang, Lei; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2012-01-01

    Initially, a poly (glycidyl methacrylate-co-acrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 μm i.d. capillary, and then was grafted with polyethylenimine (Mw, ~25,000) for adsorbing Cu(2+), followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/μL) was completed within 50 s via such immobilized enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.

  15. Lack of any association between insertion/deletion (I/D) polymorphisms in the angiotensin-converting enzyme gene and digestive system cancer risk: a meta-analysis.

    PubMed

    Liu, Jin-Fei; Xie, Hao-Jun; Cheng, Tian-Ming

    2013-01-01

    To investigate the association between the gene polymorphisms of angiotensin-converting enzyme (ACE) and digestive system cancer risk. A search was performed in Pubmed, Medline, ISI Web of Science and Chinese Biomedical (CBM) databases, covering all studies until Sep 1st, 2013. Statistical analysis was performed by using Revman5.2 and STATA 12.0. A total of 15 case-control studies comprising 2,390 digestive system cancer patients and 9,706 controls were identified. No significant association was found between the I/D polymorphism and digestive cancer risk (OR =0.93, 95%CI = (0.75, 1.16), P =0.53 for DD+DI vs. II). In the subgroup analysis by ethnicity and cancer type, no significant associations were found for the comparison of DD+DI vs. II. Results from other comparative genetic models also indicated a lack of associations between this polymorphism and digestive system cancer risks. This meta-analysis suggested that the ACE D/I polymorphism might not contribute to the risk of digestive system cancer.

  16. 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.

  17. Real-time enzyme-digesting identification of double-strand DNA in a resonance-cantilever embedded micro-chamber.

    PubMed

    Xu, Tiegang; Yu, Haitao; Xu, Pengcheng; Xu, Wangjie; Chen, Wenqing; Chen, Chuanzhao; Li, Xinxin

    2014-03-21

    A novel direct identification of double-strand DNA is proposed by using real-time enzyme-digestion in a resonant-cantilever embedded microfluidic chip. The new gene-level detection method is expected to replace the conventional DNA-hybridization based gene-detection that suffers from not only nonspecific adsorption induced false-positives but also complicated single-strand DNA preparation and hybridization. Since a detected DNA chain features a unique cutting site for a certain restriction-enzyme, the accurately cut-off mass (representing the length of the digested segment) can be online recorded by the frequency-shift signal of the resonant micro-cantilever sensor. This enzyme-digestion technique is confirmed by experimental identification of the stx2 gene of E. coli O157:H7. The direct-PCR sample is directly analyzed by using our lab-made cantilever-embedded microfluidic-chip. The 3776 bp DNA is immobilized via biotin-streptavidin binding and the added mass is recorded by a frequency-decrease of 15.9 kHz within 10 min. Then, with EcoRV-enzyme digestion at the site of 2635 bp, the cut-off mass is real-time detected by a frequency-increase of 10.2 kHz within 6 min. The detected frequency-shift ratio of 15.9/10.2 = 64.2% is consistent with the length ratio between the cut-off fragment and the whole DNA chain (2635/3776 = 69.8%). Hence, the simple and accurate double-strand detection method is verified experimentally.

  18. 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. © 2014 Japanese Society of Animal Science.

  19. Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

    PubMed Central

    Lukk, Tiit; Sakai, Ayano; Kalyanaraman, Chakrapani; Brown, Shoshana D.; Imker, Heidi J.; Song, Ling; Fedorov, Alexander A.; Fedorov, Elena V.; Toro, Rafael; Hillerich, Brandan; Seidel, Ronald; Patskovsky, Yury; Vetting, Matthew W.; Nair, Satish K.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, John A.; Jacobson, Matthew P.

    2012-01-01

    The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion. PMID:22392983

  20. 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

  1. Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay.

    PubMed Central

    Belay, T; Cherniak, R; Shinoda, T

    1993-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs. Images PMID:7685739

  2. Digestion of Nucleic Acids Starts in the Stomach.

    PubMed

    Liu, Yu; Zhang, Yanfang; Dong, Ping; An, Ran; Xue, Changhu; Ge, Yinlin; Wei, Liangzhou; Liang, Xingguo

    2015-07-14

    The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3'-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology.

  3. Digestion of Nucleic Acids Starts in the Stomach

    PubMed Central

    Liu, Yu; Zhang, Yanfang; Dong, Ping; An, Ran; Xue, Changhu; Ge, Yinlin; Wei, Liangzhou; Liang, Xingguo

    2015-01-01

    The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3′-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology. PMID:26168909

  4. Effects of copper-bearing montmorillonite on growth performance, digestive enzyme activities, and intestinal microflora and morphology of male broilers.

    PubMed

    Xia, M S; Hu, C H; Xu, Z R

    2004-11-01

    Avian commercial male broiler chicks (n = 240), 1 d of age, were used to investigate the effects of copper-bearing montmorillonite (Cu-MMT) on growth performance, digestive enzyme activities, and intestinal microflora and morphology. The chicks were allocated to 4 treatments, each of which had 5 pens of 12 chicks per pen. The 4 treatments were basal diet only (control group), basal diet + 1.5 g/kg montmorillonite (MMT), basal diet + 36.75 mg/kg Cu, in the form of CuSO4, and basal diet + 1.5 g/kg Cu-MMT. The results showed that supplementation with Cu-MMT significantly improved growth performance compared with the control diet, and that chicks fed with Cu-MMT had higher average daily gain (ADG) than those fed with MMT or CuSO4. Supplementation with Cu-MMT significantly reduced the total viable counts of Escherichia coli and Clostridium in the small intestine and cecum. Supplementation with MMT or CuSO4 had no influence on intestinal microflora. Chicks fed with Cu-MMT had lower viable counts of E. coli in cecal contents than those fed with MMT or CuSO4. The addition of either MMT or Cu-MMT to the diet improved the activities of total protease, amylase, and lipase in the small intestinal contents but had no effect on those in the pancreas. Morphological measurements of the small intestinal mucosa of chicks indicated that dietary addition of MMT or Cu-MMT improved intestinal mucosal morphology.

  5. 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

  6. [Regularities of organ-specific expression of enzyme systems in cattle].

    PubMed

    Tatarenko, O F; Glazko, V I

    1992-01-01

    The organ specificity of creatine kinase, esterase, isocitrate dehydrogenase lactate dehydrogenase, nucleoside phosphorylase, adenylate kinase, hexokinase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase of black-white cattle has been studied. Esterases, creatine kinase, adenylate kinase, hexokinase and glucose-6-phosphate dehydrogenase have a very wide spectrum of the organ variabilities. Liver and heart have the largest specificity of enzymes activity. Some peculiarities of isozyme spectrum are found in ovaries and spleen.

  7. 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.

  8. Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.

    PubMed

    MacGregor, E A; Janecek, S; Svensson, B

    2001-03-09

    The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.

  9. Impact of UV-B radiation on the digestive enzymes and immune system of larvae of Indian major carp Catla catla.

    PubMed

    Sharma, Jaigopal; Rao, Y Vasudeva; Kumar, S; Chakrabarti, Rina

    2010-03-01

    Ultraviolet radiation is a potent threat to the aquatic animals. Exposure to such stressor affects metabolic and immunological processes. The present investigation aims to study the effect of UV-B radiation on digestive enzymes and immunity of larvae of Catla catla. Larvae were exposed to ultraviolet-B (UV-B, 280-320 nm) radiation (145 microW/cm(2)) for three different exposure times of 5, 10 and 15 min on every other day. After 55 days, important digestive enzymes were assayed. For immunological study, lysozyme, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels were measured. Then the fish were kept for one month without radiation and lysozyme level was measured. Protein concentration varied directly with the duration of exposure and was highest among fish that had received the 15 min UV-B irradiation. Significantly higher amylase, protease, trypsin and chymotrypsin activities were found in 5 min exposed fish compared to others. Lysozyme level was significantly higher in control group compared to the UV-B treated fish. The lysozyme level decreased with the increasing duration of UV-B radiation. When fish were kept without UV-B radiation for one month, lysozyme level was brought to the normal level in all treatments, except 15 min exposed fish. The GOT and GPT levels were significantly higher in the 15 min exposed group than others. The effects of UV-B radiation on the digestive physiology and immune system of catla have been clearly observed in the present study. The decreased enzyme activities in UV-B radiated fish results into improper digestion and poor growth.

  10. Specific and non-specific enzymes for furanosyl-containing conjugates: biosynthesis, metabolism, and chemo-enzymatic synthesis.

    PubMed

    Chlubnova, Ilona; Legentil, Laurent; Dureau, Rémy; Pennec, Alizé; Almendros, Mélanie; Daniellou, Richard; Nugier-Chauvin, Caroline; Ferrières, Vincent

    2012-07-15

    There is no doubt now that the synthesis of compounds of varying complexity such as saccharides and derivatives thereof continuously grows with enzymatic methods. This review focuses on recent basic knowledge on enzymes specifically involved in the biosynthesis and degradation of furanosyl-containing polysaccharides and conjugates. Moreover, and when possible, biocatalyzed approaches, alternative to standard synthesis, will be detailed in order to strengthen the high potential of these biocatalysts to go further with the preparation of rare furanosides. Interesting results will be also proposed with chemo-enzymatic processes based on nonfuranosyl-specific enzymes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Enzymic synthesis of steroid sulfates XVI. Specificity and regulation of human adrenal hydroxysteroid sulfotransferase.

    PubMed

    Adams, J B; McDonald, D

    1983-05-01

    Pure hydroxysteroid sulfotransferase (EC 2.8.2.2) of human adrenal glands possesses a wide substrate specificity towards steroids. This wide specificity has now been found to extend to simple alcohols; normal aliphatic alcohols from C3 onwards acting as substrates with C9 showing the highest rate. Increased rate was accompanied by a decrease in Km. In marked contrast to the sulfurylation of steroids such as dehydroepiandrosterone, which exhibit wave-like kinetics, the kinetics with simple alcohols were of the normal Michaelis-Menten type. By means of enzyme antibody and enzyme stability studies evidence was provided that one and the same enzyme was responsible for sulfurylation of hydroxyls on the 3- and 17- positions of steroids and simple alcohols. The data lend support to previous evidence that the enzyme controls the secretion of dehydroepiandrosterone sulfate via steroid-specific binding sites, enabling self-regulation in response to ACTH action.

  12. In vitro digestion of rice bran proteins produces peptides with potent inhibitory effects on α-glucosidase and angiotensin I converting enzyme.

    PubMed

    Uraipong, Chatchaporn; Zhao, Jian

    2017-07-05

    The bioactivities of peptides released from the digestion of rice bran protein under in vitro simulated human digestive conditions were investigated. Four protein fractions extracted from rice bran were digested and the hydrolysates were fractionated by ultrafiltration and anion exchange chromatography. α-Glucosidase and angiotensin converting enzyme (ACE) inhibitory activities of the crude hydrolysates and their fractions were determined. Peptides with molecular weight (MW) < 3 kDa gave markedly higher α-glucosidase inhibitory activities than the crude digests, while the ACE inhibitory activities of the MW < 3 kDa fractions were similar to those of the crude extracts. Peptides from albumin and glutelin exhibited the greatest inhibitory effects on α-glucosidase and ACE at 52.43 ± 2 mg acarbose equivalent and 170.13 ± 2.2 nmol captopril equivalent g(-1) peptide respectively. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis identified 39 peptides in the most active fraction of the hydrolysates, and 37 of these contained peptide sequences that are known to exhibit antidiabetic or antihypertensive activities. Consumption of rice bran proteins can potentially lead to generation of bioactive peptides in the digestive tract with substantial health benefits. ACE and α-glucosidase inhibitory activities of the digests of rice bran proteins, and albumin and glutelin in particular, were especially strong, comparable to that of the standard drugs. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

    PubMed

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

    2015-08-07

    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.

  14. Zymographic approach to determine the intrinsic enzyme specific activity of diamine oxidase in presence of interfering enzymes.

    PubMed

    Ahmadifar, Samaneh; Le, Tien Canh; Marcocci, Lucia; Pietrangeli, Paola; Mateescu, Mircea Alexandru

    2017-07-04

    The purpose of this investigation was to elaborate a fast zymographic assay of oxidase enzymes in the presence of interfering enzymes as catalase (which disturbs current dosages based on H2O2 detection). This method also allows the determination of intrinsic specific activity (ISA) of oxidases, such as diamine oxidase (DAO) or glucose oxidase (GOD). The SDS-PAGE gels with entrapped peroxidase have been obtained by polymerization of acrylamide and bis-acrylamide in the presence of horse-radish peroxidase. The entrapped peroxidase was uniformly distributed in the PolyacrylAmide (PAA) material and did not migrate during electrophoresis. The obtained PAA gels allow the electrophoretic separation of various oxidases from contaminating proteins. As an example, to reveal DAO, the resulting PAA-gel should be incubated after the electrophoretic run in the developing solution containing putrescine (a DAO substrate) and o-phenylenediamine (a HRP substrate) to give coloured bands on the gel in the presence of DAO-generated H2O2. The results showed that is possible to determine the DAO in the presence of interfering catalase because they migrate differently. Thus, the H2O2 released in situ by DAO is no more decomposed by catalase because of its different mobility. It was also found that the same electrophoretic gel, after zymography, can be restained by Coomassie Blue for quantitation of proteins corresponding to the zymographic bands. With the obtained enzyme units and protein concentration it is also possible to calculate the intrinsic specific activity of DAO directly from the intensities of enzyme bands in zymography and from those of protein bands (Coomassie Blue staining), quantified by densitometry. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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.

  17. 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

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

    PubMed

    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.

  19. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

    PubMed

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

  20. 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.

  1. Substrate Specificity and Diastereoselectivity of Strictosidine Glucosidase, a Key Enzyme in Monoterpene Indole Alkaloid Biosynthesis

    PubMed Central

    Yerkes, Nancy; Wu, Jia; McCoy, Elizabeth; Galan, M. Carmen; Chen, Shi; O’Connor, Sarah E.

    2008-01-01

    Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted. PMID:18061449

  2. Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis.

    PubMed

    Yerkes, Nancy; Wu, Jia Xin; McCoy, Elizabeth; Galan, M Carmen; Chen, Shi; O'Connor, Sarah E

    2008-05-15

    Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady-state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.

  3. Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.

    PubMed

    Behrendorff, James B Y H; Huang, Weiliang; Gillam, Elizabeth M J

    2015-04-01

    Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few examples to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.

  4. Prediction of detailed enzyme functions and identification of specificity determining residues by random forests.

    PubMed

    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.

  5. 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

  6. Cryoenzymology in mixed solvents without cosolvent effects on enzyme specific activity.

    PubMed Central

    Douzou, P; Balny, C

    1977-01-01

    Water-soluble polyelectrolytes in interaction with proteins are described. These polyelectrolytes make it possible to investigate enzyme-catalyzed reactions in cooled mixed solvents without the usual effects of their organic solvent component on enzyme specific activity. The applicability of techniques developed is illustrated by results obtained on several systems. The possibility of an electrostatic "sorting out" of solvents and its potentialities in cryoenzymology are discussed. PMID:18734

  7. 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. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  8. Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes.

    PubMed

    Stewart, Alan J; Mukherjee, Joy; Roberts, Scott J; Lester, Douglas; Farquharson, Colin

    2005-01-01

    Phosphoinositol (PhoIns)-specific phospholipase C enzymes (PLCs) are central to the inositol lipid signaling pathways and contribute to intracellular Ca2+ release and protein kinase C activation. Five distinct classes of PhoIns-specific PLCs are known to exist in mammals, which are activated by membrane receptor-mediated events. Here we have identified a sixth class of PhoIns-specific PLC with a novel domain structure, which we have termed PLC-eta. Two putative PLC-eta enzymes were identified in humans and in mice. Sequence analysis revealed that residues implicated in substrate binding and catalysis from other PhoIns-specific PLCs are conserved in the novel enzymes. PLC-eta enzymes are most closely related to the PLC-delta class and share a close evolutionary relationship with other PLC isozymes. EST analysis and RT-PCR data suggest that PLC-eta enzymes are expressed in several cell types and, by analogy with other mammalian PhoIns-specific PLCs, are likely to be involved in signal transduction pathways.

  9. Site-Specific Management of Miscanthus Genotypes for Combustion and Anaerobic Digestion: A Comparison of Energy Yields

    PubMed Central

    Kiesel, Andreas; Nunn, Christopher; Iqbal, Yasir; Van der Weijde, Tim; Wagner, Moritz; Özgüven, Mensure; Tarakanov, Ivan; Kalinina, Olena; Trindade, Luisa M.; Clifton-Brown, John; Lewandowski, Iris

    2017-01-01

    In Europe, the perennial C4 grass miscanthus is currently mainly cultivated for energy generation via combustion. In recent years, anaerobic digestion has been identified as a promising alternative utilization pathway. Anaerobic digestion produces a higher-value intermediate (biogas), which can be upgraded to biomethane, stored in the existing natural gas infrastructure and further utilized as a transport fuel or in combined heat and power plants. However, the upgrading of the solid biomass into gaseous fuel leads to conversion-related energy losses, the level of which depends on the cultivation parameters genotype, location, and harvest date. Thus, site-specific crop management needs to be adapted to the intended utilization pathway. The objectives of this paper are to quantify (i) the impact of genotype, location and harvest date on energy yields of anaerobic digestion and combustion and (ii) the conversion losses of upgrading solid biomass into biogas. For this purpose, five miscanthus genotypes (OPM 3, 6, 9, 11, 14), three cultivation locations (Adana, Moscow, Stuttgart), and up to six harvest dates (August–March) were assessed. Anaerobic digestion yielded, on average, 35% less energy than combustion. Genotype, location, and harvest date all had significant impacts on the energy yield. For both, this is determined by dry matter yield and ash content and additionally by substrate-specific methane yield for anaerobic digestion and moisture content for combustion. Averaged over all locations and genotypes, an early harvest in August led to 25% and a late harvest to 45% conversion losses. However, each utilization option has its own optimal harvest date, determined by biomass yield, biomass quality, and cutting tolerance. By applying an autumn green harvest for anaerobic digestion and a delayed harvest for combustion, the conversion-related energy loss was reduced to an average of 18%. This clearly shows that the delayed harvest required to maintain biomass

  10. Site-Specific Management of Miscanthus Genotypes for Combustion and Anaerobic Digestion: A Comparison of Energy Yields.

    PubMed

    Kiesel, Andreas; Nunn, Christopher; Iqbal, Yasir; Van der Weijde, Tim; Wagner, Moritz; Özgüven, Mensure; Tarakanov, Ivan; Kalinina, Olena; Trindade, Luisa M; Clifton-Brown, John; Lewandowski, Iris

    2017-01-01

    In Europe, the perennial C4 grass miscanthus is currently mainly cultivated for energy generation via combustion. In recent years, anaerobic digestion has been identified as a promising alternative utilization pathway. Anaerobic digestion produces a higher-value intermediate (biogas), which can be upgraded to biomethane, stored in the existing natural gas infrastructure and further utilized as a transport fuel or in combined heat and power plants. However, the upgrading of the solid biomass into gaseous fuel leads to conversion-related energy losses, the level of which depends on the cultivation parameters genotype, location, and harvest date. Thus, site-specific crop management needs to be adapted to the intended utilization pathway. The objectives of this paper are to quantify (i) the impact of genotype, location and harvest date on energy yields of anaerobic digestion and combustion and (ii) the conversion losses of upgrading solid biomass into biogas. For this purpose, five miscanthus genotypes (OPM 3, 6, 9, 11, 14), three cultivation locations (Adana, Moscow, Stuttgart), and up to six harvest dates (August-March) were assessed. Anaerobic digestion yielded, on average, 35% less energy than combustion. Genotype, location, and harvest date all had significant impacts on the energy yield. For both, this is determined by dry matter yield and ash content and additionally by substrate-specific methane yield for anaerobic digestion and moisture content for combustion. Averaged over all locations and genotypes, an early harvest in August led to 25% and a late harvest to 45% conversion losses. However, each utilization option has its own optimal harvest date, determined by biomass yield, biomass quality, and cutting tolerance. By applying an autumn green harvest for anaerobic digestion and a delayed harvest for combustion, the conversion-related energy loss was reduced to an average of 18%. This clearly shows that the delayed harvest required to maintain biomass quality

  11. Effects of different salinities on growth performance, survival, digestive enzyme activity, immune response, and muscle fatty acid composition in juvenile American shad (Alosa sapidissima).

    PubMed

    Liu, Zhi-Feng; Gao, Xiao-Qiang; Yu, Jiu-Xiang; Qian, Xiao-Ming; Xue, Guo-Ping; Zhang, Qiao-Yun; Liu, Bao-Liang; Hong, Lei

    2016-12-24

    The effects of salinity on survival, growth, special activity of digestive enzymes, nonspecific immune response, and muscle fatty acid composition were evaluated in the American shad (Alosa sapidissima). Juveniles of 35 days after hatching were reared at 0 (control), 7, 14, 21, and 28 ppt for 60 days. At the end of the experiment, juvenile American shad presented higher survival and specific growth rate (SGR) in salinity group (7, 14, and 21 ppt) than control group (P < 0.05). The special activity of trypsin and chymotrypsin was highest in fish reared at 21 ppt, while the highest lipase special activity was obtained in control group (P < 0.05). The special activity of alkaline phosphatase (ALP), lysozyme (LZM), superoxide dismutase (SOD), and catalase (CAT) showed significant increases in salinity group (14 and 21 ppt) compared to control group (P < 0.05). Lower muscle ash contents were detected in salinity group (14, 21, and 28 ppt) than control group (P < 0.05), while the contents of crude lipid and crude protein were significantly higher than control group (P < 0.05). The level of monounsaturated fatty acids (MUFA) exhibited a decreasing trend, while an increased level of polyunsaturated fatty acids (PUFA) was detected with the increase of salinity. Among the PUFA, the content of n-3 fatty acids in muscle tissue was found to be increasing with the increasing salinity, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Results indicate that appropriate increase in salinity was reasonable and beneficial for juvenile American shad culture after a comprehensive consideration, especially salinity range from 14 to 21 ppt.

  12. Combining structure and sequence information allows automated prediction of substrate specificities within enzyme families.

    PubMed

    Röttig, Marc; Rausch, Christian; Kohlbacher, Oliver

    2010-01-08

    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/.

  13. 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

  14. Effects of dietary protein and energy levels on digestive enzyme activities and electrolyte composition in the small intestinal fluid of geese.

    PubMed

    Yang, Jing; Yang, Lin; Wang, Yongchang; Zhai, Shuangshuang; Wang, Shenshen; Yang, Zhipeng; Wang, Wence

    2017-02-01

    The present study was conducted to evaluate the effects of dietary protein and energy levels on digestive enzymes and electrolyte composition in jejunum of geese. A 3×3 factorial and completely randomized design was adopted with three protein levels and three energy levels. The experiment included four replicates for each treatment, and three geese for each replicate. Isovolumetric supernate from centrifugal jejuna fluid were mixed in each replicate. Activities of digestive enzymes and ions were analyzed. The results showed trypsin and chymotrypsin activities were significantly increased with increasing of dietary protein and energy levels (P<0.05). The concentrations of Ca(2+) and pH value were significantly decreased by increased dietary protein and energy levels. However, no significant differences were found for the activities of amylase and cellulase, as well as the concentration of Na(+) among groups with different protein and energy levels. In conclusion, digesta enzymes and electrolytes in the small intestine adapted to the protein and energy levels. The activities of protease, rather than amylase and cellulase were induced with increasing of protein and energy levels. The imbalance of positive and negative ions was possibly adjusted by the fluctuant concentrations of K(+) , Cl(-) and Ca(2+) for maintaining normal physiological function.

  15. Quantitative assessment of the association between the angiotensin-converting enzyme gene insertion/deletion polymorphism and digestive system cancer risk.

    PubMed

    Wang, J; Yang, S; Guo, F H; Mao, X; Zhou, H; Dong, Y Q; Wang, Z M; Luo, F

    2015-11-13

    The angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism has been reported to be associated with digestive system cancer; however, the results from previous studies have been conflicting. The present study aimed to investigate the association between the ACE I/D polymorphism and the risk of digestive system cancer using a meta-analysis of previously published studies. Databases were systematically searched to identify relevant studies published prior to December 2014. We estimated the pooled OR with its 95%CI to assess the association. The meta-analysis consisted of thirteen case-control studies that included 2557 patients and 4356 healthy controls. Meta-analysis results based on all the studies showed no significant association between the ACE I/D polymorphism and the risk of digestive system cancer (DD vs II: OR = 0.85, 95%CI = 0.59-1.24; DI vs II: OR = 0.94, 95%CI = 0.78-1.15; dominant model: OR = 0.96, 95%CI = 0.81- 1.15; recessive model: OR = 1.06, 95%CI = 0.76-1.48). Subgroup analyses by race and cancer type did not detect an association between the ACE I/D polymorphism and digestive system cancer risk. However, when the analyses were restricted to smaller studies (N < 500 patients), the summary OR of DI vs II was 0.80 (95%CI = 0.66-0.97). Our analyses detected a possibility of publication bias with a misestimate of the true association by smaller studies. Overall, meta-analysis results suggest the ACE I/D polymorphism might not be associated with susceptibility to digestive system cancer. Further large and well-designed studies are needed to confirm these conclusions.

  16. Intraruminal supplementation with increasing levels of exogenous polysaccharide-degrading enzymes: effects on nutrient digestion in cattle fed a barley grain diet.

    PubMed

    Hristov, A N; McAllister, T A; Cheng, K J

    2000-02-01

    The effects of supplying increasing ruminal doses of exogenous polysaccharide-degrading enzymes (EPDE) on rumen fermentation and nutrient digestion were studied using eight ruminally cannulated heifers, four of which were also duodenally cannulated, in a replicated Latin square. The heifers were fed a diet of 85.5% rolled barley grain and 14% barley silage (DM basis), and once daily they were given intraruminal doses of 0 (Control), 100, 200, or 400 g of a preparation containing polysaccharide-degrading enzymes. Enzyme treatment decreased ruminal pH (linear, P<.001) and increased ammonia N (quadratic, P<.001) concentration. The ruminally soluble fraction and effective degradability of feed DM in situ were increased (quadratic response, P<.001) by enzyme treatment. Ruminal administration of EPDE increased ruminal fluid carboxymethylcellulase and xylanase activities linearly (P<.001) and beta-glucanase activity quadratically (P<.01), decreased (quadratic response, P<.05) ruminal fluid viscosity, and did not affect (P>.05) ruminal fluid amylase activity. Elevated levels of fibrolytic activities in the rumen resulted in increased (quadratic, P<.001) carboxymethylcellulase, xylanase, and beta-glucanase (P<.01) activities in duodenal digesta. Duodenal amylase activity and reducing sugar concentration were also increased (quadratic responses, P<.001 and P<.05, respectively) by EPDE. Xylanase activity of fecal DM was increased linearly (P<.05) with increasing ruminal EPDE levels. Apparent digestibilities of DM, crude protein, and NDF were not affected by EPDE supplementation. Enzyme treatment did not affect (P>.05) urinary excretion of allantoin and uric acid, or concentrations of glucose and urea in blood.

  17. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Effect of Potential Probiotic Lactococcus lactis Subsp. lactis on Growth Performance, Intestinal Microbiota, Digestive Enzyme Activities, and Disease Resistance of Litopenaeus vannamei.

    PubMed

    Adel, Milad; El-Sayed, Abdel-Fattah M; Yeganeh, Sakineh; Dadar, Maryam; Giri, Sib Sankar

    2016-11-07

    The aims of this study were to evaluate the effects of Lactococcus lactis subsp. lactis on the growth, intestinal microbiota, digestive enzyme activity, and disease resistance of Litopenaeus vannamei. Diets containing four different concentrations of L. lactis (0 [basal diet], 10(6), 10(7), and 10(8) CFU g(-1)) were fed to white shrimps L. vannamei (average weight 5.89 ± 0.36 g) for 8 weeks. At the end of the feeding trial, shrimps were immersed in Caspian Seawater (10.8 ppt) contaminated with 10(6) CFU ml(-1) pathogenic V. anguillarum for 2 h. Results revealed that growth rate, survival, and body protein level were increased with dietary supplementation of L. lactis. The activities of digestive enzymes (cellulose, lipase, amylase, and protease) were significantly higher in the groups fed with diets containing 10(7) or 10(8) CFU g(-1) L. lactis than those in the control. The Lactobacillus and Bacillus counts were higher (P < 0.05) in the intestine of shrimps fed with L. lactis-supplemented diets. In addition, higher level of L. lactis supplementation decreased the Vibrio counts. Moreover, L. vannamei fed diet supplemented with 10(8) CFU g(-1) of L. lactis exhibited significantly the highest hematocyte count and post-challenge survival rate (79.2 %). Collectively, these results suggest that dietary supplementation of L. lactis subsp. lactis at 10(8) CFU g(-1) can promote growth performance, digestive enzyme activity, and disease resistance of L. vannamei.

  19. The effects of prebiotics on the digestive enzymes and gut histomorphology of red drum (Sciaenops ocellatus) and hybrid striped bass (Morone chrysops × M. saxatilis).

    PubMed

    Anguiano, Maritza; Pohlenz, Camilo; Buentello, Alejandro; Gatlin, Delbert M

    2013-02-28

    The effects of four prebiotics (fructo-oligosaccharide, Bio-MOS, transgalacto-oligosaccharide and GroBiotic-A) on digestive enzymes and intestinal morphology were studied in juvenile hybrid striped bass (Morone chrysops × M. saxatilis) and red drum (Sciaenops ocellatus) using two separate 8-week feeding trials. Red drum were fed experimental diets with the four prebiotics each individually supplemented at 1% and hybrid striped bass were fed diets supplemented with GroBiotic-A at 1 and 2%. Both trials were conducted with each diet fed to apparent satiation twice per d to three replicate groups of fifteen juvenile fish. For histomorphological analysis, gastrointestinal tract (GIT) samples from three randomly selected fish per tank were taken at 4 and 8 weeks for hybrid striped bass and at 8 weeks for red drum. For both trials, GIT samples from two randomly selected fish per tank were taken at 4 and 8 weeks and analysed for pepsin, trypsin, chymotrypsin, aminopeptidase, α-amylase, lipase, and both acid and alkaline phosphatase activities. The results of the histological evaluation indicated that the inclusion of prebiotics was adequate to elicit structural changes in the GIT of both species. On the other hand, no significant changes in the enzyme activities were detected at week 8 in both species. However, there was a transient effect of Bio-MOS supplementation on the activities of aminopeptidase, α-amylase and alkaline phosphatase at week 4 in red drum only. Thus, previously observed improvements in nutrient digestibility by these fish in response to prebiotic supplementation appear to be mostly related to changes in GIT structure as opposed to the enhancement of digestive enzyme activity.

  20. 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

    2015-10-02

    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.

  1. Specific point mutations in key redox enzymes are associated with chemoresistance in epithelial ovarian cancer.

    PubMed

    Fletcher, Nicole M; Belotte, Jimmy; Saed, Mohammed G; Memaj, Ira; Diamond, Michael P; Morris, Robert T; Saed, Ghassan M

    2017-01-01

    Oxidative stress plays an important role in the pathophysiology of ovarian cancer. Resistance to chemotherapy presents a significant challenge for ovarian cancer treatment. Specific single nucleotide polymorphisms (SNPs) in key redox enzymes have been associated with ovarian cancer survival and progression. The objective of this study was to determine whether chemotherapy induces point mutations in key redox enzymes that lead to the acquisition of chemoresistance in epithelial ovarian cancer (EOC). Human EOC cell lines and their chemoresistant counterpart were utilized for this study. Specific SNPs in key redox enzymes were analyzed by TaqMan SNP Genotyping. Activities and levels of key redox enzymes were determined by real-time RT-PCR, ELISA and a greiss assay. Point mutations in key redox enzymes were introduced into sensitive EOC cells via the CRISPR/Cas9 system. Cell viability and IC50 for cisplatin were determined by the MTT Cell Proliferation Assay. Data was analyzed with SPSS using Student's two-tailed t-tests and One-way ANOVA followed by Dunnett's or Tukey's post hoc tests, p<0.05. Here, we demonstrate that chemoresistant EOC cells are characterized by a further enhancement in oxidative stress as compared to sensitive counterparts. Additionally, chemoresistant EOC cells manifested specific point mutations, which are associated with altered enzymatic activity, in key redox enzymes that are not detected in sensitive counterparts. Supplementation of an antioxidant was able to successfully sensitize EOC cells to chemotherapeutics. Causality was established by the induction of these point mutations in sensitive EOC cells, which resulted in a significant increase in the level of chemoresistance. These findings indicate that chemotherapy induces specific point mutations in key redox enzymes that contribute to the acquisition of chemoresistance in EOC cells, highlighting a potential novel mechanism. Identification of targets for chemoresistance with either

  2. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme.

    PubMed

    Avalos, José L; Bever, Katherine M; Wolberger, Cynthia

    2005-03-18

    Sir2 enzymes form a unique class of NAD(+)-dependent deacetylases required for diverse biological processes, including transcriptional silencing, regulation of apoptosis, fat mobilization, and lifespan regulation. Sir2 activity is regulated by nicotinamide, a noncompetitive inhibitor that promotes a base-exchange reaction at the expense of deacetylation. To elucidate the mechanism of nicotinamide inhibition, we determined ternary complex structures of Sir2 enzymes containing nicotinamide. The structures show that free nicotinamide binds in a conserved pocket that participates in NAD(+) binding and catalysis. Based on our structures, we engineered a mutant that deacetylates peptides by using nicotinic acid adenine dinucleotide (NAAD) as a cosubstrate and is inhibited by nicotinic acid. The characteristics of the altered specificity enzyme establish that Sir2 enzymes contain a single site that participates in catalysis and nicotinamide regulation and provides additional insights into the Sir2 catalytic mechanism.

  3. Influence of time, storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (Sparus aurata).

    PubMed

    Solovyev, Mikhail; Gisbert, Enric

    2016-10-01

    In this study, we tested the effects of long-term storage (2 years) at -20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine-alanine peptidase) enzymes between 140 and 270 days of storage at -20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at -20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.

  4. 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. © 2015 Blackwell Verlag GmbH.

  5. Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray.

    PubMed

    Cornett, E M; Dickson, B M; Vaughan, R M; Krishnan, S; Trievel, R C; Strahl, B D; Rothbart, S B

    2016-01-01

    The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the "histone code" hypothesis, we reveal a strong influence of adjacent and, surprisingly, distant histone PTMs on the ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes.

  6. Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray

    PubMed Central

    Cornett, E.M.; Dickson, B.M.; Vaughan, R.M.; Krishnan, S.; Trievel, R.C.; Strahl, B.D.; Rothbart, S.B.

    2017-01-01

    The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the “histone code” hypothesis, we reveal a strong influenceof adjacent and,surprisingly,distant histonePTMs onthe ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes. PMID:27423856

  7. 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

  8. 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.

  9. [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.

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

    PubMed

    Jeyakanthan, Jeyaraman; Drevland, Randy M; Gayathri, Dasara Raju; Velmurugan, Devadasan; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Graham, David E

    2010-03-30

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of alpha-hydroxy acids to beta-hydroxy acids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of alpha,beta-dicarboxylates with hydrophobic gamma-chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-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 lead 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 alpha2 and alpha3 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

  11. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. Enzyme-synthesized highly branched maltodextrins have slow glucose generation at the mucosal α-glucosidase level and are slowly digestible in vivo.

    PubMed

    Lee, Byung-Hoo; Yan, Like; Phillips, Robert J; Reuhs, Bradley L; Jones, Kyra; Rose, David R; Nichols, Buford L; Quezada-Calvillo, Roberto; Yoo, Sang-Ho; Hamaker, Bruce R

    2013-01-01

    For digestion of starch in humans, α-amylase first hydrolyzes starch molecules to produce α-limit dextrins, followed by complete hydrolysis to glucose by the mucosal α-glucosidases in the small intestine. It is known that α-1,6 linkages in starch are hydrolyzed at a lower rate than are α-1,4 linkages. Here, to create designed slowly digestible carbohydrates, the structure of waxy corn starch (WCS) was modified using a known branching enzyme alone (BE) and an in combination with β-amylase (BA) to increase further the α-1,6 branching ratio. The digestibility of the enzymatically synthesized products was investigated using α-amylase and four recombinant mammalian mucosal α-glucosidases. Enzyme-modified products (BE-WCS and BEBA-WCS) had increased percentage of α-1,6 linkages (WCS: 5.3%, BE-WCS: 7.1%, and BEBA-WCS: 12.9%), decreased weight-average molecular weight (WCS: 1.73×10(8) Da, BE-WCS: 2.76×10(5) Da, and BEBA-WCS 1.62×10(5) Da), and changes in linear chain distributions (WCS: 21.6, BE-WCS: 16.9, BEBA-WCS: 12.2 DPw). Hydrolysis by human pancreatic α-amylase resulted in an increase in the amount of branched α-limit dextrin from 26.8% (WCS) to 56.8% (BEBA-WCS). The α-amylolyzed samples were hydrolyzed by the individual α-glucosidases (100 U) and glucogenesis decreased with all as the branching ratio increased. This is the first report showing that hydrolysis rate of the mammalian mucosal α-glucosidases is limited by the amount of branched α-limit dextrin. When enzyme-treated materials were gavaged to rats, the level of postprandial blood glucose at 60 min from BEBA-WCS was significantly higher than for WCS or BE-WCS. Thus, highly branched glucan structures modified by BE and BA had a comparably slow digesting property both in vitro and in vivo. Such highly branched α-glucans show promise as a food ingredient to control postprandial glucose levels and to attain extended glucose release.

  14. Influence of low protein diets on gene expression of digestive enzymes and hormone secretion in the gastrointestinal tract of young weaned piglets*

    PubMed Central

    Tian, Zhi-mei; Ma, Xian-yong; Yang, Xue-fen; Fan, Qiu-li; Xiong, Yun-xia; Qiu, Yue-qin; Wang, Li; Wen, Xiao-lu; Jiang, Zong-yong

    2016-01-01

    To investigate dietary protein level effects on digestive mechanisms, weaned piglets were fed for 45 d with diets containing 20%, 17%, or 14% crude protein (CP) supplemented to meet requirements for essential amino acids. This article describes the influence of dietary protein on gastrointestinal hormones and expression of an array of digestive enzymes in the gastrointestinal tract and pancreas. Results indicated that there were no significant differences in expression of enzymes involved in carbohydrate digestion, except for maltase in the duodenum. In the jejunum, amylase expression in pigs fed 20% CP was much higher than that in pigs fed other diets (P<0.05) and maltase expression in those fed 17% CP was higher than that in other treatments (P<0.05). Although there were no remarkable differences in expression of aminopeptidase in the small intestine or carboxypeptidase in the pancreas (P>0.05), there was a trend towards higher expression of various proteases in pigs fed 17% CP. The duodenal expression of enteropeptidase in diets with 14% and 17% CP was significantly higher than that with 20% CP (P<0.05), but treatment differences did not existed in jejunum (P>0.05). The expression of GPR93 as a nutrient-responsive G protein-coupled receptor in 14% and 17% CP diets was significantly higher than that in 20% CP diet in the small intestine (P<0.05). The expressions of genes for pancreatic enzymes, lipase and elastase, were significantly higher in pigs fed diets with low CP, while similar trends occurred for carboxypeptidase, chymotrypsin and amylase. Conversely, the gastric expressions of pepsinogen A and progastricsin were lower with the 17% CP diet. Differences between treatments were found in the gastric antral contents of cholecystokinin and somatostatin: both increased in pigs fed 17% CP, accompanied by decreased content of motilin, which was also seen in plasma concentrations. These patterns were not reflected in duodenal contents. In general, 17% dietary CP

  15. Pigs that are divergent in feed efficiency, differ in intestinal enzyme and nutrient transporter gene expression, nutrient digestibility and microbial activity.

    PubMed

    Vigors, S; Sweeney, T; O'Shea, C J; Kelly, A K; O'Doherty, J V

    2016-11-01

    Feed efficiency is an important trait in the future sustainability of pig production, however, the mechanisms involved are not fully elucidated. The objective of this study was to examine nutrient digestibility, organ weights, select bacterial populations, volatile fatty acids (VFA's), enzyme and intestinal nutrient transporter gene expression in a pig population divergent in feed efficiency. Male pigs (n=75; initial BW 22.4 kg SEM 2.03 kg) were fed a standard finishing diet for 43 days before slaughter to evaluate feed intake and growth for the purpose of calculating residual feed intake (RFI). Phenotypic RFI was calculated as the residuals from a regression model regressing average daily feed intake (ADFI) on average daily gain (ADG) and midtest BW0.60 (MBW). On day 115, 16 pigs (85 kg SEM 2.8 kg), designated as high RFI (HRFI) and low RFI (LRFI) were slaughtered and digesta was collected to calculate the coefficient of apparent ileal digestibility (CAID), total tract nutrient digestibility (CATTD), microbial populations and VFA's. Intestinal tissue was collected to examine intestinal nutrient transporter and enzyme gene expression. The LRFI pigs had lower ADFI (P<0.001), improved feed conversion ratio (P<0.001) and an improved RFI value relative to HRFI pigs (0.19 v. -0.14 SEM 0.08; P<0.001). The LRFI pigs had an increased CAID of gross energy (GE), and an improved CATTD of GE, nitrogen and dry matter compared to HRFI pigs (P<0.05). The LRFI pigs had higher relative gene expression levels of fatty acid binding transporter 2 (FABP2) (P<0.01), the sodium/glucose co-transporter 1 (SGLT1) (P<0.05), the glucose transporter GLUT2 (P<0.10), and the enzyme sucrase-isomaltase (SI) (P<0.05) in the jejunum. The LRFI pigs had increased populations of lactobacillus spp. in the caecum compared with HRFI pigs. In colonic digesta HRFI pigs had increased acetic acid concentrations (P<0.05). Differences in nutrient digestibility, intestinal microbial populations and gene

  16. Growth, nutrient digestibility, ileal digesta viscosity, and energy metabolizability of growing turkeys fed diets containing malted sorghum sprouts supplemented with enzyme or yeast.

    PubMed

    Oke, F O; Oso, A O; Oduguwa, O O; Jegede, A V; Südekum, K-H; Fafiolu, A O; Pirgozliev, V

    2017-06-01

    Growth, apparent nutrient digestibility, ileal digesta viscosity, and energy metabolizability of growing turkeys fed diets containing malted sorghum sprouts (MSP) supplemented with enzyme or yeast were investigated using 120, 28-day-old male turkeys. Six treatments were laid out in a 3 × 2 factorial arrangement of treatments with three dietary inclusion levels of MSP (0, 50, and 100 g/kg) and supplemented with 200 mg/kg yeast (Saccharomyces cerevisiae) or 200 mg/kg of a commercial enzyme. The experiment lasted for the starter (day 28-56) and grower phases (day 57-84) of the birds. Each treatment group consisted of 20 turkeys replicated four times with five birds each. Data were analysed using analysis of variance while polynomial contrast was used to determine the trends (linear and quadratic) of MSP inclusion levels. Irrespective of dietary supplementation with enzyme or yeast, final body weight (BW), total BW gain, and feed intake for turkey poults from day 29-56 was reduced (p < 0.05) with increasing inclusion level of MSP. Dietary supplementation with yeast resulted in increased (p < 0.05) feed intake while enzyme supplementation improved (p < 0.05) feed conversion ratio of the poults. Turkeys fed enzyme-supplemented MSP diets had higher (p < 0.05) BW gain than their counterparts fed yeast-supplemented MSP diets. Apparent ash digestibility reduced linearly (p < 0.05) with increasing inclusion levels of MSP. Apparent metabolizable energy (AME) did not vary significantly (p > 0.05) with MSP inclusion levels. Enzyme supplementation reduced (p < 0.05) ileal viscosity but had no effect (p > 0.05) on AME. Inclusion of MSP resulted in poor growth performance. This confirms earlier studies that utilization of MSP by poultry is rather poor. Supplementation with enzyme or yeast did not lead to any appreciable improvement in performance of turkeys in this study. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

  17. 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.

  18. 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.

  19. 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. © 2015 Wiley Periodicals, Inc.

  20. Effect of dietary histamine supplementation on growth, digestive enzyme activities and morphology of intestine and hepatopancreas in the Chinese mitten crab Eriocheir sinensis.

    PubMed

    Zhao, Liulan; Yang, Xiaozhen; Cheng, Yongxu; Yang, Song

    2016-01-01

    A 28-days feeding experiment was conducted to investigate the effect of histamine on digestive physiology of the Chinese mitten crab, Eriocheir sinensis. Four experimental diets were supplemented with histamine at 0, 1, 2, 4 g/kg. Histamine supplementation had no effect on growth. The activities of digestive enzyme decreased significantly at first (days 7 and 14) (p < 0.05) and then increased or finally slightly recovered in the hepatopancreas and intestinal tract on the 28th day. Tryptase and amylase activities were inhibited significantly in each histamine-treated group on day 7 as compared to the control (p < 0.05). On day 7, 14 and 28, tryptase mRNA relative expression in the histamine treatments correlated positively with the histamine concentration (p < 0.05). Histopathologic analyses showed serious alterations in hepatopancreas, moderate alterations in the hindgut and intestinal bulb, and no alterations in the midgut. In hepatopancreas, low levels (1 g/kg) of histamine caused an increase in the number of B-cells. High levels (4 g/kg) of histamine increased the number of R-cells, which were also highly vacuolized. In extreme cases, the basal lamina was detached from the tubule. In the intestinal bulb and hindgut, high levels of histamine (4 g/kg) decreased the density of reserve inclusion cells. Thus, this indicated that histamine had dose-dependent effect on the activity of digestive enzymes and the morphology of the intestine and hepatopancreas.

  1. Allosteric Inhibitory Molecular Recognition of a Photochromic Dye by a Digestive Enzyme: Dihydroindolizine makes α-chymotrypsin Photo-responsive

    PubMed Central

    Bagchi, Damayanti; Ghosh, Abhijit; Singh, Priya; Dutta, Shreyasi; Polley, Nabarun; Althagafi, Ismail.I.; Jassas, Rabab S.; Ahmed, Saleh A.; Pal, Samir Kumar

    2016-01-01

    The structural-functional regulation of enzymes by the administration of an external stimulus such as light could create photo-switches that exhibit unique biotechnological applications. However, molecular recognition of small ligands is a central phenomenon involved in all biological processes. We demonstrate herein that the molecular recognition of a photochromic ligand, dihydroindolizine (DHI), by serine protease α-chymotrypsin (CHT) leads to the photo-control of enzymatic activity. We synthesized and optically characterized the photochromic DHI. Light-induced reversible pyrroline ring opening and a consequent thermal back reaction via 1,5-electrocyclization are responsible for the photochromic behavior. Furthermore, DHI inhibits the enzymatic activity of CHT in a photo-controlled manner. Simultaneous binding of the well-known inhibitors 4-nitrophenyl anthranilate (NPA) or proflavin (PF) in the presence of DHI displays spectral overlap between the emission of CHT-NPA or CHT-PF with the respective absorption of cis or trans DHI. The results suggest an opportunity to explore the binding site of DHI using Förster resonance energy transfer (FRET). Moreover, to more specifically evaluate the DHI binding interactions, we employed molecular docking calculations, which suggested binding near the hydrophobic site of Cys-1-Cys-122 residues. Variations in the electrostatic interactions of the two conformers of DHI adopt unfavorable conformations, leading to the allosteric inhibition of enzymatic activity. PMID:27677331

  2. Allosteric Inhibitory Molecular Recognition of a Photochromic Dye by a Digestive Enzyme: Dihydroindolizine makes α-chymotrypsin Photo-responsive

    NASA Astrophysics Data System (ADS)

    Bagchi, Damayanti; Ghosh, Abhijit; Singh, Priya; Dutta, Shreyasi; Polley, Nabarun; Althagafi, Ismail. I.; Jassas, Rabab S.; Ahmed, Saleh A.; Pal, Samir Kumar

    2016-09-01

    The structural-functional regulation of enzymes by the administration of an external stimulus such as light could create photo-switches that exhibit unique biotechnological applications. However, molecular recognition of small ligands is a central phenomenon involved in all biological processes. We demonstrate herein that the molecular recognition of a photochromic ligand, dihydroindolizine (DHI), by serine protease α-chymotrypsin (CHT) leads to the photo-control of enzymatic activity. We synthesized and optically characterized the photochromic DHI. Light-induced reversible pyrroline ring opening and a consequent thermal back reaction via 1,5-electrocyclization are responsible for the photochromic behavior. Furthermore, DHI inhibits the enzymatic activity of CHT in a photo-controlled manner. Simultaneous binding of the well-known inhibitors 4-nitrophenyl anthranilate (NPA) or proflavin (PF) in the presence of DHI displays spectral overlap between the emission of CHT-NPA or CHT-PF with the respective absorption of cis or trans DHI. The results suggest an opportunity to explore the binding site of DHI using Förster resonance energy transfer (FRET). Moreover, to more specifically evaluate the DHI binding interactions, we employed molecular docking calculations, which suggested binding near the hydrophobic site of Cys-1-Cys-122 residues. Variations in the electrostatic interactions of the two conformers of DHI adopt unfavorable conformations, leading to the allosteric inhibition of enzymatic activity.

  3. Allosteric Inhibitory Molecular Recognition of a Photochromic Dye by a Digestive Enzyme: Dihydroindolizine makes α-chymotrypsin Photo-responsive.

    PubMed

    Bagchi, Damayanti; Ghosh, Abhijit; Singh, Priya; Dutta, Shreyasi; Polley, Nabarun; Althagafi, Ismail I; Jassas, Rabab S; Ahmed, Saleh A; Pal, Samir Kumar

    2016-09-28

    The structural-functional regulation of enzymes by the administration of an external stimulus such as light could create photo-switches that exhibit unique biotechnological applications. However, molecular recognition of small ligands is a central phenomenon involved in all biological processes. We demonstrate herein that the molecular recognition of a photochromic ligand, dihydroindolizine (DHI), by serine protease α-chymotrypsin (CHT) leads to the photo-control of enzymatic activity. We synthesized and optically characterized the photochromic DHI. Light-induced reversible pyrroline ring opening and a consequent thermal back reaction via 1,5-electrocyclization are responsible for the photochromic behavior. Furthermore, DHI inhibits the enzymatic activity of CHT in a photo-controlled manner. Simultaneous binding of the well-known inhibitors 4-nitrophenyl anthranilate (NPA) or proflavin (PF) in the presence of DHI displays spectral overlap between the emission of CHT-NPA or CHT-PF with the respective absorption of cis or trans DHI. The results suggest an opportunity to explore the binding site of DHI using Förster resonance energy transfer (FRET). Moreover, to more specifically evaluate the DHI binding interactions, we employed molecular docking calculations, which suggested binding near the hydrophobic site of Cys-1-Cys-122 residues. Variations in the electrostatic interactions of the two conformers of DHI adopt unfavorable conformations, leading to the allosteric inhibition of enzymatic activity.

  4. Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family.

    PubMed

    Park, K H; Kim, T J; Cheong, T K; Kim, J W; Oh, B H; Svensson, B

    2000-05-23

    Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.

  5. Effects of dissolved oxygen on glycolytic enzyme specific activities in liver and skeletal muscle of Fundulus heteroclitus.

    PubMed

    Abbaraju, Naga V; Rees, Bernard B

    2012-06-01

    Many aquatic habitats are characterized by variable concentrations of dissolved oxygen (DO), and fish that occur in these habitats respond to changes in DO through behavioral, physiological, and biochemical adjustments. The goal of the present study was to measure the effects of an ecologically relevant range of DO treatments, from severe hypoxia to moderate hyperoxia, on the maximal activities of nine glycolytic enzymes during chronic exposure of the mummichog, Fundulus heteroclitus. Over the 28 days of exposure period, specific activity was significantly affected by DO for three enzymes in liver and one enzyme in white skeletal muscle, although at specific times of exposure three other muscle enzymes were affected by DO. In general, exposure of fish to severe hypoxia led to higher specific activities in liver, but lower specific activities in skeletal muscle. Exposure to hyperoxia did not elicit changes in enzyme specific activities in either tissue. Surprisingly, exposure duration had strong effects on glycolytic enzyme specific activities in both liver and white skeletal muscle, with specific activities increasing with exposure duration regardless of DO treatment. The results demonstrate that the effects of DO on enzyme specific activities were restricted to a subset of the glycolytic enzymes in liver and white skeletal muscle of F. heteroclitus and that the directions of the changes were opposite in these two tissues. These observations suggest that the mechanisms resulting in these alterations are enzyme- and tissue specific, rather than applying uniformly to all enzymes within the glycolytic pathway.

  6. 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.

  7. The effect of enzyme and substrate levels on the specific hydrolysis rate of pretreated poplar wood

    SciTech Connect

    Nutor, J.R.K.; Converse, A.O.

    1991-12-31

    The hydrolysis of pretreated poplar wood was carried out with initial concentrations of 1.26, 2.52, 5.04 mg protein/mL of GC123 Trichoderma reesei cellulose and substrate concentrations of 2.5% w/v, 5% w/v, and 10% w/v at pH 4.8 and 40{degrees}C. The concentration of enzyme protein remaining in solution, the glucose concentration, and the total potential glucose concentrations were measured as a function of time during the hydrolysis. The enzyme rapidly adsorbed initially, reaching a maximum in about 30 min. About 55-75% of the cellulose returned to solution as the remaining cellulose was hydrolyzed. Dilution of the unhydrolyzed residue, largely lignin, did not cause additional desorption of the cellulose. The specific hydrolysis rate (i.e., the rate/amount of adsorbed enzyme) declined significantly with increased conversion, even when corrected for glucose inhibition. At a given initial substrate concentration, the specific rate was found to be largely independent of the total enzyme concentration. However, at a given fractional conversion, the specific rate was found to be reduced by increased substrate concentration.

  8. Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

    PubMed

    Litman, G W; Berger, L; Jahn, C L

    1982-06-11

    High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions.

  9. 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.

  10. 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).

  11. Between-habitat comparison of digestive enzymes activities and energy reserves in the SW Atlantic euryhaline burrowing crab Neohelice granulata.

    PubMed

    Pinoni, Silvina A; Iribarne, Oscar; Mañanes, Alejandra A López

    2011-04-01

    The digestive and metabolic characteristics at the biochemical level underlying between-habitat dietary shift of the SW Atlantic euryhaline burrowing crab Neohelice granulata under natural conditions are unknown. We made studies on adult males of N. granulata from the open mudflat and the vegetated saltmarsh in a SW Atlantic costal lagoon (Mar Chiquita, 37°32'-37°45'S; 57°19'-57°26'W, Argentina). We determined and compared amylase, maltase, sucrase, proteolytic, lipase and alkaline phosphatases activities in the hepatopancreas; glycemia, and glycogen, free glucose, triglycerides and protein concentrations in hepatopancreas, chela muscle, and anterior and posterior gills. The results show that N. granulata exhibits characteristics and between-habitat differences at the biochemical level (i.e. high amylase and disaccharidase activities, differences in total proteolytic, lipase and levamisole-insensitive AP activities in the hepatopancreas, and in the concentrations of glycogen in the gills, triglycerides in the hepatopancreas and of protein in the chela muscle) which could represent adaptive digestive and metabolic strategies to face the differences in environmental conditions (i.e. food availability). The possible relationship between digestive and metabolic characteristics and feeding patterns, type of food available and environmental conditions in each habitat is discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

    PubMed

    MacKenzie, C R; Henrich, B; Hadding, U

    1996-11-01

    The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.

  13. Defining host-symbiont collaboration in termite lignocellulose digestion

    PubMed Central

    Karl, Zachary J.; Sethi, Amit; Sen, Ruchira; Raychoudhury, Rhitoban; Boucias, Drion G.

    2011-01-01

    Termites have the unique ability to exploit lignocellulose as a primary nutrition source. Traditionally, termite lignocellulose digestion has been considered as a gut-symbiont-mediated process; however, in recent years the importance of host digestive capabilities have become apparent. Despite this growing understanding, how digestive enzymes from different origins specifically collaborate (i.e., additively or synergistically) has remained largely unknown. In a recent study, we undertook translational-genomic studies to address these questions in the lower termite Reticulitermes flavipes (Isoptera: Rhinotermitidae) and its symbiotic gut fauna. We used a combination of native gut tissue preparations and recombinant enzymes derived from the host gut transcriptome to identify synergistic collaborations between host and symbiont, and also among enzymes produced exclusively by the host termite. These findings provided important new evidence of synergistic collaboration among enzymes in the release of fermentable monosaccharides from wood lignocellulose, and laid a foundation for future integrative studies into termite digestion, symbiosis and eusociality. PMID:22446549

  14. Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet.

    PubMed

    Giraldo, L A; Tejido, M L; Ranilla, M J; Ramos, S; Carro, M D

    2008-07-01

    Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of BW(0.75). Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of NH(3)-N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction.

  15. 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

    2017-01-01

    Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of different 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.

  16. Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages.

    PubMed

    Di Raimo, Tania; Leopizzi, Martina; Mangino, Giorgio; Rocca, Carlo Della; Businaro, Rita; Longo, Lucia; Lo Vasco, Vincenza Rita

    2016-12-01

    Macrophages' phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs' expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes' mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes' expression and PLC proteins' presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

  17. 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.

  18. Chitinosanase: A fungal chitosan hydrolyzing enzyme with a new and unusually specific cleavage pattern.

    PubMed

    Kohlhoff, Markus; Niehues, Anna; Wattjes, Jasper; Bénéteau, Julie; Cord-Landwehr, Stefan; El Gueddari, Nour Eddine; Bernard, Frank; Rivera-Rodriguez, Gustavo R; Moerschbacher, Bruno M

    2017-10-15

    The biological activities of partially acetylated chitosan oligosaccharides (paCOS) depend on their degree of polymerization (DP), fraction of acetylation (FA), and potentially their pattern of acetylation (PA). Therefore, analyzing structure-function relationships require fully defined paCOS, but these are currently unavailable. A promising approach for obtaining at least partially defined paCOS is using chitosanolytic enzymes. Here we purified and characterized a novel chitosan-hydrolyzing enzyme from the fungus Alternaria alternata possessing an absolute cleavage specificity, yielding fully defined paCOS. It cleaves specifically after GlcN-GlcNAc pairs and is most active towards moderately acetylated chitosans, but shows no activity against fully acetylated or fully deacetylated substrates. These unique properties match neither those of chitinases nor chitosanases. Therefore, the enzyme represents the first member of a new class of chitosanolytic enzymes that will allow for the production of fully defined paCOS. Additionally, it represents a highly valuable tool for fingerprinting analyses of chitosan polymers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. 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

  20. Developmental changes in digestive physiology of nestling house sparrows, Passer domesticus.

    PubMed

    Caviedes-Vidal, E; Karasov, W H

    2001-01-01

    Six decades of studies have speculated that digestive capacity might limit avian growth rate or that developmental changes in the gut might determine developmental changes in digestive efficiency. However, there are no studies on digestive enzymes during avian development, except for studies on mainly domestic birds that exhibit the precocial mode of development. We studied alimentary organ masses, intestinal enzyme activities (sucrase, maltase, isomaltase, aminopeptidase-N), and pancreatic enzyme activities (amylase, trypsin, chymotrypsin) during development of a wild passerine bird exhibiting the altricial mode of development. Wild nestling house sparrows were studied immediately after removal from the nest (days 0, 3, 6 of age; day 0=hatch), whereas captives were raised in the laboratory beginning day 3 on a formulated casein/starch-based diet until fledging age (after day 12). Digestive biochemistry was dynamic. Tissue-specific activities of some digestive enzymes continued to increase through fledging, by >10 times in some cases (e.g., sucrase and maltase in midintestine). Total pancreatic amylase activity increased 100 times between hatch and day 12 through a combination of increases in tissue-specific activity and pancreas mass. House sparrows differ from poultry, in whom after about 2 wk of age the specific activity of intestinal and pancreatic digestive enzymes is generally constant or declines during development. The data on intestinal and pancreatic enzymes help explain why digestive efficiency of nestling house sparrows improves with age, and the data seem consistent with the idea that digestive capacity might limit feeding rate and hence growth rate.

  1. Effects of dietary amylose/amylopectin ratio on growth performance, feed utilization, digestive enzymes, and postprandial metabolic responses in juvenile obscure puffer Takifugu obscurus.

    PubMed

    Liu, Xiang-he; Ye, Chao-xia; Ye, Ji-dan; Shen, Bi-duan; Wang, Chun-yan; Wang, An-li

    2014-10-01

    The effect of dietary amylose/amylopectin (AM/AP) ratio on growth, feed utilization, digestive enzyme activities, plasma parameters, and postprandial blood glucose responses was evaluated in juvenile obscure puffer, Takifugu obscurus. Five isonitrogenous (430 g kg(-1) crude protein) and isolipidic (90 g kg(-1) crude lipid) diets containing an equal starch level (250 g kg(-1) starch) with different AM/AP ratio diets of 0/25, 3/22, 6/19, 9/16 and 12/13 were formulated. Each experimental diet was fed to triplicate groups (25 fish per tank), twice daily during a period of 60 days. After the growth trial, a postprandial blood response test was carried out. Fish fed diet 6/19 showed best growth, feed efficiency and protein efficiency ratio. Hepatosomatic index, plasma total cholesterol concentration, liver glycogen and lipid content, and gluconokinase, pyruvate kinase and fructose-1,6-bisphosphatase activities were lower in fish fed highest AM/AP diet (12/13) than in fish fed the low-amylose diets. Activities of liver and intestinal trypsin in fish fed diet 3/22 and diet 6/19 were higher than in fish fed diet 9/16 and diet 12/13. Activities of liver and intestinal amylase and intestinal lipase, and starch digestibility were negatively correlated with dietary AM/AP ratio. Fish fed diet 3/22 and diet 6/19 showed higher plasma total amino acid concentration than fish fed the other diets, while plasma urea nitrogen concentration and activities of alanine aminotransferase and aspartate aminotransferase showed the opposite trend. Equal values were found for viscerosomatic index and condition factor, whole body and muscle composition, plasma high-density and low-density lipoprotein cholesterol concentrations, and activities of lipase and hexokinase and glucose-6-phosphatase in liver. Postprandial plasma glucose and triglyceride peak value of fish fed diet 12/13 were lower than in fish fed the low-amylose diets, and the peak time of plasma glucose was later than in fish fed the

  2. Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems.

    PubMed

    Parales, R E; Emig, M D; Lynch, N A; Gibson, D T

    1998-05-01

    Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.

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

    PubMed

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

    2016-01-07

    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.

  4. Substrate Specificities of Hybrid Naphthalene and 2,4-Dinitrotoluene Dioxygenase Enzyme Systems

    PubMed Central

    Parales, Rebecca E.; Emig, Matthew D.; Lynch, Nancy A.; Gibson, David T.

    1998-01-01

    Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (α and β). To assess the contributions of the α and β subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different β subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes. PMID:9573183

  5. 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

  6. 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.

  7. 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

  8. 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

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

    PubMed

    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.

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

    PubMed

    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.

  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 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

  13. 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

    2013-11-05

    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.

  14. Demonstration of specific neuronal cell groups in rat brain by beta-galactosidase enzyme histochemistry.

    PubMed

    Hatton, J D; Lin, L

    1992-12-01

    beta-Galactosidase activity as illuminated by the indigogenic X-gal staining method has been used to demonstrate the presence of genetically modified cells carrying the reporter gene lacZ, coding for the E. coli enzyme. Endogenous activity has been assumed to be minimal since the pH optimum for the mammalian enzyme is 3.5-5.5, while the pH optimum for the E. coli enzyme (and thus of the staining procedure usually employed) is 7.3. Background staining has been reported to be limited to pericytes and a few specific neuronal cell groups. In contrast, our investigations of normal rat brain anatomy demonstrate that many specific neuronal cell groups possess endogenous beta-galactosidase activity when staining is performed at physiological pH. This suggests that background staining of endogenous beta-galactosidase activity in the rat brain has been underestimated. In addition, such specific activity would afford an additional means of identification and illustration of these cells.

  15. Specific xyloglucanases as a new class of polysaccharide-degrading enzymes.

    PubMed

    Grishutin, Sergey G; Gusakov, Alexander V; Markov, Alexander V; Ustinov, Boris B; Semenova, Margarita V; Sinitsyn, Arkady P

    2004-11-01

    Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes