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Sample records for wall degrading enzymes

  1. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides

    NARCIS (Netherlands)

    Vries, de R.P.; Visser, J.

    2001-01-01

    Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a m

  2. Effects of processing technologies combined with cell wall degrading enzymes on in vitro degradability of barley.

    Science.gov (United States)

    de Vries, S; Pustjens, A M; Schols, H A; Hendriks, W H; Gerrits, W J J

    2012-12-01

    Effects of processing technologies and cell wall degrading enzymes on in vitro degradation of barley were tested in a 5 × 2 factorial arrangement: 5 technologies (unprocessed, wet-milling, extrusion, autoclaving, and acid-autoclaving), with or without enzymes. Upper gastrointestinal tract digestion (Boisen incubation) and large intestinal fermentation (gas production technique) were simulated in duplicate. All technologies increased digestion of DM (13 to 43% units) and starch (22 to 51% units) during Boisen incubation, compared with the unprocessed control (P starch (≈ 20% units), and CP (≈ 10% units) in unprocessed and autoclaved barley (P starch present in the Boisen residues. In conclusion, wet-milling, extrusion, and acid-autoclaving improved in vitro starch and CP digestion in barley, which is related to the cell wall matrix disruption. Addition of xylanases and β-glucanases improved in vitro starch and CP digestion only in unprocessed barley or barley poorly affected by processing.

  3. Cell wall degrading enzymes in Trichoderma asperellum grown on wheat bran

    DEFF Research Database (Denmark)

    Bech, Lasse; Busk, Peter Kamp; Lange, Lene

    2015-01-01

    Trichoderma asperellum is a filamentous fungus that is able to produce and secrete a wide range of extracellular hydrolytic enzymes used for plant cell wall degradation. The Trichoderma genus has attracted considerable attention from the biorefinery industry due to the production of cell wall...... degrading enzymes and strong secretion ability of this genus. Here we report extensive transcriptome analysis of plant cell wall degrading enzymes in T. asperellum. The production of cell wall degrading enzymes by T. asperellum was tested on a range of cellulosic materials under various conditions. When T...... the theory that the glycoside hydrolases have evolved from a common ancestor, followed by a specialization in which saprotrophic fungi such as T. reesei and T. longibrachiatum lost a significant number of genes including several glycoside hydrolases....

  4. Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

    Science.gov (United States)

    Discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly c...

  5. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi.

    Science.gov (United States)

    Kubicek, Christian P; Starr, Trevor L; Glass, N Louise

    2014-01-01

    Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.

  6. An efficient treatment for detoxification process of cassava starch by plant cell wall-degrading enzymes.

    Science.gov (United States)

    Sornyotha, Somphit; Kyu, Khin Lay; Ratanakhanokchai, Khanok

    2010-01-01

    The objective of this work was to remove linamarin in starch from cassava (Manihot esculenta Crantz cv. KU-50) roots, a high-cyanogen variety by using plant cell wall-degrading enzymes, xylanase and cellulase. The combination of xylanase from Bacillus firmus K-1 and xylanase and cellulase from Paenibacillus curdlanolyticus B-6 at the ratio of 1:9 showed the maximum synergism at 1.8 times for hydrolyzing cassava cortex cell walls and releasing linamarase. Combined enzyme treatment enhanced linamarin liberation from the parenchyma by 90%. In addition, when the combined enzymes were applied for detoxification during cassava starch production, a low-cyanide-product was obtained with decreased linamarin concentration (96%) compared to non-enzyme treated tissues. Based on these results, xylanase and cellulase treatment is a good method for low-cyanide-cassava starch production and could be applied for detoxification of cassava products during processing.

  7. Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

    Directory of Open Access Journals (Sweden)

    Bergstrom Gary C

    2011-02-01

    Full Text Available Abstract Background The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides. Results Large-scale screening identified a range of hydrolytic activities among 348 unique isolates representing 156 species of plant pathogenic and non-pathogenic fungi. Hierarchical clustering was used to identify groups of species with similar hydrolytic profiles. Among moderately and highly active species, plant pathogenic species were found to be more active than non-pathogens on six of eight substrates tested, with no significant difference seen on the other two substrates. Among the pathogenic fungi, greater hydrolysis was seen when they were tested on biomass and hemicellulose derived from their host plants (commelinoid monocot or dicot. Although T. reesei has a hydrolytic profile that is highly active on cellulose and pretreated biomass, it was less active than some natural isolates of fungi when tested on xylans and untreated biomass. Conclusions Several highly active isolates of plant pathogenic fungi were identified, particularly when tested on xylans and untreated biomass. There were statistically significant preferences for biomass type reflecting the monocot or dicot host preference of the

  8. Role of cell wall degrading enzymes in the interaction of poplar and Melampsora larici-populina Kleb.

    Institute of Scientific and Technical Information of China (English)

    Chengming TIAN; Peng ZHAO; Zhimin CAO

    2009-01-01

    The activity of cell wall-degrading enzymes,produced in poplar cultivars infected Melampsora larici-populina Kleb., was studied. The results show that PMG,PMTE, Cx and fl-glucosidase played roles during the infection. After inoculation, the activity of PMG in both susceptible and resistant cultivars had two peak values in 2 dpi and 5 dpi. The activities of PMTE and β-glucosidase had a peak value in 3 dpi, and Cx in 2 dpi. Among these cell wall-degrading enzymes, the activities of PMG and PMTE were higher and the activities of Cx and β-glucosidase were relatively lower. The activities of these cell wall-degrading enzymes were significantly higher in susceptible cultivars than those in resistant cultivars. All these demonstrated that these cell wall-degrading enzymes played certain roles in the infection ofM. larici-populina.

  9. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

    Science.gov (United States)

    Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

    2012-01-01

    A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

  10. ASSOCIATION BETWEEN SPORULATION AND CELL-WALL DEGRADING ENZYMES IN THE WHEAT PATHOGEN MYCOSPHAERELLA GRAMINICOLA.

    Science.gov (United States)

    Ors, M; Siah, A; Randoux, B; Selim, S; Couleaud, G; Maumene, C; Reignault, Ph; Halama, P

    2015-01-01

    Mycosphaerella graminicola is a hemibiotrophic fungus that causes Septoria tritici blotch (STB), one of the most serious foliar diseases of wheat. STB can occur with a wide range of disease levels on the host, which depend not only on the pathogenicity of fungal strains, but also on the resistance of host cultivars. Here, we investigated the association between the disease level and fungal cell-wall degrading enzyme and protease activities in three wheat cultivars differing in their resistance levels against M. graminicola. The experiments were carried out in the greenhouse using artificial inoculations with the M. graminicola strain T01193. Disease symptoms scored at 21-day post-inoculation (dpi) were significantly higher on the susceptible and moderately resistant cultivars, Alixan and Premio (48% and 42% of diseased leaf area, respectively), than in the resistant one, Altigo (28% of diseased leaf area). Regarding sporulation, the rate of pycnidial density was significantly higher on Alixan (2.9) compared to Premio and Altigo (1.1 and 1.0, respectively). Further biochemical investigations revealed, by 17 dpi, significant fungal beta-1,4-endoxylanase, beta-1,4-endoglucanase and protease activities, whose amounts increased according to the pycnidial density recorded on the infected leaves. At 21 dpi, the amounts of these activities were significantly higher on Alixan compared to Premio and Altigo (0.36 U/mg, 0.63 U/mg and 2.70 mU/mg total proteins on Alixan, 0.09 U/mg, 0.19 U/mg and 0.72 mU/mg total proteins on Premio and 0.05 U/mg, 0.15 U/mg and 0.52 mU/mg total proteins on Altigo for beta-1,4-endoxylanase, beta-1,4-endoglucanase and protease activities, respectively). These results confirm the importance of CWDE and protease activities in the process of fungal sporulation during the necrotrophic phase of M. graminicola.

  11. Adaptive expression of host cell wall degrading enzymes in fungal disease: an example from Fusarium root rot of medicinal Coleus.

    Science.gov (United States)

    Bhattacharya, A

    2013-12-15

    Quantity of extracellular proteins and activities two cell wall degrading enzymes pectinase and cellulase were determined in the culture filtrate of Fusarium solani, the causal organism of root rot of Coleus forskohlii. Substitution of carbon source in the medium with either pectin or carboxymethyl cellulose led to the increased production of extracellular proteins by the fungus. Pectinase and cellulase activity in the culture filtrate was detected only when the growth medium contained substituted carbon source in the form of pectin and CMC, respectively. Pectinase activity was highest after 5 days incubation and then decreased gradually with time but cellulase activity showed a steady time dependent increase. In vitro virulence study showed the requirement of both the enzymes for complete expression of rot symptoms on Coleus plants. Thus the present study established the adaptive, substrate dependent expression of the two enzymes by the fungus and also their involvement in the root rot disease of Coleus forskohlii.

  12. Synergistic effect of different plant cell wall degrading enzymes is important for virulence of Fusarium graminearum.

    Science.gov (United States)

    Paccanaro, Maria Chiara; Sella, Luca; Castiglioni, Carla; Giacomello, Francesca; Martinez-Rocha, Ana Lilia; D'Ovidio, Renato; Schäfer, Wilhelm; Favaron, Francesco

    2017-08-11

    Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, whilst their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single and double disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared to wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild-type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG and cellulase activities, but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

  13. Carbohydrate-active enzymes in pythium and their role in plant cell wall and storage polysaccharide degradation.

    Directory of Open Access Journals (Sweden)

    Marcelo M Zerillo

    Full Text Available Carbohydrate-active enzymes (CAZymes are involved in the metabolism of glycoconjugates, oligosaccharides, and polysaccharides and, in the case of plant pathogens, in the degradation of the host cell wall and storage compounds. We performed an in silico analysis of CAZymes predicted from the genomes of seven Pythium species (Py. aphanidermatum, Py. arrhenomanes, Py. irregulare, Py. iwayamai, Py. ultimum var. ultimum, Py. ultimum var. sporangiiferum and Py. vexans using the "CAZymes Analysis Toolkit" and "Database for Automated Carbohydrate-active Enzyme Annotation" and compared them to previously published oomycete genomes. Growth of Pythium spp. was assessed in a minimal medium containing selected carbon sources that are usually present in plants. The in silico analyses, coupled with our in vitro growth assays, suggest that most of the predicted CAZymes are involved in the metabolism of the oomycete cell wall with starch and sucrose serving as the main carbohydrate sources for growth of these plant pathogens. The genomes of Pythium spp. also encode pectinases and cellulases that facilitate degradation of the plant cell wall and are important in hyphal penetration; however, the species examined in this study lack the requisite genes for the complete saccharification of these carbohydrates for use as a carbon source. Genes encoding for xylan, xyloglucan, (galacto(glucomannan and cutin degradation were absent or infrequent in Pythium spp.. Comparative analyses of predicted CAZymes in oomycetes indicated distinct evolutionary histories. Furthermore, CAZyme gene families among Pythium spp. were not uniformly distributed in the genomes, suggesting independent gene loss events, reflective of the polyphyletic relationships among some of the species.

  14. Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

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    Margret E Berg Miller

    Full Text Available BACKGROUND: Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. METHODOLOGY/PRINCIPAL FINDINGS: The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb, and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs, polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs. Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315 exhibited the highest levels of up-regulation. CONCLUSIONS/SIGNIFICANCE: The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional

  15. The snf1 gene of Ustilago maydis acts as a dual regulator of cell wall degrading enzymes.

    Science.gov (United States)

    Nadal, Marina; Garcia-Pedrajas, Maria D; Gold, Scott E

    2010-12-01

    Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.

  16. Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, Kátia F., E-mail: katia@icb.ufg.br [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Cortijo-Triviño, David [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Batista, Karla A.; Ulhoa, Cirano J. [Departamento de Bioquímica e Biologia Molecular, Instituo de Ciências Biológicas, Universidade Federal de Goiás, Cx. Postal 131, 74001-970 Goiânia, GO (Brazil); García-Ruiz, Pedro A. [Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain)

    2013-07-01

    In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na{sub 2}SO{sub 4}. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na{sub 2}SO{sub 4} was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO–TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO–TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO–TCWDE retained 100% activity after 3 h incubation at 55 °C. TCNSO–TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity. - Highlights: • TCWDE immobilized on TCNSO, a support with highly hydrophobic character • New immobilization strategy for immobilization on a hydrophobic support • TCNSO–TCWDE were retained during washes and during incubation at 55 °C for 3 h.

  17. Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads.

    Science.gov (United States)

    Fernandes, Kátia F; Cortijo-Triviño, David; Batista, Karla A; Ulhoa, Cirano J; García-Ruiz, Pedro A

    2013-07-01

    In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na2SO4. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na2SO4 was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO-TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO-TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO-TCWDE retained 100% activity after 3h incubation at 55 °C. TCNSO-TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 μg of N-acetylglucosamine (NAG) maintaining 83% of initial activity.

  18. Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

    Directory of Open Access Journals (Sweden)

    Kirsch Roy

    2012-11-01

    Full Text Available Abstract Background The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs. The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH families: GH11 (xylanases, GH28 (polygalacturonases or pectinases, and GH45 (β-1,4-glucanases or cellulases. Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs families as well as polygalacturonase-inhibiting proteins (PGIPs were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome

  19. AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production.

    Science.gov (United States)

    Kõiv, Viia; Andresen, Liis; Mäe, Andres

    2010-06-01

    Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239-246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.

  20. PLASMALEMMA PATCH CLAMP EXPERIMENTS IN PLANT-ROOT CELLS - PROCEDURE FOR FAST ISOLATION OF PROTOPLASTS WITH MINIMAL EXPOSURE TO CELL-WALL DEGRADING ENZYMES

    NARCIS (Netherlands)

    VOGELZANG, SA; PRINS, HBA

    1992-01-01

    A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments. was developed for root cells of tomato (Lycopersicon esculentum and Plantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme

  1. Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

    Institute of Scientific and Technical Information of China (English)

    LIU Mei (刘梅); SUN Zong-xiu (孙宗修); ZHU Jie (朱洁); XU Tong (徐同); HARMAN Gary E.; LORITO Matteo

    2004-01-01

    Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

  2. Effect of combined cell wall degrading enzyme treatment on the total dissolved solids and sugars of soymilk

    Directory of Open Access Journals (Sweden)

    Chijioke Maduka OSUJI

    2013-08-01

    Full Text Available Soymilk from different varieties of soybean was treated with combinations of cell wall hydrolyzing enzymes (glucanase, cellulose, arabanase, hemicellulase and xylanase. Treated samples and control were evaluated for Total Dissolved Solids (TDS and different sugars (glucose, raffinose, sucrose, fructose, xylose, maltose, lactose, stachyose, starch, galactose, cellulose using HPLC. Mean TDS of all enzyme-treated soymilk samples (235.8-268.3 ppm was significantly (p≤0.05 higher than the control (167.8 ppm, it also increased significantly (p≤0.05 after sterilization. Sugars present in the enzyme-hydrolyzed soymilk were significantly (p≤0.05 different from the control. Sucrose content was depleted after enzyme treatment. The change in content of glucose, xylose, fructose, maltose, raffinose, starchyose had high correlation with TDS. Possible chemical modification of sugars impaired their detection despite increases in TDS. Use of TDS for rapid monitoring of enzyme hydrolyses of soymilk cell-wall sugars is feasible.

  3. Plant Wall Degradative Compounds and Systems

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The present invention relates to cell wall degradative systems, in particular to systems containing enzymes that bind to and/or depolymerize cellulose. These systems...

  4. In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina, the causative agent of charcoal rot in corn.

    Science.gov (United States)

    Ramos, Araceli M; Gally, Marcela; Szapiro, Gala; Itzcovich, Tatiana; Carabajal, Maira; Levin, Laura

    Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  5. Regulation of three genes encoding cell-wall-degrading enzymes of Trichoderma aggressivum during interaction with Agaricus bisporus.

    Science.gov (United States)

    Abubaker, Kamal S; Sjaarda, Calvin; Castle, Alan J

    2013-06-01

    Members of the genus Trichoderma are very effective competitors of a variety of fungi. Cell-wall-degrading enzymes, including proteinases, glucanases, and chitinases, are commonly secreted as part of the competitive process. Trichoderma aggressivum is the causative agent of green mould disease of the button mushroom, Agaricus bisporus. The structures of 3 T. aggressivum genes, prb1 encoding a proteinase, ech42 encoding an endochitinase, and a β-glucanase gene, were determined. Promoter elements in the prb1 and ech42 genes suggested that transcription is regulated by carbon and nitrogen levels and by stress. Both genes had mycoparasitism-related elements indicating potential roles for the protein products in competition. The promoter of the β-glucanase gene contained CreA and AreA binding sites indicative of catabolite regulation but contained no mycoparasitism elements. Transcription of the 3 genes was measured in mixed cultures of T. aggressivum and A. bisporus. Two A. bisporus strains, U1, which is sensitive to green mould disease, and SB65, which shows some resistance, were used in co-cultivation tests to assess possible roles of the genes in disease production and severity. prb1 and ech42 were coordinately upregulated after 5 days, whereas β-glucanase transcription was upregulated from day 0 with both Agaricus strains. Upregulation was much less pronounced in mixed cultures of T. aggressivum with the resistant strain, SB65, than with the sensitive strain, U1. These observations suggested that the proteins encoded by these genes have roles in both nutrition and in severity of green mould disease.

  6. Nutritive values of corn, soybean meal, canola meal, and peas for broiler chickens as affected by a multicarbohydrase preparation of cell wall degrading enzymes.

    Science.gov (United States)

    Meng, X; Slominski, B A

    2005-08-01

    The effect of a new multicarbohydrase supplement of cell wall degrading activities on the nutritive value of corn, soybean meal (SBM), canola meal (CM), and peas for broiler chickens was investigated. Four isoenergetic and isonitrogenous corn (69% corn), SBM (30% SBM, 59% corn), CM (30% CM, 54% corn), and pea (30% peas, 52% corn) diets, without or with enzyme supplementation, were formulated to meet NRC specifications for broiler chickens (except for AME and CP, which were at 95 and 92% of NRC requirements, respectively). The enzyme supplement supplied 1,000 U of xylanase, 400 U of glucanase, 1,000 U of pectinase, 120 U of cellulase, 280 U of mannanase, and 180 U of galactanase per kilogram of diet. Each diet was fed in a mash form to 9 replicate pens of 5 broilers from 5 to 18 d. When compared with the control treatment, enzyme addition to the corn diet improved (P enzyme-supplemented corn diet. An improvement (P enzyme supplementation was observed for the SBM diet. However, nutrient digestibilities and AMEn of CM and pea diets were not affected (P > 0.05) by enzyme addition even though the NSP digestibilities increased significantly (P enzyme-supplemented diets. It would appear from this study that the nutrient utilization of corn-SBM diet by broilers could be enhanced by using an appropriate multicarbohydrase enzyme supplement. The nutrient encapsulating effect of cell wall polysaccharides in SBM, CM, and peas may not be the only factor responsible for incomplete nutrient utilization. The improvement in feed efficiency and starch availability in birds fed corn diet likely resulted from the cell wall degrading activity of the enzyme supplement.

  7. Suppression of Cell Wall Degrading Enzymes and their Encoding Genes in Button Mushrooms (Agaricus bisporus) by CaCl2 and Citric Acid.

    Science.gov (United States)

    Khan, Zia Ullah; Jiayin, Li; Khan, Nasir Mehmood; Mou, Wangshu; Li, Dongdong; Wang, Yansheng; Feng, Simin; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

    2017-03-01

    Fresh button mushrooms (Agaricus bisporus) were harvested and treated with a solution of 1.5% CaCl2 + 0.5% citric acid and stored for 16 days at 12 °C. The effects of this treatment on firmness, weight, color, cell wall compositions (cellulose and chitin) and cell wall degrading enzymes (cel1ulase, beta-1, 3 glucanase, chitinase and phenylalanine ammonialyase) were investigated during post-harvest storage. The expressions of major genes (Cel1, Glu1, Chi1 and PAL1) involved in cell wall degradation during post-harvest storage were also monitored. The results revealed that the post-harvest chemical treatment maintained better firmness, weight, color and inhibited cellulase, beta-1, 3 glucanase, chitinase and phenylalanine ammonialyase activities. These findings showed that the down-regulation of cell wall degrading enzymes is a possible mechanism that delays the softening of button mushrooms by the application of combined chemical treatment.

  8. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

    Science.gov (United States)

    Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

    2015-01-01

    Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

  9. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

    Directory of Open Access Journals (Sweden)

    Paolo Longoni

    2015-01-01

    Full Text Available Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

  10. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels.

    Science.gov (United States)

    Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad

    2014-01-01

    Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

  11. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

    Directory of Open Access Journals (Sweden)

    Shazia Rehman

    2014-12-01

    Full Text Available Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE, in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase using a novel substrate, Banana Peels (BP for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

  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.

    Science.gov (United States)

    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. The use of plant cell wall-degrading enzymes from newly isolated Penicillium ochrochloron Biourge for viscosity reduction in ethanol production with fresh sweet potato tubers as feedstock.

    Science.gov (United States)

    Huang, Yuhong; Jin, Yanling; Shen, Weiliang; Fang, Yang; Zhang, Guohua; Zhao, Hai

    2014-01-01

    Penicillium ochrochloron Biourge, which was isolated from rotten sweet potato, can produce plant cell wall-degrading enzymes (PCWDEs) with high viscosity reducing capability for ethanol production using fresh sweet potato tubers as feedstock. The enzyme preparation was characterized by a broad enzyme spectrum including 13 kinds of enzymes with the activity to hydrolyze cellulose, hemicellulose, pectin, starch, and protein. The maximum viscosity-reducing capability was observed when the enzyme preparation was obtained after 5 days of fermentation using 20 g/L corncob as a sole carbon source, 4.5 g/L NH4 NO3 as a sole nitrogen source, and an initial medium pH of 6.5. The sweet potato mash treated with the enzyme preparation exhibited much higher fermentation efficiency (92.58%) compared with commercial cellulase (88.06%) and control (83.5%). The enzyme production was then scaled up to 0.5, 5, and 100 L, and the viscosity-reducing rates were found to be 85%, 90%, and 91%, respectively. Thus, P. ochrochloron Biourge displays potential viscosity-reducing capability for ethanol production.

  14. RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

    Science.gov (United States)

    Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R

    2015-01-01

    RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1

  15. Abeta-degrading enzymes in Alzheimer's disease.

    Science.gov (United States)

    Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth

    2008-04-01

    In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.

  16. Production of cellulose- and xylan-degrading enzymes by a koji mold, aspergillus oryzae, and their contribution to the maceration of rice endosperm cell wall.

    Science.gov (United States)

    Yamane, Yu-Ichi; Fujita, Jin; Shimizu, Ryu-Ichi; Hiyoshi, Akira; Fukuda, Hisashi; Kizaki, Yasuzo; Wakabayashi, Saburo

    2002-01-01

    The production of cellulose- (CEL), xylan- (XYL), and pectin-degrading enzymes (PEC) by a koji mold, Aspergillus oryzae, was studied, and their contributions to the maceration of the rice endosperm cell wall were investigated with regard to the utilization of available rice in the sake mash. The sake koji mold showed higher CEL and XYL productivities, whereas the miso and soy sauce koji molds showed higher PEC productivity. Statistical analyses indicated that CEL and XYL contribute predominantly and synergistically to the maceration of the rice endosperm cell wall. A. oryzae produced at least three kinds of CEL (Cel-1, 2, 3) and two kinds of XYL (Xyl-1, 2) when cultured in a wheat bran medium. In the solid-state culture, the production of Cel-3 and Xyl-2 was markedly stimulated by decreasing the moisture content of the solid substrate, although the production levels of Cel-1 and Xyl-1 were almost the same. These data suggest that the production of Cel-3 and Xyl-2 is strongly influenced by culture conditions, and that water activity is one of the dominant factors in the regulation of their production.

  17. An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

    Science.gov (United States)

    King, Brian C; Donnelly, Marie K; Bergstrom, Gary C; Walker, Larry P; Gibson, Donna M

    2009-03-01

    Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.

  18. Search for cell-wall-degrading enzymes of world-wide rice grains by PCR and their effects on the palatability of rice.

    Science.gov (United States)

    Nakamura, Sumiko; Machida, Keisuke; Ohtsubo, Ken'ichi

    2012-01-01

    Such rice cultivars as Japonica, Japonica-Indica hybrid, Javanica and Indica, were evaluated for their main chemical components (amylose content and protein content), pasting property of rice flour (consistency), physical property of the cooked rice grains (adhesion, L3), and enzyme activities (cellulase and xylanase). The amylose content, cellulase activity and xylanase activity showed significant positive or negative correlation with the pasting property (consistency) of rice flour (r = 0.89, r = 0.58, r = 0.70, respectively) and with the physical property of the cooked rice grains (adhesion, L3: r = -0.51, r = -0.61, r = -0.71, respectively) at the level of 1%. Endogenous xylanase and cellulase played important roles to determine the texture of the cooked rice grains similarly to the amylose content. Part of the DNA sequences of the α-glucosidase gene differed among the Japonica, Japonica-Indica hybrid and Indica subspecies. We found discriminative DNA bands appearing by PCR, corresponding to 1,4-β-xylanase and endo-1,4-β-glucanase 13 in the case of Indica rice, Indica-Japonica hybrid rice, and Javanica rice (non-Japonica subspecies). The equation for estimating the physical property (adhesion) of cooked rice grains by PCR was improved by adding novel primers related to the cell-wall-degrading enzymes.

  19. An Optimized Microplate Assay System for Quantitative Evaluation of Plant Cell Wall Degrading Enzyme Activity of Fungal Culture Extracts

    Science.gov (United States)

    Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospe...

  20. Novel Enzymes for Targeted Hydrolysis of Algal Cell Walls

    DEFF Research Database (Denmark)

    Schultz-Johansen, Mikkel

    are incapable of breaking the complex polysaccharides found in seaweed cell walls. Therefore, new enzymes are needed for degradation of seaweed biomass. Bacteria that colonize the surfaces of seaweed secrete enzymes that allow them to degrade and utilize seaweed polysaccharides as energy. In addition, sea...... urchins are known algae-eaters and may therefore be inhabited by endosymbiotic bacteria that help in degradation of algal cell wall constituents. This thesis work investigated bacteria associated with seaweed, seagrass and sea urchins for their enzymatic activities against algal cell wall polysaccharides...

  1. Action of Multiple Cell Wall-Degrading Enzymes Is Required for Elicitation of Innate Immune Responses During Xanthomonas oryzae pv. oryzae Infection in Rice.

    Science.gov (United States)

    Tayi, Lavanya; Maku, Roshan; Patel, Hitendra Kumar; Sonti, Ramesh V

    2016-08-01

    Xanthomonas oryzae pv. oryzae secretes a number of plant cell wall-degrading enzymes (CWDEs) whose purified preparations induce defense responses in rice. These defense responses are suppressed by X. oryzae pv. oryzae using type 3 secretion system (T3SS) effectors and a type 3 secretion system mutant (T3SS(-)) of X. oryzae pv. oryzae is an inducer of rice defense responses. We assessed the role of individual CWDEs in induction of rice defense responses during infection, by mutating them in the genetic background of a T3SS(-). We mutated the genes for five different plant CWDEs secreted by X. oryzae pv. oryzae, including two cellulases (clsA and cbsA), one xylanase (xyn), one pectinase (pglA), and an esterase (lipA), singly in a T3SS(-) background. We have demonstrated that, as compared with a T3SS(-) of X. oryzae pv. oryzae, a cbsA(-)T3SS(-), a clsA(-)T3SS(-), and a xyn(-)T3SS(-) are deficient in induction of rice immune responses such as callose deposits and programmed cell death. In comparison, a lipA(-) T3SS(-) and a pglA(-)T3SS(-) is as efficient in induction of host defense responses as a T3SS(-). Overall, these results indicate that the collective action of X. oryzae pv. oryzae-secreted ClsA, CbsA, and Xyn proteins is required for induction of rice defense responses during infection.

  2. Controlled enzyme catalyzed heteropolysaccharide degradation

    DEFF Research Database (Denmark)

    Rasmussen, Louise Enggaard

    The work presented in this PhD thesis has provided a better understanding of the enzyme kinetics and quantitative phenomena of the hydrolysis of xylan substrates by selected pure enzyme preparations. Furthermore, the options for producing specific substituted xylooligosaccharides from selected...... substrates by specific xylanase treatment have been examined. The kinetics of the enzymatic degradation of water-extractable wheat arabinoxylan (WE-AX) during designed treatments with selected monocomponent enzymes was investigated by monitoring the release of xylose and arabinose. The results of different...... between -xylosidase and the α-L-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ~25 times in 30 minutes. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate...

  3. Rice transformation with cell wall degrading enzyme genes from Trichoderma atroviride and its effect on plant growth and resistance to fungal pathogens

    Institute of Scientific and Technical Information of China (English)

    Liu Mei; Sun Zong-Xiu; Zhu Jie; Xu Tong; Gary E Harman; Matteo Lorito; Sheri Woo

    2004-01-01

    @@ Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag70 and gluc78from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305. 2 singly and in all possible combinations. The coding sequences were placed downstream of the rice actin promoter and all vectors were used to transform rice plants. A total of more than 1,800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Expression in plant was obtained for all the fungal genes used singly or in combinations. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had a lesser effect. The expression level of endochitinase but not of the exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the positive effect of endochitinase on disease resistance when two genes were co-expressed in transgenic rice. Improved resistance to Magnaporthe grisea was found in all types of regenerated plants, including those with the gluc78 gene alone, while a few lines expressing either ech42 or nag70 appeared to be immune to this pathogen. Transgenic plants expressing the gluc78 gene alone were stunted and only few of them survived, even though they showed resistance to M. grisea. However, combination with either one of the two other genes ( ech42, nag70 ) as included in the same T-DNA region, reduced the negative effect of gluc78 on plant growth. This is the first report of single or multiple of expression of transgens encoding CWDEs that results in resistance to blast and sheath blight in rice.

  4. Short-Term Treatment with Cell Wall Degrading Enzymes Increases the Activity of the Inositol Phospholipid Kinases and the Vanadate-Sensitive ATPase of Carrot Cells 1

    Science.gov (United States)

    Chen, Qiuyun; Boss, Wendy F.

    1990-01-01

    Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [3H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed. Images Figure 2 Figure 6 PMID:16667922

  5. Short-term treatment with cell wall degrading enzymes increases the activity of the inositol phospholipid kinases and the vanadate-sensitive ATPase of carrot cells.

    Science.gov (United States)

    Chen, Q; Boss, W F

    1990-12-01

    Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [(3)H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed.

  6. Impact of cell wall-degrading enzymes on water-holding capacity and solubility of dietary fibre in rye and wheat bran.

    Science.gov (United States)

    Petersson, Karin; Nordlund, Emilia; Tornberg, Eva; Eliasson, Ann-Charlotte; Buchert, Johanna

    2013-03-15

    Rye and wheat bran were treated with several xylanases and endoglucanases, and the effects on physicochemical properties such as solubility, viscosity, water-holding capacity and particle size as well as the chemical composition of the soluble and insoluble fractions of the bran were studied. A large number of enzymes with well-defined activities were used. This enabled a comparison between enzymes of different origins and with different activities as well as a comparison between the effects of the enzymes on rye and wheat bran. The xylanases derived from Bacillus subtilis were the most effective in solubilising dietary fibre from wheat and rye bran. There was a tendency for a higher degree of degradation of the soluble or solubilised dietary fibre in rye bran than in wheat bran when treated with most of the enzymes. None of the enzymes increased the water-holding capacity of the bran or the viscosity of the aqueous phase. The content of insoluble material decreased as the dietary fibre was solubilised by the enzymes. The amount of material that may form a network to retain water in the system was thereby decreased. © 2012 Society of Chemical Industry.

  7. CELLULOSE DEGRADATION BY OXIDATIVE ENZYMES

    Directory of Open Access Journals (Sweden)

    Maria Dimarogona

    2012-09-01

    Full Text Available Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs, cellobiose dehydrogenases (CDHs and members of carbohydrate-binding module family 33 (CBM33. PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

  8. Cellulose degradation by oxidative enzymes

    Directory of Open Access Journals (Sweden)

    Maria Dimarogona

    2012-09-01

    Full Text Available Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs, cellobiose dehydrogenases (CDHs and members of carbohydrate-binding module family 33 (CBM33. PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

  9. The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae.

    Science.gov (United States)

    Molina-Hidalgo, Francisco J; Franco, Antonio R; Villatoro, Carmen; Medina-Puche, Laura; Mercado, José A; Hidalgo, Miguel A; Monfort, Amparo; Caballero, José Luis; Muñoz-Blanco, Juan; Blanco-Portales, Rosario

    2013-04-01

    Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

  10. On-Site Enzyme Production by Trichoderma asperellum for the Degradation of Duckweed

    DEFF Research Database (Denmark)

    Bech, Lasse; Herbst, Florian-Alexander; Grell, Morten Nedergaard

    2015-01-01

    The on-site production of cell wall degrading enzymes is an important strategy for the development of sustainable bio-refinery processes. This study concerns the optimization of production of plant cell wall-degrading enzymes produced by Trichoderma asperellum. A comparative secretome analysis...

  11. On-Site Enzyme Production by Trichoderma asperellum for the Degradation of Duckweed

    DEFF Research Database (Denmark)

    Bech, Lasse; Herbst, Florian-Alexander; Grell, Morten Nedergaard

    2015-01-01

    The on-site production of cell wall degrading enzymes is an important strategy for the development of sustainable bio-refinery processes. This study concerns the optimization of production of plant cell wall-degrading enzymes produced by Trichoderma asperellum. A comparative secretome analysis...

  12. Multiple functions of insulin-degrading enzyme

    DEFF Research Database (Denmark)

    Tundo, Grazia R; Sbardella, Diego; Ciaccio, Chiara

    2017-01-01

    Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled...

  13. Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture : Response to Cell Wall-Degrading Enzymes.

    Science.gov (United States)

    Tan, Z; Boss, W F

    1992-12-01

    Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation.

  14. Alfalfa stem tissues: Cell wall deposition, composition, and degradability

    NARCIS (Netherlands)

    Jung, H.G.; Engels, F.M.

    2002-01-01

    Declining cell wall degradability of alfalfa (Medicago sativa L.) stems with maturation limits the nutritional value of alfalfa for ruminants. This study characterized changes in cell wall concentration, composition, and degradability by rumen microbes resulting from alfalfa stem tissue proliferatio

  15. Alfalfa stem tissues: Cell wall deposition, composition, and degradability

    NARCIS (Netherlands)

    Jung, H.G.; Engels, F.M.

    2002-01-01

    Declining cell wall degradability of alfalfa (Medicago sativa L.) stems with maturation limits the nutritional value of alfalfa for ruminants. This study characterized changes in cell wall concentration, composition, and degradability by rumen microbes resulting from alfalfa stem tissue

  16. Alfalfa stem tissues: Cell wall deposition, composition, and degradability

    NARCIS (Netherlands)

    Jung, H.G.; Engels, F.M.

    2002-01-01

    Declining cell wall degradability of alfalfa (Medicago sativa L.) stems with maturation limits the nutritional value of alfalfa for ruminants. This study characterized changes in cell wall concentration, composition, and degradability by rumen microbes resulting from alfalfa stem tissue proliferatio

  17. Purification of highly chlorinated dioxins degrading enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Ishii, K.; Furuichi, T.; Koike, K.; Kuboshima, M. [Hokkaido Univ. (Japan). Division of Environment Resource Engineering, Graduate School of Engineering

    2004-09-15

    Soil contamination caused by dioxins in and around sites of incinerators for municipal solid waste (MSW) is a concern in Japan. For example, scattering wastewater from a wet gas scrubber at an MSW incinerator facility in Nose, Osaka caused soil and surface water contamination. The concentration of dioxins in the soil was about 8,000 pg-TEQ/g. Other contamination sites include soils on which fly ash has been placed directly or improperly stored and landfill sites that have received bottom and fly ash over a long period. Some countermeasures are required immediately at these dioxins-contaminated sites. We have previously developed bioreactor systems for dioxin-contaminated water and soil. We have shown that a fungus, Pseudallescheria boydii (P. boydii), isolated from activated sludge treating wastewater that contained dioxins, has the ability to degrade highly chlorinated dioxins. A reaction product of octachlorinated dibenzo-p-dioxin (OCDD) was identified as heptachlorinated dibenzo-p-dioxin. Therefore, one of the pathways for degradation of OCDD by this fungus was predicted to be as follows: OCDD is transformed by dechlorination and then one of the remaining aromatic rings is oxidized. To apply P. boydii to on-site technologies (e.g., bioreactor systems), as well as in situ technologies, enzyme treatment using a dioxin-degrading enzyme from P. boydii needs to be developed because P. boydii is a weak pathogenic fungus, known to cause opportunistic infection. As a result, we have studied enzyme purification of nonchlorinated dioxin, namely, dibenzo-pdioxin (DD). However, we did not try to identify enzymes capable of degrading highly chlorinated dioxins. This study has elucidated a method of enzyme assay for measuring OCDD-degrading activity, and has attempted to purify OCDD-degrading enzymes from P. boydii using enzyme assay. In addition, as first step toward purifying 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,7,8-TCDD degradation tests were carried out

  18. Redox regulation of insulin degradation by insulin-degrading enzyme.

    Directory of Open Access Journals (Sweden)

    Crystal M Cordes

    Full Text Available Insulin-degrading enzyme (IDE is a thiol sensitive peptidase that degrades insulin and amyloid β, and has been linked to type 2 diabetes mellitus and Alzheimer's disease. We examined the thiol sensitivity of IDE using S-nitrosoglutathione, reduced glutathione, and oxidized glutathione to distinguish the effects of nitric oxide from that of the redox state. The in vitro activity of IDE was studied using either partially purified cytosolic enzyme from male Sprague-Dawley rats, or purified rat recombinant enzyme. We confirm that nitric oxide inhibits the degrading activity of IDE, and that it affects proteasome activity through this interaction with IDE, but does not affect the proteasome directly. Oxidized glutathione inhibits IDE through glutathionylation, which was reversible by dithiothreitol but not by ascorbic acid. Reduced glutathione had no effect on IDE, but reacted with partially degraded insulin to disrupt its disulfide bonds and accelerate its breakdown to trichloroacetic acid soluble fragments. Our results demonstrate the sensitivity of insulin degradation by IDE to the redox environment and suggest another mechanism by which the cell's oxidation state may contribute to the development of, and the link between, type 2 diabetes and Alzheimer's disease.

  19. Cell wall degradation is required for normal starch mobilisation in barley endosperm.

    Science.gov (United States)

    Andriotis, Vasilios M E; Rejzek, Martin; Barclay, Elaine; Rugen, Michael D; Field, Robert A; Smith, Alison M

    2016-09-13

    Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.

  20. Cell wall degradation is required for normal starch mobilisation in barley endosperm

    Science.gov (United States)

    Andriotis, Vasilios M. E.; Rejzek, Martin; Barclay, Elaine; Rugen, Michael D.; Field, Robert A.; Smith, Alison M.

    2016-01-01

    Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and β-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth. PMID:27622597

  1. Degradation of various dyes using Laccase enzyme.

    Science.gov (United States)

    Dhaarani, S; Priya, A K; Rajan, T Vel; Kartic, D Navamani

    2012-10-01

    Disposal of untreated dyeing effluent in water bodies, from textile industries, cause serious environmental and health hazards. The chemical structures of dye molecules are designed to resist fading on exposure to light or chemical attack, and they prove to be quite resistant towards microbial degradation. Therefore, current conventional biological processes may not be able to meet wastewater discharge criteria and reuse. An enzymatic treatment undergoes oxidative cleavage avoiding formation of toxic amines. Laccase is a multi-copper containing protein that catalyzes the oxidation of a wide range of aromatic substrates concomitantly with the reduction of molecular oxygen to water. UV visible spectral analysis of various synthetic dyes was performed in the study and wavelengths of maximum absorbance determined. Laccase enzyme was obtained from the fungi Pleorotus ostreatus. The enzyme showed high efficiency against Malachite Green, Basic Red and Acid Majanta with decolorization capacities of 97%, 94% and 94% respectively. Further, these dyes can be used for optimization of degradation parameters and analysis of degradation products.

  2. Bisphenol A degradation in water by ligninolytic enzymes.

    Science.gov (United States)

    Gassara, Fatma; Brar, Satinder K; Verma, M; Tyagi, R D

    2013-08-01

    Many endocrine disruptor compounds, such as bisphenol A (BPA) are used today and released into the environment at low doses but they are barely degraded in wastewater treatment plants. One of the potential alternatives to effectively degrade endocrine disruptor compounds is based on the use of the oxidative action of extracellular fungal enzymes. The aim of this work is to study the ability of free and encapsulated enzymes (manganese peroxidase, lignin peroxidase and laccase) to degrade BPA. Higher degradation of BPA (90%) by ligninolytic enzymes encapsulated on polyacrylamide hydrogel and pectin after 8h was obtained. The degradation of BPA while using the free enzyme (26%) was lower than the value obtained with encapsulated enzymes. The presence of pectin in the formulation significantly (p>0.05) enhanced the activity of enzymes. Kinetics of BPA degradation showed an increase in Vm, while Km remained constant when enzymes were encapsulated. Hence, encapsulation protected the enzymes from non-competitive inhibition.

  3. Enzyme-catalyzed degradation of carbon nanomaterials

    Science.gov (United States)

    Kotchey, Gregg P.

    Carbon nanotubes and graphene, the nanoscale sp 2 allotropes of carbon, have garnered widespread attention as a result of their remarkable electrical, mechanical, and optical properties and the promise of new technologies that harness these properties. Consequently, these carbon nanomaterials (CNMs) have been employed for diverse applications such as electronics, sensors, composite materials, energy conversion devices, and nanomedicine. The manufacture and eventual disposal of these products may result in the release of CNMs into the environment and subsequent exposure to humans, animals, and vegetation. Given the possible pro-inflammatory and toxic effects of CNMs, much attention has been focused on the distribution, toxicity, and persistence of CNMs both in living systems and the environment. This dissertation will guide the reader though recent studies aimed at elucidating fundamental insight into the persistence of CNMs such as carbon nanotubes (CNTs) and graphene derivatives (i.e., graphene oxide and reduced graphene oxide). In particular, in-testtube oxidation/degradation of CNMs catalyzed by peroxidase enzymes will be examined, and the current understanding of the mechanisms underlying these processes will be discussed. Finally, an outlook of the current field including in vitro and in vivo biodegradation experiments, which have benefits in terms of human health and environmental safety, and future directions that could have implications for nanomedical applications such as imaging and drug delivery will be presented. Armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation/biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. For example, in nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. Also, the incorporation of CNMs with defect sites in consumer

  4. Novel Enzymes for Targeted Hydrolysis of Algal Cell Walls

    DEFF Research Database (Denmark)

    Schultz-Johansen, Mikkel

    . These enzymes degraded fucoidan extracted from brown algae of the order Fucales, but displayed individual substrate preference and degradation pattern. This work adds substantial information to a protein family which is largely undiscovered to date. Several of the enzyme activities discovered in this thesis...

  5. THERMOSTABLE ALGINATE DEGRADING ENZYMES AND THEIR METHODS OF USE

    NARCIS (Netherlands)

    Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

    2015-01-01

    The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

  6. Thermostable Alginate degrading enzymes and their methods of use

    NARCIS (Netherlands)

    Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

    2015-01-01

    The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

  7. Enzymes and other agents that enhance cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  8. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  9. An Extended Polyanion Activation Surface in Insulin Degrading Enzyme.

    Directory of Open Access Journals (Sweden)

    Eun Suk Song

    Full Text Available Insulin degrading enzyme (IDE is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ, glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898 within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity.

  10. Cell wall degradation in the autolysis of filamentous fungi.

    Science.gov (United States)

    Perez-Leblic, M I; Reyes, F; Martinez, M J; Lahoz, R

    1982-12-27

    A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes. Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present. In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.

  11. Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

    Science.gov (United States)

    Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

    2011-11-01

    There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

  12. Type IV collagen-degrading enzyme activity in human serum.

    Directory of Open Access Journals (Sweden)

    Hashimoto,Noriaki

    1988-02-01

    Full Text Available Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful for estimating the degradation of type IV collagen.

  13. Degradation of glyphosate and other pesticides by ligninolytic enzymes.

    Science.gov (United States)

    Pizzul, Leticia; Castillo, María del Pilar; Stenström, John

    2009-11-01

    The ability of pure manganese peroxidase (MnP), laccase, lignin peroxidase (LiP) and horseradish peroxidase (HRP) to degrade the widely used herbicide glyphosate and other pesticides was studied in separate in vitro assays with addition of different mediators. Complete degradation of glyphosate was obtained with MnP, MnSO4 and Tween 80, with or without H2O2. In the presence of MnSO4, with or without H(2)O(2), MnP also transformed the herbicide, but to a lower rate. Laccase degraded glyphosate in the presence of (a) 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), (b) MnSO(4) and Tween 80 and (c) ABTS, MnSO4 and Tween 80. The metabolite AMPA was detected in all cases where degradation of glyphosate occurred and was not degraded. The LiP was tested alone or with MnSO4, Tween 80, veratryl alcohol or H2O2 and in the HRP assay the enzyme was added alone or with H2O2 in the reaction mixture. However, these enzymes did not degrade glyphosate. Further experiments using MnP together with MnSO4 and Tween 80 showed that the enzyme was also able to degrade glyphosate in its commercial formulation Roundup Bio. The same enzyme mixture was tested for degradation of 22 other pesticides and degradation products present in a mixture and all the compounds were transformed, with degradation percentages ranging between 20 and 100%. Our results highlight the potential of ligninolytic enzymes to degrade pesticides. Moreover, they suggest that the formation of AMPA, the main metabolite of glyphosate degradation found in soils, can be a result of the activity of lignin-degrading enzymes.

  14. Technological Implications of Modifying the Extent of Cell Wall-Proanthocyanidin Interactions Using Enzymes

    Directory of Open Access Journals (Sweden)

    Ana Belén Bautista-Ortín

    2016-01-01

    Full Text Available The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing of the fruits. Therefore, the effective extraction of proanthocyanidins from fruits to their juices or derived products will depend on the ability to manage these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the role of pure hydrolytic enzymes (polygalacturonase and cellulose and a commercial enzyme containing these two activities on the extent of proanthocyanidin-cell wall interactions. The results showed that the modification promoted by enzymes reduced the amount of proanthocyanidins adsorbed to cell walls since they contributed to the degradation and release of the cell wall polysaccharides, which diffused into the model solution. Some of these released polysaccharides also presented some reactivity towards the proanthocyanidins present in a model solution.

  15. Type IV collagen-degrading enzyme activity in human serum.

    OpenAIRE

    Hashimoto, Noriaki; Kobayashi, Michio; Watanabe,Akiharu; Higashi, Toshiro; Tsuji, Takao

    1988-01-01

    Type IV collagen-degrading enzyme activity was detected in human serum. Serum was preincubated with 4-aminophenylmercuric acetate and trypsin to activate the enzyme prior to assay. Type IV collagen, purified from human placentas and radiolabeled with [1-14C] acetic anhydride, was used as the substrate. The enzyme activity was measured at pH 7.5 and inhibited by treatment with ethylenediaminetetraacetic acid or heat. The assay of type IV collagen-degrading enzyme in human serum might be useful...

  16. Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa)

    Science.gov (United States)

    Gibeaut, David M.; Karuppiah, Nadarajah; Chang, S.-R.; Brock, Thomas G.; Vadlamudi, Babu; Kim, Donghern; Ghosheh, Najati S.; Rayle, David L.; Carpita, Nicholas C.; Kaufman, Peter B.

    1990-01-01

    The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response and asymmetric processes involving degradation of starch and cell wall synthesis. Cellular and biochemical events were studied by investigation of the activities of related enzymes and changes in cell walls and their constituents. It is suggested that an osmotic potential gradient acts as the driving factor for growth, while wall extensibility is a limiting factor in pulvinus growth.

  17. Novel enzymes for the degradation of cellulose

    Directory of Open Access Journals (Sweden)

    Horn Svein

    2012-07-01

    Full Text Available Abstract The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix.

  18. Cell wall degrading enzyme induced rice innate immune responses are suppressed by the type 3 secretion system effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Sinha, Dipanwita; Gupta, Mahesh Kumar; Patel, Hitendra Kumar; Ranjan, Ashish; Sonti, Ramesh V

    2013-01-01

    Innate immune responses are induced in plants and animals through perception of Damage Associated Molecular Patterns. These immune responses are suppressed by pathogens during infection. A number of studies have focussed on identifying functions of plant pathogenic bacteria that are involved in suppression of Pathogen Associated Molecular Pattern induced immune responses. In comparison, there is very little information on functions used by plant pathogens to suppress Damage Associated Molecular Pattern induced immune responses. Xanthomonasoryzae pv. oryzae, a gram negative bacterial pathogen of rice, secretes hydrolytic enzymes such as LipA (Lipase/Esterase) that damage rice cell walls and induce innate immune responses. Here, we show that Agrobacterium mediated transient transfer of the gene for XopN, a X. oryzae pv. oryzae type 3 secretion (T3S) system effector, results in suppression of rice innate immune responses induced by LipA. A xopN (-) mutant of X. oryzae pv. oryzae retains the ability to suppress these innate immune responses indicating the presence of other functionally redundant proteins. In transient transfer assays, we have assessed the ability of 15 other X. oryzae pv. oryzae T3S secreted effectors to suppress rice innate immune responses. Amongst these proteins, XopQ, XopX and XopZ are suppressors of LipA induced innate immune responses. A mutation in any one of the xopN, xopQ, xopX or xopZ genes causes partial virulence deficiency while a xopN (-) xopX (-) double mutant exhibits a greater virulence deficiency. A xopN (-) xopQ (-) xopX (-) xopZ (-) quadruple mutant of X. oryzae pv. oryzae induces callose deposition, an innate immune response, similar to a X. oryzae pv. oryzae T3S(-) mutant in rice leaves. Overall, these results indicate that multiple T3S secreted proteins of X. oryzae pv. oryzae can suppress cell wall damage induced rice innate immune responses.

  19. Discovery and Characterization of Enzymes for Degradation of Xyloglucan and Extensin

    DEFF Research Database (Denmark)

    Feng, Tao; Mikkelsen, Jørn Dalgaard

    The aim of this PhD study is based on the concept ‘lean green food’ where application of enzymes replace chemicals in the biomass refinery process to recover labile value-added components and produce biofuels. To achieve this task valuable products like pectin should be isolated from the biomass...... before the residual polymers are used in the bioethanol production. Therefore, mono-component, substrate-specific enzymes that could selectively degrade or modify plant cell wall components are required. In this PhD study, three enzymes, including two xyloglucan-specific endoglucanases and one...

  20. Discovery of novel algae-degrading enzymes from marine bacteria

    DEFF Research Database (Denmark)

    Schultz-Johansen, Mikkel; Bech, Pernille Kjersgaard; Hennessy, Rosanna Catherine

    and functional screening. This resulted in the discovery of a novel marine bacterium which displays a large enzymatic potential for degradation of red algal polysaccharides e.g. agar and carrageenan. In addition, we searched metagenome sequence data and identified new enzyme candidates for degradation...

  1. Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture.

    Science.gov (United States)

    English, P D; Jurale, J B; Albersheim, P

    1971-01-01

    The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more alpha-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal alpha-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal alpha-arabinosidase activity. Galactose, when used as the carbon source, stimulates alpha-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and alpha-arabinosidase are secreted first, followed by beta-xylosidase and cellulase, then beta-glucosidase, and, finally, alpha-galactosidase.

  2. A Simple Endpoint Assay for Starch-Degrading Enzymes.

    Science.gov (United States)

    Kroen, William K.

    1998-01-01

    Since many of the important energy-transferring pathways involve synthesis or degradation of biological macromolecules, observations of the enzymes responsible for starch breakdown provide a useful case study. Provides a short, one-step assay for the enzymes amylase and amyloglucosidase. Topics covered include goals, preparation, assay procedure,…

  3. Downstream processing of polysaccharide degrading enzymes by affinity chromatography.

    NARCIS (Netherlands)

    Somers, W.A.C.

    1992-01-01

    The objective of this study was the development of affinity matrices to isolate and purify a number of polysaccharide degrading enzymes and the application of these adsorbents in the large- scale purification of the enzymes from fermentation broths. Affinity adsorbents were developed for endo-polyga

  4. Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

    OpenAIRE

    Burke, TP; Loukitcheva, A; Zemansky, J; Wheeler, R; Boneca, IG; Portnoy, DA

    2014-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward geneti...

  5. Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

    OpenAIRE

    Burke, Thomas P.; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G.; Portnoy, Daniel A.

    2014-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward geneti...

  6. A highly sensitive peptide substrate for detecting two Aß-degrading enzymes: neprilysin and insulin-degrading enzyme.

    Science.gov (United States)

    Chen, Po-Ting; Liao, Tai-Yan; Hu, Chaur-Jong; Wu, Shu-Ting; Wang, Steven S-S; Chen, Rita P-Y

    2010-06-30

    Neprilysin has been singled out as the most promising candidate for use in the degradation of Abeta as a therapy for Alzheimer's disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the Abeta peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin and 0.2 nM of insulin-degrading enzyme can be detected.

  7. Bacterial enzymes involved in lignin degradation

    NARCIS (Netherlands)

    de Gonzalo, Gonzalo; Colpa, Dana I; Habib, Mohamed H M; Fraaije, Marco W

    2016-01-01

    Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the (bio)p

  8. Bacterial enzymes involved in lignin degradation

    NARCIS (Netherlands)

    de Gonzalo, Gonzalo; Colpa, Dana I; Habib, Mohamed H M; Fraaije, Marco W

    2016-01-01

    Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the

  9. Recent advances in microbial raw starch degrading enzymes.

    Science.gov (United States)

    Sun, Haiyan; Zhao, Pingjuan; Ge, Xiangyang; Xia, Yongjun; Hao, Zhikui; Liu, Jianwen; Peng, Ming

    2010-02-01

    Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.

  10. Targeting amyloid-degrading enzymes as therapeutic strategies in neurodegeneration.

    Science.gov (United States)

    Turner, Anthony J; Fisk, Lilia; Nalivaeva, Natalia N

    2004-12-01

    The levels of amyloid beta-peptides (Abeta) in the brain represent a dynamic equilibrium state as a result of their biosynthesis from the amyloid precursor protein (APP) by beta- and gamma-secretases, their degradation by a team of amyloid-degrading enzymes, their subsequent oligomerization, and deposition into senile plaques. While most therapeutic attention has focused on developing inhibitors of secretases to prevent Abeta formation, enhancing the rate of Abeta degradation represents an alternative and viable strategy. Current evidence both in vivo and in vitro suggests that there are three major players in amyloid turnover: neprilysin, endothelin converting enzyme(s), and insulin-degrading enzyme, all of which are zinc metallopeptidases. Other proteases have also been implicated in amyloid metabolism, including angiotensin-converting enzyme, and plasmin but for these the evidence is less compelling. Neprilysin and endothelin converting enzyme(s) are homologous membrane proteins of the M13 peptidase family, which normally play roles in the biosynthesis and/or metabolism of regulatory peptides. Insulin-degrading enzyme is structurally and mechanistically distinct. The regional, cellular, and subcellular localizations of these enzymes differ, providing an efficient and diverse mechanism for protecting the brain against the normal accumulation of toxic Abeta peptides. Reduction in expression levels of some of these proteases following insults (e.g., hypoxia and ischemia) or aging might predispose to the development of Alzheimer's disease. Conversely, enhancement of their levels by gene delivery or pharmacological means could be neuroprotective. Even a relatively small enhancement of Abeta metabolism could slow the inexorable progression of the disease. The relative merits of targeting these enzymes for the treatment of Alzheimer's disease will be reviewed and possible side-effects of enhancing their activity evaluated.

  11. Changes in free polyamines and related enzymes during stipule and pod wall development in Pisum sativum.

    Science.gov (United States)

    Chattopadhyay, Soumen; Lahiri, Kajari; Bharati, Ghosh

    2002-08-01

    Level of free polyamines, their key metabolic enzymes, and other features related to ageing were examined during stipule and pod wall development in pea (Pisum sativum). Free polyamine titre (per unit fresh mass) in both the organs, the specific activities of arginine decarboxylase and ornithine decarboxylase in the pod wall, gradually decreased with maturation. In stipule, these enzymes attained peak activity at 15 days after pod emergence and declined thereafter. Ornithine decarboxylase activity was greater in pod wall than in stipule; while, arginine decarboxylase activity was higher in stipule. Activity of degradative enzyme diamine oxidase increased with the onset of senescence in both the organs. Chlorophyll and electrical conductance had a inverse relationship throughout the experimental period, whereas, the chlorophyll content was directly related with polyamine levels in both stipule and pod wall during aging. On the other hand, protein and RNA contents were positively correlated with free polyamines throughout the test period in stipule, but in the pod wall this was true only for the later stages of development.

  12. Structural biology of starch-degrading enzymes and their regulation

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Svensson, Birte

    2016-01-01

    Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant...... disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley limit dextrinase inhibitor binding...... to the debranching enzyme, limit dextrinase using a new binding mode for cereal protein inhibitors....

  13. Effect of solvents on the enzyme mediated degradation of copolymers

    Science.gov (United States)

    Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

    2015-09-01

    The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 °C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

  14. Structural biology of starch-degrading enzymes and their regulation

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Svensson, Birte

    2016-01-01

    Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant...... disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley limit dextrinase inhibitor binding...

  15. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Directory of Open Access Journals (Sweden)

    Lori B Huberman

    Full Text Available Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  16. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Science.gov (United States)

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  17. Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium Burds

    Energy Technology Data Exchange (ETDEWEB)

    Tien, M.; Kirk, T.K.

    1983-08-12

    The extracellular fluid of ligninolytic cultures of the wood-decomposing basidiomycete Phanerochaete chrysosporium Burds contains an enzyme that degrades lignin substructure model compounds as well as spruce and birch lignins. It has a molecular size of 42,000 daltons and requires hydrogen peroxide for activity. (Refs. 24).

  18. Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

    Science.gov (United States)

    Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

    2013-06-01

    A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

  19. Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in Phlebia tremellosa.

    Science.gov (United States)

    Kum, Hyunwoo; Lee, Sungsuk; Ryu, Sunhwa; Choi, Hyoung T

    2011-10-01

    A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.

  20. A Cell Wall-degrading Endopolygalacturonase Secreted by Colletotrichum lindemuthianum.

    Science.gov (United States)

    English, P D; Maglothin, A; Keegstra, K; Albersheim, P

    1972-03-01

    Cultures of Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner have been induced to secrete an endopolygalacturonase (polygalacturonide glycanohydrolase EC3.2. 1.15). This enzyme has been brought to a high state of purity by ion exchange, gel filtration, and agarose affinity chromatography. The enzyme has optimal activity at pH 5, has an apparent molecular weight as determined by gel filtration of about 70,000, and prefers polygalacturonic acid to pectin as its substrate. The enzyme, while hydrolyzing only 1% of the glycosidic bonds, reduces the viscosity of a polygalacturonic solution by 50%. Nevertheless, the initial as well as the final products of polygalacturonic acid hydrolysis are predominantly tri- and digalacturonic acid and, to a lesser extent, monogalacturonic acid. The purified enzyme catalyzes the removal of about 80% of the galacturonic acid residues of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus) as well as from the walls isolated from 8-day-old Red Kidney bean (Phaseolus vulgaris) hypocotyls.

  1. Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

    Science.gov (United States)

    Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

    2015-09-23

    The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

  2. Extracellular entrapment and degradation of single-walled carbon nanotubes

    Science.gov (United States)

    Farrera, Consol; Bhattacharya, Kunal; Lazzaretto, Beatrice; Andón, Fernando T.; Hultenby, Kjell; Kotchey, Gregg P.; Star, Alexander; Fadeel, Bengt

    2014-05-01

    Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials.Neutrophils extrude neutrophil extracellular traps (NETs) consisting of a network of chromatin decorated with antimicrobial proteins to enable non-phagocytic killing of microorganisms. Here, utilizing a model of ex vivo activated human neutrophils, we present evidence of entrapment and degradation of carboxylated single-walled carbon nanotubes (SWCNTs) in NETs. The degradation of SWCNTs was catalyzed by myeloperoxidase (MPO) present in purified NETs and the reaction was facilitated by the addition of H2O2 and NaBr. These results show that SWCNTs can undergo acellular, MPO-mediated biodegradation and imply that the immune system may deploy similar strategies to rid the body of offending microorganisms and engineered nanomaterials. Electronic supplementary information (ESI) available: Suppl. Fig. 1 - length distribution of SWCNTs; suppl. Fig. 2 - characterization of pristine vs. oxidized SWCNTs; suppl. Fig. 3 - endotoxin evaluation; suppl. Fig. 4 - NET characterization; suppl. Fig. 5 - UV-Vis/NIR analysis of biodegradation of oxidized SWCNTs; suppl. Fig. 6 - cytotoxicity of partially degraded SWCNTs. See DOI: 10.1039/c3nr06047k

  3. Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts

    Directory of Open Access Journals (Sweden)

    Normand Philippe

    2008-01-01

    genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

  4. Production of an extracellular polyethylene-degrading enzyme(s) by Streptomyces species.

    Science.gov (United States)

    Pometto, A L; Lee, B T; Johnson, K E

    1992-01-01

    Extracellular culture concentrates were prepared from Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2 shake flask cultures. Ten-day-heat-treated (70 degrees C) starch-polyethylene degradable plastic films were incubated with shaking with active or inactive enzyme for 3 weeks (37 degrees C). Active enzyme illustrated changes in the films' Fourier transform infrared spectra, mechanical properties, and polyethylene molecular weight distributions. PMID:1610196

  5. Activity of cell wall degrading glycanases in methyl jasmonate-induced leaf abscission in Kalanchoe blossfeldiana

    Directory of Open Access Journals (Sweden)

    Marian Saniewski

    2013-12-01

    Full Text Available It was found previously that methyl jasmonate (JA-Me induced leaf abscission in Kalanchoe blossfeldiana. In present studies it was shown that JA-Me markedly increased the total activities of cellulase, polygalacturonase, pectinase and xylanase in petioles, but did not affect activities of these enzymes in the blades and apical part of shoots of K. blossfeldiana. These results suggest that methyl jasmonate promotes the degradation of cell wall polysaccharides in the abscission zone and in this way induces leaf abscission in Kalanchoe blossfeldiana.

  6. Diversity screening for novel enzymes degrading synthetic polymers

    DEFF Research Database (Denmark)

    Lezyk, Mateusz Jakub

    The objective of this PhD study was to evaluate the feasibility of enzymatic degradation of synthetic polymers used as binder materials in marine coatings. Enzymatic modification of synthetic polymers like epoxy resin, polyurethanes and various acrylics is desirable in several industrial processes...... is reported. First, a collection of fungal strains was screened for the capability to degrade several compounds of synthetic origin. Strains with the ability to modify colloidal polyester polyurethane, as well as various commercial emulsions of acrylates were identified. Secondly, we have used metagenomic...... and the choice of proper substrates for identification of promising enzyme candidates. Several genes coding for enzymes with the capability to oxidize various natural and synthetic substrates were identified, expressed heterologously and characterized. Three multicopper oxidases (MCOs) were identified encoded...

  7. Acceleration of Fibrous Biomass Degradation by Bacterial Enzymes

    OpenAIRE

    大宮, 邦雄; 河津, 哲; 孫, 嘉琳; 木村, 哲哉; 苅田, 修一; 粟冠, 和郎; Ohmiya,Kunio; Kawazu, Tetsu; Sun, Jialin; KIMURA, TETSUYA; Karita, Shuichi; Sakka, Kazuo

    1997-01-01

    Since biomass photosynthesized from C02 and H2O is one of the most predominant storage sites of solar energy, its effective utilization will be essential to overcome the shortage of foods and energy in future. Relaxation of biomass tissue is focused to enhance solubilization by expressing fiber‐degrading enzyme genes in plants. A xylanase gene from Clostridium thermocellum was highly expressed in tobacco plant (4%) without apparent defects in the growth. Another xylanase from Clostridium ste...

  8. Recent advances in azo dye degrading enzyme research.

    Science.gov (United States)

    Chen, Huizhong

    2006-04-01

    Azo dyes, which are characterized by one or more azo bonds, are a predominant class of colorants used in tattooing, cosmetics, foods, and consumer products. These dyes are mainly metabolized by bacteria to colorless aromatic amines, some of which are carcinogenic, by azoreductases that catalyze a NAD(P)H-dependent reduction. The resulting amines are further degraded aerobically by bacteria. Some bacteria have the ability to degrade azo dyes both aerobically and anaerobically. Plant-degrading white rot fungi can break down azo dyes by utilizing a number of oxidases and peroxidases as well. In yeast, a ferric reductase system participates in the extracellular reduction of azo dyes. Recently, two types of azoreductases have been discovered in bacteria. The first class of azoreductases is monomeric flavin-free enzymes containing a putative NAD(P)H binding motif at their N-termini; the second class is polymeric flavin dependent enzymes which are studied more extensively. Azoreductases from bacteria represent novel families of enzymes with little similarity to other reductases. Dissociation and reconstitution of the flavin dependent azoreductases demonstrate that the non-covalent bound flavin prosthetic group is required for the enzymatic functions. In this review, structures and carcinogenicity of azo colorants, protein structure, enzymatic function, and substrate specificity, as well as application of the azo dyes and azoreductases will be discussed.

  9. Micropollutant degradation via extracted native enzymes from activated sludge.

    Science.gov (United States)

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  10. Cell Wall Degrading Enzymes Involved in Mycoparasitism of the Biocontrol Agent Chaetomium spirale ND35%生防因子螺旋毛壳ND35的细胞壁降解酶与重寄生作用

    Institute of Scientific and Technical Information of China (English)

    高克祥; 刘晓光; Dana Friesem; Leonid Chernin; 时呈奎

    2005-01-01

    Some Chaetomium spp. Are capable of antagonizing several plant pathogenic fungi through production of antibiotics and mycoparasitism. Secretion of lytic enzymes, mainly including glucanases and chitinases, is considered the most important step in the mycoparasitic process. In this study, an about 110kDa exo - β - 1,3 - glucanase from C. Spirale ND35 was detected both in culture filtrate and directly on PAGE and IEF gels, as well as chitinases, although protease was not detectable on Litmus milk agar plates. Coiling and penetrating the hyphae of host fungus Valsa mali were observed by scanning electron microscope (SEM), which may be related to the synergistic interaction between β - 1,3 - glucanase and chitinases. Β - 1,3 - glucanase activity of C. Spirale ND35 varied considerably when C. Spirale ND35 was grown in different carbon sources during various incubation time, and might be subjected to both induction by substrate and catabolite repression.

  11. Novel Aflatoxin-Degrading Enzyme from Bacillus shackletonii L7

    Directory of Open Access Journals (Sweden)

    Liang Xu

    2017-01-01

    Full Text Available Food and feed contamination by aflatoxin (AFB1 has adverse economic and health consequences. AFB1 degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil samples for AFB1-reducing activity. The higher activity was detected in isolate L7, which was identified as Bacillus shackletonii. L7 reduced AFB1, AFB2, and AFM1 levels by 92.1%, 84.1%, and 90.4%, respectively, after 72 h at 37 °C. The L7 culture supernatant degraded more AFB1 than viable cells and cell extracts; and the degradation activity was reduced from 77.9% to 15.3% in the presence of proteinase K and sodium dodecyl sulphate. A thermostable enzyme purified from the boiled supernatant was designated as Bacillus aflatoxin-degrading enzyme (BADE. An overall 9.55-fold purification of BADE with a recovery of 39.92% and an activity of 3.85 × 103 U·mg−1 was obtained using chromatography on DEAE-Sepharose. BADE had an estimated molecular mass of 22 kDa and exhibited the highest activity at 70 °C and pH 8.0, which was enhanced by Cu2+ and inhibited by Zn2+, Mn2+, Mg2+, and Li+. BADE is the major protein involved in AFB1 detoxification. This is the first report of a BADE isolated from B. shackletonii, which has potential applications in the detoxification of aflatoxins during food and feed processing.

  12. Novel Aflatoxin-Degrading Enzyme from Bacillus shackletonii L7

    Science.gov (United States)

    Xu, Liang; Eisa Ahmed, Mohamed Farah; Sangare, Lancine; Zhao, Yueju; Selvaraj, Jonathan Nimal; Xing, Fuguo; Wang, Yan; Yang, Hongping; Liu, Yang

    2017-01-01

    Food and feed contamination by aflatoxin (AF)B1 has adverse economic and health consequences. AFB1 degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil samples for AFB1-reducing activity. The higher activity was detected in isolate L7, which was identified as Bacillus shackletonii. L7 reduced AFB1, AFB2, and AFM1 levels by 92.1%, 84.1%, and 90.4%, respectively, after 72 h at 37 °C. The L7 culture supernatant degraded more AFB1 than viable cells and cell extracts; and the degradation activity was reduced from 77.9% to 15.3% in the presence of proteinase K and sodium dodecyl sulphate. A thermostable enzyme purified from the boiled supernatant was designated as Bacillus aflatoxin-degrading enzyme (BADE). An overall 9.55-fold purification of BADE with a recovery of 39.92% and an activity of 3.85 × 103 U·mg−1 was obtained using chromatography on DEAE-Sepharose. BADE had an estimated molecular mass of 22 kDa and exhibited the highest activity at 70 °C and pH 8.0, which was enhanced by Cu2+ and inhibited by Zn2+, Mn2+, Mg2+, and Li+. BADE is the major protein involved in AFB1 detoxification. This is the first report of a BADE isolated from B. shackletonii, which has potential applications in the detoxification of aflatoxins during food and feed processing. PMID:28098812

  13. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease.

  14. Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

  15. A Suite of Activity-Based Probes for Cellulose Degrading Enzymes

    Science.gov (United States)

    Chauvigné-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-01-01

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose degrading systems, and facilitates a greater understanding of the organismal role associated with biofuel development. PMID:23176123

  16. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts.

    Directory of Open Access Journals (Sweden)

    Eric C Martens

    2011-12-01

    Full Text Available Symbiotic bacteria inhabiting the human gut have evolved under intense pressure to utilize complex carbohydrates, primarily plant cell wall glycans in our diets. These polysaccharides are not digested by human enzymes, but are processed to absorbable short chain fatty acids by gut bacteria. The Bacteroidetes, one of two dominant bacterial phyla in the adult gut, possess broad glycan-degrading abilities. These species use a series of membrane protein complexes, termed Sus-like systems, for catabolism of many complex carbohydrates. However, the role of these systems in degrading the chemically diverse repertoire of plant cell wall glycans remains unknown. Here we show that two closely related human gut Bacteroides, B. thetaiotaomicron and B. ovatus, are capable of utilizing nearly all of the major plant and host glycans, including rhamnogalacturonan II, a highly complex polymer thought to be recalcitrant to microbial degradation. Transcriptional profiling and gene inactivation experiments revealed the identity and specificity of the polysaccharide utilization loci (PULs that encode individual Sus-like systems that target various plant polysaccharides. Comparative genomic analysis indicated that B. ovatus possesses several unique PULs that enable degradation of hemicellulosic polysaccharides, a phenotype absent from B. thetaiotaomicron. In contrast, the B. thetaiotaomicron genome has been shaped by increased numbers of PULs involved in metabolism of host mucin O-glycans, a phenotype that is undetectable in B. ovatus. Binding studies of the purified sensor domains of PUL-associated hybrid two-component systems in conjunction with transcriptional analyses demonstrate that complex oligosaccharides provide the regulatory cues that induce PUL activation and that each PUL is highly specific for a defined cell wall polymer. These results provide a view of how these species have diverged into different carbohydrate niches by evolving genes that target

  17. Biosurfactant and Degradative Enzymes Mediated Crude Oil Degradation by Bacterium Bacillus subtilis A1

    Science.gov (United States)

    Parthipan, Punniyakotti; Preetham, Elumalai; Machuca, Laura L.; Rahman, Pattanathu K. S. M.; Murugan, Kadarkarai; Rajasekar, Aruliah

    2017-01-01

    In this work, the biodegradation of the crude oil by the potential biosurfactant producing Bacillus subtilis A1 was investigated. The isolate had the ability to synthesize degradative enzymes such as alkane hydroxylase and alcohol dehydrogenase at the time of biodegradation of hydrocarbon. The biosurfactant producing conditions were optimized as pH 7.0, temperature 40°C, 2% sucrose and 3% of yeast extract as best carbon and nitrogen sources for maximum production of biosurfactant (4.85 g l-1). Specifically, the low molecular weight compounds, i.e., C10–C14 were completely degraded, while C15–C19 were degraded up to 97% from the total hydrocarbon pools. Overall crude oil degradation efficiency of the strain A1 was about 87% within a short period of time (7 days). The accumulated biosurfactant from the biodegradation medium was characterized to be lipopeptide in nature. The strain A1 was found to be more robust than other reported biosurfactant producing bacteria in degradation efficiency of crude oil due to their enzyme production capability and therefore can be used to remove the hydrocarbon pollutants from contaminated environment. PMID:28232826

  18. Metagenomics for the discovery of pollutant degrading enzymes.

    Science.gov (United States)

    Ufarté, Lisa; Laville, Élisabeth; Duquesne, Sophie; Potocki-Veronese, Gabrielle

    2015-12-01

    Organic pollutants, including xenobiotics, are often persistent and toxic organic compounds resulting from human activities and released in large amounts into terrestrial, fluvial and marine environments. However, some microbial species which are naturally exposed to these compounds in their own habitat are capable of degrading a large range of pollutants, especially poly-aromatic, halogenated and polyester molecules. These microbes constitute a huge reservoir of enzymes for the diagnosis of pollution and for bioremediation. Most are found in highly complex ecosystems like soils, activated sludge, compost or polluted water, and more than 99% have never been cultured. Meta-omic approaches are thus well suited to retrieve biocatalysts from these environmental samples. In this review, we report the latest advances in functional metagenomics aimed at the discovery of enzymes capable of acting on different kinds of polluting molecules. Copyright © 2015. Published by Elsevier Inc.

  19. High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

    Directory of Open Access Journals (Sweden)

    K. M. Singh

    2014-01-01

    Full Text Available The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs. We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs, carbohydrate binding module (CBM: 23 contigs, glycosyl transferase (GT: 373 contigs, carbohydrate esterases (CE: 259 contigs, and polysaccharide lyases (PE: 16 contigs. The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

  20. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  1. Microbial degradation of n-hexadecane in mineral salt medium as mediated by degradative enzymes.

    Science.gov (United States)

    Mishra, Shweta; Singh, S N

    2012-05-01

    In the present study, n-hexadecane degradation in MSM was investigated by three bacteria identified as Pseudomonas aeruginosa PSA5, Rhodococcus sp. NJ2 and Ochrobactrum intermedium P2, isolated from petroleum sludge. During 10 days of incubation, n-hexadecane was degraded to 99% by P. aeruginosa PSA5, 95% by Rhodococcus sp. NJ2 and 92% by O. intermedium P2. During degradation process, the induction of catabolic enzymes alkane hydroxylase, alcohol dehydrogenase and lipase were also examined. Among these enzymes, the highest activities of alkane hydroxylase (185 μmol mg(-1) protein) and alcohol dehydrogenase (75.78 μmol mg(-1) protein) were recorded in Rhodococcus sp. NJ2 while lipase activity was highly induced in P. aeruginosa PSA5 (48.71 μmol mg(-1) protein). Besides, accumulation of n-hexadecane in inclusion bodies was found to be maximum 60.8 g l(-1) in P. aeruginosa PSA5, followed by Rhodococcus sp. NJ2 (56.1 g l(-1)) and the least (51.6 g l(-1)) was found in O. intermedium P2. Biosurfactant production by bacterial strains was indicated by the reduction in surface tension and induction of cell surface hydrophobicity and pseudosolubilization which facilitated n-hexadecane degradation.

  2. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Directory of Open Access Journals (Sweden)

    Peter K Busk

    Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

  3. Typical Lignocellulose-degrading Enzymes: a Synthesis of Kinetic Properties

    Science.gov (United States)

    Wang, G.; Post, W. M.; Mayes, M. A.; Frerichs, J.; Jagadamma, S.

    2011-12-01

    While soil enzymes have been explicitly included in the soil organic carbon (SOC) decomposition models, there are big concerns on the model parameterization. Our object is to study the kinetic parameters of five typical lignocellulose-degrading enzymes through literature research and data synthesis. The kinetic parameters refer to the maximum specific enzyme activity (Vmax) and half-saturation constant (Km) in the Michaelis-Menton equation. The Activation energy (Ea) and the pH optimum and sensitivity (pHopt and pHsen) were also analyzed. pHsen was estimated by curve fitting of an exponential-quadratic function. The Vmax values in different units under various conditions were converted into the same units at a reference temperature (20°C) and optimum pH. The scaling issue on Vmax and Km and the effects of soil temperature, pH, and SWC were discussed later. Major findings are summarized as follows. (i) Both Vmax and Km are log-normal distributed. (ii) No significant difference in Vmax is found between groups (ligninases and cellulases). The one-standard-deviation interval of Vmax falls within 10-1000 (mean ≈ 100) mg C mg^-1 Enz h^-1. However, there is significant difference in Km between groups. (iii) Significant difference in activation energy, i.e., 53±17 and 37±15 kJ mol^-1 is found for ligninases and cellulases, respectively. (iv) Both ligninases and cellulases prefer to acid environment. The average ratio of pHsen to pHopt ranges 0.3-0.4 and the optimum pH for ligninases is significantly lower than pHopt for cellulases. (v) A preliminary analysis of Vmax indicates a scaling factor 0.01-0.1 for transforming the Vmax from lab measurements to SOC decomposition models. This study provides useful information for the parameterization of enzyme-driven SOC decomposition models.

  4. Antifirming effects of starch degrading enzymes in bread crumb.

    Science.gov (United States)

    Goesaert, Hans; Leman, Pedro; Bijttebier, Annabel; Delcour, Jan A

    2009-03-25

    Antifirming properties of amylases in bread crumb were evaluated in straight dough breadmaking and related to the amylolytically modified starch structure. Amylase properties and action mechanisms determine starch structure in the breads and, hence, how amylopectin recrystallization, starch network formation, water redistribution, and water mobility occur during breadmaking and storage. A bacterial endo-alpha-amylase mainly hydrolyzed the longer starch polymer chains internally. It thus reduced the number of connections between the crystallites in the starch networks, resulting in a softer bread crumb. However, because the enzyme had only little impact on the outer amylopectin chains, amylopectin recrystallization and the concomitant water immobilization presumably were not hindered. The loss of plasticizing water as a result of recrystallization presumably reduces the flexibility of the gluten network and results in poor crumb resilience. In contrast, in breadmaking, the Bacillus stearothermophilus maltogenic alpha-amylase acted as an exoacting amylase with more pronounced endoaction at higher temperatures. This enzyme caused extensive degradation of the crystallizable amylopectin side chains and thus limited amylopectin recrystallization and network formation during storage. As a result, it prevented the incorporation of water in the amylopectin crystallites. In this way, the different starch and gluten networks kept their flexibility, resulting in a softer crumb with good resilience.

  5. Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    Science.gov (United States)

    2013-01-01

    Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also

  6. Changes in Activities of Three Enzymes Degrading Galactomannan During and Following Rice Seed Germination

    Institute of Scientific and Technical Information of China (English)

    REN Yan-fang; HE Jun-yu; WANG Xiao-feng

    2007-01-01

    To investigate the relationships among β-mannanase, β-mannosidase and α-galactosidase required for degrading galactomannan in cell wall during and following rice seed germination, the activities of the three enzymes and the effects of ABA and GA3 on them were surveyed. The activities of β-mannosidase and α-galactosidase presented in dry and pre-germinated rice seeds, and increased slowly during and following germination. However, the activity of β-mannanase was detected only after germination. GA3 could promote the activities of β-mannanase and α-galactosidase. ABA had little effect on the activities of β-mannosidase and α-galactosidase, but it could seriously inhibit the activity of β-mannanase.

  7. Transcriptional directionality of the human insulin-degrading enzyme promoter.

    Science.gov (United States)

    Zhang, Lang; Wang, Pan; Ding, Qingyang; Wang, Zhao

    2013-10-01

    Unidirectional promoters dominate among mammalian genomes. However, the mechanism through which the transcriptional directionality of promoters is accomplished remains to be clarified. Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc metalloprotease, whose promoter contains a CpG island. We previously showed that the basal promoter region of mouse IDE has bidirectional transcriptional activity, but an upstream promoter element blocks its antisense transcription. Therefore, we wonder whether the human IDE promoter contains an analogous element. Similarly, the basal promoter region of human IDE (-102 ~ +173 and -196 ~ +173 relative to the transcription start site) showed bidirectional transcriptional activity. However, the region from -348 to +173 could only be transcribed from the normal orientation, implying that an upstream promoter element between -348 and -196 blocks the antisense transcription of the human IDE promoter. Through promoter deletion and mutagenesis analysis, we mapped this element precisely and found that the upstream promoter element locates between -318 and -304. Furthermore, the transcription-blocking elements in the mouse and human IDE promoters inhibited the transcription of the SV40 promoter when put downstream of it. In conclusion, we identify an upstream promoter element which blocks the antisense transcription of the human IDE promoter. Our studies are helpful to clarify the transcriptional directionality of promoters.

  8. Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes.

    Science.gov (United States)

    Song, Eun Suk; Jang, HyeIn; Guo, Hou-Fu; Juliano, Maria A; Juliano, Luiz; Morris, Andrew J; Galperin, Emilia; Rodgers, David W; Hersh, Louis B

    2017-04-04

    Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

  9. Effect of commercial enzymes on berry cell wall deconstruction in the context of intravineyard ripeness variation under winemaking conditions

    DEFF Research Database (Denmark)

    Gao, Yu; Fangel, Jonatan Ulrik; Willats, William George Tycho;

    2016-01-01

    at the berry cell wall polymer level and occurred within the experimental vineyard block. Furthemore, all enzyme treatments reduced cell wall variation via depectination. Interestingly, cell wall esterification levels were unaffected by enzyme treatments. This study provides clear evidence that enzymes can...

  10. Hemicellulose biosynthesis and degradation in tobacco cell walls

    NARCIS (Netherlands)

    Compier, M.G.M.

    2005-01-01

    Natural fibres have a wide range of technological applications, such as in paper and textile industries. The basic properties and the quality of plant fibres are determined by the composition of the plant cell wall. Characteristic for fibres are thick secondary cell walls, which consist of cellulose

  11. Hemicellulose biosynthesis and degradation in tobacco cell walls

    NARCIS (Netherlands)

    Compier, M.G.M.

    2005-01-01

    Natural fibres have a wide range of technological applications, such as in paper and textile industries. The basic properties and the quality of plant fibres are determined by the composition of the plant cell wall. Characteristic for fibres are thick secondary cell walls, which consist of cellulose

  12. Significance of the insulin degrading enzyme in the pathogenesis of the type 2 diabetes mellitus

    OpenAIRE

    Pivovarova, Olga

    2010-01-01

    The insulin degrading enzyme (IDE) is an ubiquitously expressed enzyme responsible for the insulin degradation. The IDE knockout in mice lead to decreased insulin degradation, hyperinsulinaemia and impaired glucose tolerance. Some association studies have identified IDE as a candidate gene for the type 2 diabetes mellitus (T2DM); however, other studies couldn’t confirm this association. In this work, the significance of the IDE in the T2DM pathogenesis was investigated on various levels. The ...

  13. Modulation of insulin degrading enzyme activity and liver cell proliferation.

    Science.gov (United States)

    Pivovarova, Olga; von Loeffelholz, Christian; Ilkavets, Iryna; Sticht, Carsten; Zhuk, Sergei; Murahovschi, Veronica; Lukowski, Sonja; Döcke, Stephanie; Kriebel, Jennifer; de las Heras Gala, Tonia; Malashicheva, Anna; Kostareva, Anna; Lock, Johan F; Stockmann, Martin; Grallert, Harald; Gretz, Norbert; Dooley, Steven; Pfeiffer, Andreas F H; Rudovich, Natalia

    2015-01-01

    Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis.

  14. Accessory enzymes from Aspergillus involved in xylan and pectin degradation

    NARCIS (Netherlands)

    Vries, de R.P.

    1999-01-01

    The xylanolytic and pectinolytic enzyme systems from Aspergillus have been the subject of study for many years. Although the main chain cleaving enzymes and their encoding genes have been studied in detail, little information is available about most of the accessory enzymes and their corresponding g

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

    Directory of Open Access Journals (Sweden)

    George Janssen

    Full Text Available 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

  16. Influence of exogenous fibrolytic enzymes on in vitro and in sacco degradation of forages for ruminants

    Directory of Open Access Journals (Sweden)

    Lorenzo Carreón

    2010-02-01

    Full Text Available An in vitro assay was carried out to evaluate the effects of exogenous fibrolytic enzymes (1, 2, 3 and 4 g/kg DM powder preparation containing xylanase and cellulase from Aspergillus niger and Trichoderma viride on DM, NDF and ADF degradation of alfalfa hay, corn silage, corn stover, elephant grass, Guinea grass and oat straw. Kinetics data of in vitro degradations were analyzed. The potentially degradable fraction and degradation rate of NDF and ADF of alfalfa increased quadratically (P<0.05 as the inclusion level of enzyme increased up to 3 g. The others forages were not affected by the enzyme. An in sacco trail was performed using four Holstein steers fitted with ruminal cannulas to evaluate the effects of the exogenous fibrolytic enzymes (3 g/kg DM on DM, NDF and ADF degradation of alfalfa hay and corn stover. Kinetics data were also analyzed. The potentially degradable fraction degradation of NDF (62.0 vs 65.7% and ADF (52.8 vs 56.9%, of alfalfa hay were increased (P<0.05 by the exogenous fibrolytic enzymes, but no differences were found for corn stover. These results suggest that the enzymes increased in vitro and in sacco fibre degradation only for alfalfa hay.

  17. Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Solomon, Kevin V.; Haitjema, Charles; Henske, John K.; Gilmore, Sean P.; Borges-Rivera, Diego; Lipzen, Anna; Brewer, Heather M.; Purvine, Samuel O.; Wright, Aaron T.; Theodorou, Michael K.; Grigoriev, Igor V.; Regev, Aviv; Thompson, Dawn; O' Malley, Michelle A.

    2016-03-11

    The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed, and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.

  18. Experimental strategy to discover microbes with gluten-degrading enzyme activities

    Science.gov (United States)

    Helmerhorst, Eva J.; Wei, Guoxian

    2014-06-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  19. Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes.

    Science.gov (United States)

    Solomon, Kevin V; Haitjema, Charles H; Henske, John K; Gilmore, Sean P; Borges-Rivera, Diego; Lipzen, Anna; Brewer, Heather M; Purvine, Samuel O; Wright, Aaron T; Theodorou, Michael K; Grigoriev, Igor V; Regev, Aviv; Thompson, Dawn A; O'Malley, Michelle A

    2016-03-11

    The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing.

  20. Survey of ectomycorrhizal, litter-degrading, and wood-degrading Basidiomycetes for dye decolorization and ligninolytic enzyme activity.

    Science.gov (United States)

    Casieri, Leonardo; Anastasi, Antonella; Prigione, Valeria; Varese, Giovanna Cristina

    2010-11-01

    Basidiomycetes are essential in forest ecology, being deeply involved in wood and litter decomposition, humification, and mineralization of soil organic matter. The fungal oxidoreductases involved in these processes are today the focus of much attention with a view to their applications. The ecological role and potential biotechnological applications of 300 isolates of Basidiomycetes were assessed, taking into account the degradation of model dyes in different culture conditions and the production of oxidoreductase enzymes. The tested isolates belong to different ecophysiological groups (wood-degrading, litter-degrading, ectomycorrhizal, and coprophilous fungi) and represent a broad systematic and functional biodiversity among Basidiomycetes occurring in deciduous and evergreen forests of northwest Italy (Piedmont Region). The high number of species tested and the use of different culture conditions allowed the investigation of the degradation activity of several novel species, neglected to date. Oxidative enzyme activities varied widely among all ecophysiological groups and laccases were the most commonly detected enzymes. A large number of isolates (86%), belonging to all ecophysiological groups, were found to be active against at least one model dye; the wood-degrading fungi represented the most efficient group. Noteworthily, also some isolates of litter-degrading and ectomycorrhizal fungi achieved good decolorization yield. The 25 best isolates were then tested against nine industrial dyes commonly employed in textile industries. Three isolates of Bjerkandera adusta efficiently decolorized the dyes on all media and can be considered important candidates for application in textile wastewater treatment.

  1. Kinetic studies of novel inhibitors of endomorphin degrading enzymes

    OpenAIRE

    Perlikowska, Renata; Fichna, Jakub; do-Rego, Jean Claude; Gach, Katarzyna; Janecka, Anna

    2011-01-01

    Endomorphins (EMs), two endogenous μ-opioid receptor selective ligands, are attractive lead compounds for opioid-based pain management studies. However, these peptides are quickly degraded by peptidases, in particular by dipeptidylpeptidase IV (DPP IV) and aminopeptidase M (APM). Targeting enzymatic degradation is one approach to prolong endomorphin activity. In this study we characterized the action of two new inhibitors of similar to endomorphins structure, Tyr-Pro-Ala-NH2 (EMDB-2) and Tyr-...

  2. Petroleum-degrading enzymes: bioremediation and new prospects.

    Science.gov (United States)

    Peixoto, R S; Vermelho, A B; Rosado, A S

    2011-01-01

    Anthropogenic forces, such as petroleum spills and the incomplete combustion of fossil fuels, have caused an accumulation of petroleum hydrocarbons in the environment. The accumulation of petroleum and its derivatives now constitutes an important environmental problem. Biocatalysis introduces new ways to improve the development of bioremediation strategies. The recent application of molecular tools to biocatalysis may improve bioprospecting research, enzyme yield recovery, and enzyme specificity, thus increasing cost-benefit ratios. Enzymatic remediation is a valuable alternative as it can be easier to work with than whole organisms, especially in extreme environments. Furthermore, the use of free enzymes avoids the release of exotic or genetically modified organisms (GMO) in the environment.

  3. Purification and Characterization of a Nonylphenol (NP)-degrading Enzyme from Bacillus cereus.Frankland

    Institute of Scientific and Technical Information of China (English)

    YANG Ge; ZHANG Ying; BAI Yanfen

    2011-01-01

    An extracellular NP-degrading enzyme secreted by Bacillus cereus.Frankland was purified to homogeneity by a combination of ammonium sulfate precipitation,Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography.On SDS(sodium dodecyl sulfate)-polyacrylamide gel electrophoresis analysis,the purified enzyme showed a relative molecular mass of 58.3 kDa.The depolymerzation of subunits was accompanied with the loss of NP-degrading enzyme activity,and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity.The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60℃,and was the most active at pH 6.0.The enzymatic activity was stable at pH 4-8 and inhibited by Cu2+.TenN-terminal amino acids were determined to be ASVNSIKIGY,demonstrating that the purified enzyme was a novel one.The hydrolysis pattern of the purified enzyme indicated that the NP-degrading enzyme was an endo NP-degrading enzyme.The extraordinary thermo-stability provided the enzyme with a good prospect to be used as a new tool for clean-production process for textile industry.

  4. Comparative analysis of the secretomes of Schizophyllum commune and other wood-decay basidiomycetes during solid-state fermentation reveals its unique lignocellulose-degrading enzyme system.

    Science.gov (United States)

    Zhu, Ning; Liu, Jiawen; Yang, Jinshui; Lin, Yujian; Yang, Yi; Ji, Lei; Li, Meng; Yuan, Hongli

    2016-01-01

    The genome of Schizophyllum commune encodes a diverse repertoire of degradative enzymes for plant cell wall breakdown. Recent comparative genomics study suggests that this wood decayer likely has a mode of biodegradation distinct from the well-established white-rot/brown-rot models. However, much about the extracellular enzyme system secreted by S. commune during lignocellulose deconstruction remains unknown and the underlying mechanism is poorly understood. In this study, extracellular proteins of S. commune colonizing Jerusalem artichoke stalk were analyzed and compared with those of two white-rot fungi Phanerochaete chrysosporium and Ceriporiopsis subvermispora and a brown-rot fungus Gloeophyllum trabeum. Under solid-state fermentation (SSF) conditions, S. commune displayed considerably higher levels of hydrolytic enzyme activities in comparison with those of P. chrysosporium, C. subvermispora and G. trabeum. During biodegradation process, this fungus modified the lignin polymer in a way which was consistent with a hydroxyl radical attack, similar to that of G. trabeum. The crude enzyme cocktail derived from S. commune demonstrated superior performance over a commercial enzyme preparation from Trichoderma longibrachiatum in the hydrolysis of pretreated lignocellulosic biomass at low enzyme loadings. Secretomic analysis revealed that compared with three other fungi, this species produced a higher diversity of carbohydrate-degrading enzymes, especially hemicellulases and pectinases acting on polysaccharide backbones and side chains, and a larger set of enzymes potentially supporting the generation of hydroxyl radicals. In addition, multiple non-hydrolytic proteins implicated in enhancing polysaccharide accessibility were identified in the S. commune secretome, including lytic polysaccharide monooxygenases (LPMOs) and expansin-like proteins. Plant lignocellulose degradation by S. commune involves a hydroxyl radical-mediated mechanism for lignocellulose modification

  5. Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes

    OpenAIRE

    2016-01-01

    The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspe...

  6. Hydrolysis of Brewers' Spent Grain by Carbohydrate Degrading Enzymes

    NARCIS (Netherlands)

    Forssell, P.; Kontkanen, H.; Schols, H.A.; Hinz, S.W.A.; Eijsink, V.G.H.; Treimo, J.; Robertson, J.A.; Waldron, K.W.; Faulds, C.B.; Buchert, J.

    2008-01-01

    In this work four commercial cellulase-hemicellulase mixtures with different activity profiles were used for solubilization of carbohydrates from brewers' spent grain (BSG). After the enzyme treatment, both the solubilised fraction and the unhydrolysed residue were characterized. Treatment with

  7. Effect of enzyme addition to forage at ensiling on silage chemical composition and NDF degradation characteristics

    DEFF Research Database (Denmark)

    Dehghani, Mohammad Reza; Weisbjerg, Martin Riis; Hvelplund, Torben

    2012-01-01

    The effect of different exogenous fibrolytic enzymes added to forages at ensiling was examined for effect on chemical composition and in vitro NDF degradability characteristics of the resulting silage. Maize stover and lucerne were used to study effect on chemical composition in experiment 1...... digestibility decreased in treated maize stover silage. Potential NDF degradability decreased due to enzyme treatments but not for all maize stover treatments. Treatments with combination of enzymes with glucanase, β-glucanase and pectinase activity mostly resulted in increases in fermentation products compared......, and two varieties of maize stover, lucerne and grass clover were used to study NDF degradation characteristics in experiment 2. Forages were treated with enzymes (500 mg crude protein of the enzyme products/kg DM) and ensiled for 60 days in vacuum-sealed bags. Samples of forage (before ensiling...

  8. Cell wall degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA species

    Institute of Scientific and Technical Information of China (English)

    Sanz L; Hermosa M R; González F J; Monte E

    2004-01-01

    @@ Species of the fungus Trichoderma, a genus of Hyphomycetes, are ubiquitous in the environment, but especially in soil. They have been used in a wide range of commercial applications including the production of hydrolases and in the biological control of plant diseases. A fundamental part of the Trichoderma antifungal system consists of a series of genes coding for a surprising variety of extracellular cell wall degrading enzymes (CWDE).Characterisation and identification of strains at the species level is the first step in utilizing the full potential of fungi in specific applications. One aim when isolating Trichoderma strains is to identify those which can be used in new agricultural and industrial applications. In the past it was not uncommon that biocontrol strains were defined as T. harzianum Rifai, due to the limited classification system of the genus Trichoderma. In recent years, several PCR-based molecular techniques have been used to detect and discriminate among microorganisms. Sequence analysis of the ITS regions of the ribosomal DNA and gene fragments as those corresponding to tef1 gene have been helpful in the neotypification, description and characterization of species in the genus Trichoderna.Another useful method for the identification of Trichoderma strains is the randomly amplified polymorphic DNA (RAPD) technique.Isozyme polymorphisms evaluation of five putative extracellular lytic enzymes loci (β-1,3-glucanase, β-1,6-glucanase, cellulase, chitinase and protease antivities) were carried out using representative strains of defined molecular groups. CWDE groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location and antifungal activities.Compiling morphological, biochemical and sequence information data into a common database would provide a useful resource that could be used to accurately name new haplotypes identified in the future and correctly place them within the genus Trichoderma.

  9. [Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes].

    Science.gov (United States)

    Zhu, Lili; Yang, Zhibang; Yang, Qian; Shi, Zhongquan; Deng, Xichuan

    2015-10-01

    In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.

  10. It’s War Out There: Fighting for life with xenobiotic degrading enzymes

    Science.gov (United States)

    It’s War Out There: Fighting for life with xenobiotic degrading enzymes Beta-lactamase enzymes are well studied because of their tremendous impact on medicine. Their prominent role is in resistance to beta-lactam (four membered lactam ring) antibiotics including the first and most famous fungally d...

  11. Degradative Enzymes from the Pharmacy or Health Food Store: Interesting Examples for Introductory Biology Laboratories

    Science.gov (United States)

    Deutch, Charles E.

    2007-01-01

    Degradative enzymes in over-the-counter products from pharmacies and health food stores provide good examples of biological catalysis. These include [beta]-galactosidase in Lactaid[TM], [alpha]-galactosidase in Beano[R], [alpha]-amylase and proteases in digestive aids, and proteases in contact lens cleaners. These enzymes can be studied…

  12. Degradative Enzymes from the Pharmacy or Health Food Store: Interesting Examples for Introductory Biology Laboratories

    Science.gov (United States)

    Deutch, Charles E.

    2007-01-01

    Degradative enzymes in over-the-counter products from pharmacies and health food stores provide good examples of biological catalysis. These include [beta]-galactosidase in Lactaid[TM], [alpha]-galactosidase in Beano[R], [alpha]-amylase and proteases in digestive aids, and proteases in contact lens cleaners. These enzymes can be studied…

  13. Petroleum-Degrading Enzymes: Bioremediation and New Prospects

    Directory of Open Access Journals (Sweden)

    R. S. Peixoto

    2011-01-01

    Full Text Available Anthropogenic forces, such as petroleum spills and the incomplete combustion of fossil fuels, have caused an accumulation of petroleum hydrocarbons in the environment. The accumulation of petroleum and its derivatives now constitutes an important environmental problem. Biocatalysis introduces new ways to improve the development of bioremediation strategies. The recent application of molecular tools to biocatalysis may improve bioprospecting research, enzyme yield recovery, and enzyme specificity, thus increasing cost-benefit ratios. Enzymatic remediation is a valuable alternative as it can be easier to work with than whole organisms, especially in extreme environments. Furthermore, the use of free enzymes avoids the release of exotic or genetically modified organisms (GMO in the environment.

  14. Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes.

    Science.gov (United States)

    Burke, Thomas P; Loukitcheva, Anastasia; Zemansky, Jason; Wheeler, Richard; Boneca, Ivo G; Portnoy, Daniel A

    2014-11-01

    Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. Understanding Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance (Presentation)

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.; Donohoe, B.; Katahira, R.; Ashutosh, M.; Beckham, G.; Himmel, M.; Decker, S.

    2014-04-01

    Fungal free enzymes and bacterial complexed cellulosomes deconstruct biomass using different physical mechanisms. Free enzymes, which typically contain a large proportion of GH7 cellobiohydrolase, diffuse throughout the substrate and hydrolyze primarily from the cellulose reducing end, resulting in 'sharpened' macrofibrils. In contrast, complexed cellulosomes contain a diverse array of carbohydrate binding modules and multiple catalytic specificities leading to delamination and physical peeling of the cellulose macrofibril structures. To investigate how cellulose structure contributes to recalcitrance, we compared the deconstruction of cellulose I, II, and III; using free and complexed enzyme systems. We also evaluated both systems on Clean Fractionation and alkaline pretreated biomass, which remove much of the lignin, to determine the impact on enzyme loading reduction. Free fungal enzymes demonstrated a swelling of the outer surface of the plant cell walls while removing localized disruptions, resulting in a smooth surface appearance. Cellulosomes produced cell wall surfaces with localized areas of disruption and little surface layer swelling. These studies contribute to the overall understanding of biomass recalcitrance and how combining different enzymatic paradigms may lead to the formulation of new enzyme cocktails to reduce the cost of producing sugars from plant cell wall carbohydrates.

  16. Contribution of AmyA, an extracellular alpha-glucan degrading enzyme, to group A streptococcal host-pathogen interaction.

    Science.gov (United States)

    Shelburne, Samuel A; Keith, David B; Davenport, Michael T; Beres, Stephen B; Carroll, Ronan K; Musser, James M

    2009-10-01

    alpha-Glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular alpha-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in alpha-glucan binding and degradation; pulA encodes a cell wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin alpha-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS alpha-glucan degradation. The DeltaamyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the DeltaamyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded alpha-glucans into beta-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared with strains causing pharyngitis. GAS proliferation in a defined alpha-glucan-containing medium was dependent on the presence of human salivary alpha-amylase. These data delineate the molecular mechanisms by which alpha-glucan degradation contributes to GAS host-pathogen interaction, including how GAS uses human salivary alpha-amylase for its own metabolic benefit.

  17. Hydrolysis of Brewers' Spent Grain by Carbohydrate Degrading Enzymes

    NARCIS (Netherlands)

    Forssell, P.; Kontkanen, H.; Schols, H.A.; Hinz, S.W.A.; Eijsink, V.G.H.; Treimo, J.; Robertson, J.A.; Waldron, K.W.; Faulds, C.B.; Buchert, J.

    2008-01-01

    In this work four commercial cellulase-hemicellulase mixtures with different activity profiles were used for solubilization of carbohydrates from brewers' spent grain (BSG). After the enzyme treatment, both the solubilised fraction and the unhydrolysed residue were characterized. Treatment with 5,00

  18. Affinity purification of polysaccharide degrading enzymes with crosslinked substrates

    NARCIS (Netherlands)

    Rozie, H.J.

    1992-01-01

    The aim of this work was to find economically favourable, affinity based, purification methods for several polysaccharide splitting bulk enzymes. The framework in which this study is done is described in Chapter 1.

    Chapter 2 describes the adsorption of endo-polygalacturonase (endoPG

  19. Affinity purification of polysaccharide degrading enzymes with crosslinked substrates

    NARCIS (Netherlands)

    Rozie, H.J.

    1992-01-01

    The aim of this work was to find economically favourable, affinity based, purification methods for several polysaccharide splitting bulk enzymes. The framework in which this study is done is described in Chapter 1.

    Chapter 2 describes the adsorption of endo-polygalacturonase

  20. Polydopamine microcapsules with different wall structures prepared by a template-mediated method for enzyme immobilization.

    Science.gov (United States)

    Shi, Jiafu; Yang, Chen; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi; Zhang, Wenyan; Song, Xiaokai; Ai, Qinghong; Tian, Chunyong

    2013-10-23

    Microcapsules with diverse wall structures may exhibit different performance in specific applications. In the present study, three kinds of mussel-inspired polydopamine (PDA) microcapsules with different wall structures have been prepared by a template-mediated method. More specifically, three types of CaCO3 microspheres (poly(allylamine hydrochloride), (PAH)-doped CaCO3; pure-CaCO3; and poly(styrene sulfonate sodium), (PSS)-doped CaCO3) were synthesized as sacrificial templates, which were then treated by dopamine to obtain the corresponding PDA-CaCO3 microspheres. Through treating these microspheres with disodium ethylene diamine tetraacetic acid (EDTA-2Na) to remove CaCO3, three types of PDA microcapsules were acquired: that was (1) PAH-PDA microcapsule with a thick (∼600 nm) and highly porous capsule wall composed of interconnected networks, (2) pure-PDA microcapsule with a thick (∼600 nm) and less porous capsule wall, (3) PSS-PDA microcapsule with a thin (∼70 nm) and dense capsule wall. Several characterizations confirmed that a higher degree in porosity and interconnectivity of the capsule wall would lead to a higher mass transfer coefficient. When serving as the carrier for catalase (CAT) immobilization, these enzyme-encapsulated PDA microcapsules showed distinct structure-related activity and stability. In particular, PAH-PDA microcapsules with a wall of highly interconnected networks displayed several significant advantages, including increases in enzyme encapsulation efficiency and enzyme activity/stability and a decrease in enzyme leaching in comparison with other two types of PDA microcapsules. Besides, this hierarchically structured PAH-PDA microcapsule may find other promising applications in biocatalysis, biosensors, drug delivery, etc.

  1. Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles.

    Science.gov (United States)

    Gao, Yuan; Truong, Yen Bach; Cacioli, Paul; Butler, Phil; Kyratzis, Ilias Louis

    2014-01-10

    Bioremediation using enzymes has become an attractive approach for removing hazardous chemicals such as organophosphate pesticides from the environment. Enzymes immobilized on solid carriers are particularly suited for such applications. In this study, the organophosphate degrading enzyme A (OpdA) was covalently immobilized on highly porous nonwoven polyester fabrics for organophosphate pesticide degradation. The fabrics were first activated with ethylenediamine to introduce free amine groups, and the enzyme was then attached using the bifunctional crosslinker glutaraldehyde. The immobilization only slightly increased the Km (for methyl parathion, MP), broadened the pH profile such that the enzyme had significant activity at acidic pH, and enhanced the stability of the enzyme. The OpdA-functionalized fabrics could be stored in a phosphate buffer or in the dry state at 4°C for at least 4 weeks without a large loss of activity. When used in batch mode, the functionalized textiles could degrade 20 μM MP in un-buffered water at liquor to fabric ratios as high as 5000:1 within 2h, and could be used repeatedly. The fabrics could also be made into columns for continuous pesticide degradation. The columns were able to degrade 50 μM MP at high flow rates, and could be used repeatedly over 2 months. These results demonstrate that OpdA immobilized on nonwoven polyester fabrics is useful in environmental remediation of organophosphate compounds.

  2. [Ligninolytic enzyme production by white rot fungi during paraquat (herbicide) degradation].

    Science.gov (United States)

    Camacho-Morales, Reyna L; Gerardo-Gerardo, José Luis; Guillén Navarro, Karina; Sánchez, José E

    Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  3. Degradation of 4-aminophenol by hydrogen peroxide oxidation using enzyme from Serratia marcescens as catalyst

    Institute of Scientific and Technical Information of China (English)

    SUN Min; YAO Risheng; YOU Yahua; DENG Shengsong; GAO Wenxia

    2007-01-01

    This paper reports on the degradation of 4-aminophenol using hydrogen peroxide as oxidizer and the enzyme from Serratia marcescens AB 90027 as catalyst.The effecting factors during degradation and the degrading mechanism were studied.Also,the location of the enzyme in the cell,which could catalyze the degradation of 4-aminophenol,was analyzed.The results showed that to degrade 50 mL of 4-aminophenol whose concentration was 500 mg/L,the optimal conditions were:volume of H2O2=3 mL,temperature=40-60℃ and pH=9-10]In the degradation process,4-aminophenol was first converted to benzo quinone and NH3,then organic acids including maleic acid,fumaleic acid,and oxalic acid were formed,and then finally CO2 and H2O were generated as final products.The enzyme that could catalyze the degradation of 4-aminophenol was mainly extracellular enzyme.

  4. Enzymes and Genes Involved in Aerobic Alkane Degradation

    Directory of Open Access Journals (Sweden)

    Zongze eShao

    2013-05-01

    Full Text Available Alkanes are major constituents of crude oil. They are also present at low concentrations in diverse non-contaminated because many living organisms produce them as chemo-attractants or as protecting agents against water loss. Alkane degradation is a widespread phenomenon in nature. The numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing alkanes as a carbon and energy source, have been isolated and characterized. This review summarizes the current knowledge of how bacteria metabolize alkanes aerobically, with a particular emphasis on the oxidation of long-chain alkanes, including factors that are responsible for chemotaxis to alkanes , transport across cell membrane of alkanes , the regulation of alkane degradation gene and initial oxidation.

  5. The 3-ureidopropionase of Caenorhabditis elegans, an enzyme involved in pyrimidine degradation.

    Science.gov (United States)

    Janowitz, Tim; Ajonina, Irene; Perbandt, Markus; Woltersdorf, Christian; Hertel, Patrick; Liebau, Eva; Gigengack, Ulrike

    2010-10-01

    Pyrimidines are important metabolites in all cells. Levels of cellular pyrimidines are controlled by multiple mechanisms, with one of these comprising the reductive degradation pathway. In the model invertebrate Caenorhabditis elegans, two of the three enzymes of reductive pyrimidine degradation have previously been characterized. The enzyme catalysing the final step of pyrimidine breakdown, 3-ureidopropionase (β-alanine synthase), had only been identified based on homology. We therefore cloned and functionally expressed the 3-ureidopropionase of C. elegans as hexahistidine fusion protein. The purified recombinant enzyme readily converted the two pyrimidine degradation products: 3-ureidopropionate and 2-methyl-3-ureidopropionate. The enzyme showed a broad pH optimum between pH 7.0 and 8.0. Activity was highest at approximately 40 °C, although the half-life of activity was only 65 s at that temperature. The enzyme showed clear Michaelis-Menten kinetics, with a K(m) of 147 ± 26 μM and a V(max) of 1.1 ± 0.1 U·mg protein(-1). The quaternary structure of the recombinant enzyme was shown to correspond to a dodecamer by 'blue native' gel electrophoresis and gel filtration. The organ specific and subcellular localization of the enzyme was determined using a translational fusion to green fluorescent protein and high expression was observed in striated muscle cells. With the characterization of the 3-ureidopropionase, the reductive pyrimidine degradation pathway in C. elegans has been functionally characterized.

  6. Integrative computational approach for genome-based study of microbial lipid-degrading enzymes.

    Science.gov (United States)

    Vorapreeda, Tayvich; Thammarongtham, Chinae; Laoteng, Kobkul

    2016-07-01

    Lipid-degrading or lipolytic enzymes have gained enormous attention in academic and industrial sectors. Several efforts are underway to discover new lipase enzymes from a variety of microorganisms with particular catalytic properties to be used for extensive applications. In addition, various tools and strategies have been implemented to unravel the functional relevance of the versatile lipid-degrading enzymes for special purposes. This review highlights the study of microbial lipid-degrading enzymes through an integrative computational approach. The identification of putative lipase genes from microbial genomes and metagenomic libraries using homology-based mining is discussed, with an emphasis on sequence analysis of conserved motifs and enzyme topology. Molecular modelling of three-dimensional structure on the basis of sequence similarity is shown to be a potential approach for exploring the structural and functional relationships of candidate lipase enzymes. The perspectives on a discriminative framework of cutting-edge tools and technologies, including bioinformatics, computational biology, functional genomics and functional proteomics, intended to facilitate rapid progress in understanding lipolysis mechanism and to discover novel lipid-degrading enzymes of microorganisms are discussed.

  7. Developments in application of light and scanning electron microscopy techniques for cell wall degradation studies.

    NARCIS (Netherlands)

    Engels, F.M.

    1996-01-01

    The results of recent technological developments in light and scanning electron microscopy closely used for research on forage cell wall degradation in ruminants, are reviewed. The indigestibility of forages by rumen microorganisms used to be ascribed mainly to an overall presence of lignin in the p

  8. Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes.

    Science.gov (United States)

    Thomsen, Malene S; Nidetzky, Bernd

    2009-01-01

    Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB.

  9. Selective degradation of the recalcitrant cell wall of Scenedesmus quadricauda CASA CC202.

    Science.gov (United States)

    Reshma, Ragini; Arumugam, Muthu

    2017-07-06

    An eco-friendly cell wall digestion strategy was developed to enhance the availability of nutritionally important bio molecules of edible microalgae and exploit them for cloning, transformation, and expression of therapeutic proteins. Microalgae are the source for many nutritionally important bioactive compounds and potential drugs. Even though edible microalgae are rich in nutraceutical, bioavailability of all these molecules is very less due to their rigid recalcitrant cell wall. For example, the cell wall of Scenedesmus quadricauda CASA CC202 is made up of three layers comprising of rigid outer pectin and inner cellulosic layer separated by a thin middle layer. In the present investigation, a comprehensive method has been developed for the selective degradation of S. quadricauda CASA CC202 cell wall, by employing both mechanical and enzymatic treatments. The efficiency of cell wall removal was evaluated by measuring total reducing sugar (TRS), tannic acid-ferric chloride staining, calcoflour white staining, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) analysis. It was confirmed that the yield of TRS increased from 129.82 mg/g in 14 h from pectinase treatment alone to 352.44 mg/g by combined sonication and enzymatic treatment within 12 h. As a result, the combination method was found to be effective for the selective degradation of S. quadricauda CASA CC202 cell wall. This study will form a base for our future works, where this will help to enhance the digestibility and availability of nutraceutically important proteins.

  10. Immobilization of oxalate-degrading enzymes into p(HEMA) for inhibiting encrustation on ureteral stents

    Science.gov (United States)

    Mellman, James Kenneth

    Ureteral stents develop calcium-bearing deposits, called encrustation, that diminish their biocompatibility due to complications, such as chronic abrasion to the lumen of the ureter wall and subsequent infection. A reduction of encrustation, namely calcium oxalate, will improve the lifetime, health care costs, and infection resistance of such devices. The purpose of this research project is to study oxalate-degrading enzymes entrapped into a coating material that will control the interface to the urinary environment for ureteral stents. The coating material was a lightly crosslinked poly(2-hydroxyethyl methacrylate) (p(HEMA)) matrix in which the active enzymes were entrapped within the bulk material's free volume. The swelling of p(HEMA) films was comparable in ddH2O and urine. This hydrophilic matrix allows oxalate anions to diffuse into the bulk so that enzyme activity against oxalate can lower its local concentration, and thereby reduce the supersaturation of calcium oxalate. Oxalate oxidase (OxO) and oxalate decarboxylase (OxDc) were the oxalate-degrading enzymes examined herein. Michaelis Menten kinetic models were applied to free and immobilized enzyme activity. A substrate inhibition model was applied to OxO. The free form of OxO had a Vmax of 1.8 +/- 0.1 muM/min-mug, a km of 1.8 +/- 0.1 mM, and a ks of 35.4 +/- 3.7 mM while the immobilized form had a Vmax of 1.2 +/- 0.2 muM/min-mug, a km of 4.1 +/- 0.6 mM, and a ks of 660 +/- 140 mM. The free form of OxDc had a Vmax of 23.5 +/- 1.4 muM/min-mug and a km of 0.5 +/- 0.1 mM while the immobilized form had a Vmax of 5.0 +/- 1.9 muM/min-mug and km of 23.2 +/- 9.1 mM. The enzyme activity was measured to indicate viable application conditions for the coating, such as storing the films in urine over time. The maximum activity was shown at pH 4.2 to 4.5 and activity drops to be negligible by pH 7.0. Storing the enzyme at pH 6.1 exhibited a larger retained activity than storing at pH 4.2, yet storing in urine showed

  11. Potential of extracellular enzymes from Trametes versicolor F21a in Microcystis spp. degradation.

    Science.gov (United States)

    Du, Jingjing; Pu, Gaozhong; Shao, Chen; Cheng, Shujun; Cai, Ji; Zhou, Liang; Jia, Yong; Tian, Xingjun

    2015-03-01

    Studies have shown that microorganisms may be used to eliminate cyanobacteria in aquatic environments. The present study showed that the white-rot fungus Trametes versicolor F21a could degrade Microcystis aeruginosa. After T. versicolor F21a and Microcystis spp. were co-incubated for 60h, >96% of Microcystis spp. cells were degraded by T. versicolor F21a. The activities of extracellular enzymes showed that cellulase, β-glucosidase, protease, and laccase were vital to Microcystis spp. degradation in the early stage (0h to 24h), while β-glucosidase, protease, laccase, and manganese peroxidase in the late stage (24h to 60h). The positive and significant correlation of the degradation rate with these enzyme activities indicated that these enzymes were involved in the degradation rate of Microcystis spp. cells at different phases. It suggested that the extracellular enzymes released by T. versicolor F21a might be vital to Microcystis spp. degradation. The results of this study may be used to develop alternative microbial control agents for cyanobacterial control.

  12. Relating Nanoscale Accessibility within Plant Cell Walls to Improved Enzyme Hydrolysis Yields in Corn Stover Subjected to Diverse Pretreatments.

    Science.gov (United States)

    Crowe, Jacob D; Zarger, Rachael A; Hodge, David B

    2017-10-04

    Simultaneous chemical modification and physical reorganization of plant cell walls via alkaline hydrogen peroxide or liquid hot water pretreatment can alter cell wall structural properties impacting nanoscale porosity. Nanoscale porosity was characterized using solute exclusion to assess accessible pore volumes, water retention value as a proxy for accessible water-cell walls surface area, and solute-induced cell wall swelling to measure cell wall rigidity. Key findings concluded that delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity and that the subsequent cell wall swelling resulted increased nanoscale porosity and improved enzyme binding and hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 Å dextran probe within the cell wall was found to be correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields.

  13. Microbial surface displayed enzymes based biofuel cell utilizing degradation products of lignocellulosic biomass for direct electrical energy.

    Science.gov (United States)

    Fan, Shuqin; Hou, Chuantao; Liang, Bo; Feng, Ruirui; Liu, Aihua

    2015-09-01

    In this work, a bacterial surface displaying enzyme based two-compartment biofuel cell for the direct electrical energy conversion from degradation products of lignocellulosic biomass is reported. Considering that the main degradation products of the lignocellulose are glucose and xylose, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) were used as anode catalysts in anode chamber with methylene blue as electron transfer mediator. While the cathode chamber was constructed with laccase/multi-walled-carbon nanotube/glassy-carbon-electrode. XDH-bacteria exhibited 1.75 times higher catalytic efficiency than GDH-bacteria. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. Moreover, the maximum power density of the cell could reach 53 μW cm(-2) when fueled with degradation products of corn stalk. Thus, this finding holds great potential to directly convert degradation products of biomass into electrical energy.

  14. Cell wall ultrastructure of flocculent and non-flocculent Schizosaccharomyces pombe strains. Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure.

    Science.gov (United States)

    Geleta, Anna; Kristóf, Z; Maráz, Anna

    2007-03-01

    Scanning and transmission electron microscopic studies revealed the presence of slime-like, amorphous material on the surface of Schizosaccahromyces pombe RIVE 4-2-1 cells, independently, whether they were in flocculated or in non-flocculated state. Close contact of the adjacent cells via the merging outermost cell wall layers was found, however, only in the case of floc formation, which was induced by cultivating the cells in the presence of 6% (v/v) ethanol. Irreversible loss of the flocculation ability of the cells by treatment with proteinases suggests that proteinaceous cell surface molecules as lectins contribute to the cell-to-cell interaction during flocculation. Both proteinase K and pronase treatments removed a distinct outer layer of the cell wall, which indicated that the protein moieties of the phosphogalactomannan outer surface layer has a crucial role in the maintenance of cell wall integrity. In the case of lysing enzyme treatment the removal of the outermost layer was also observed as the first step of the cell wall digestion, while driselase treatment resulted in almost complete digestion of the cell wall.

  15. Interaction and modulation of two antagonistic cell wall enzymes of mycobacteria.

    Directory of Open Access Journals (Sweden)

    Erik C Hett

    Full Text Available Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB, a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA, an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1, as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein-protein interactions between enzymes with antagonistic functions.

  16. Peroxides and peroxide-degrading enzymes in the thyroid.

    Science.gov (United States)

    Schweizer, Ulrich; Chiu, Jazmin; Köhrle, Josef

    2008-09-01

    Iodination of thyroglobulin is the key step of thyroid hormone biosynthesis. It is catalyzed by thyroid peroxidase and occurs within the follicular space at the apical plasma membrane. Hydrogen peroxide produced by thyrocytes as an oxidant for iodide may compromise cellular and genomic integrity of the surrounding cells, unless these are sufficiently protected by peroxidases. Thus, peroxidases play two opposing roles in thyroid biology. Both aspects of peroxide biology in the thyroid are separated in space and time and respond to the different physiological states of the thyrocytes. Redox-protective peroxidases in the thyroid are peroxiredoxins, glutathione peroxidases, and catalase. Glutathione peroxidases are selenoenzymes, whereas selenium-independent peroxiredoxins are functionally linked to the selenoenzymes of the thioredoxin reductase family through their thioredoxin cofactors. Thus, selenium impacts directly and indirectly on protective enzymes in the thyroid, a link that has been supported by animal experiments and clinical observations. In view of this relationship, it is remarkable that rather little is known about selenoprotein expression and their potential functional roles in the thyroid. Moreover, selenium-dependent and -independent peroxidases have rarely been examined in the same studies. Therefore, we review the relevant literature and present expression data of both selenium-dependent and -independent peroxidases in the murine thyroid.

  17. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    Science.gov (United States)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  18. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    Science.gov (United States)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-01-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  19. Magnetite-alginate beads for purification of some starch degrading enzymes.

    Science.gov (United States)

    Teotia, Sunita; Gupta, M N

    2002-03-01

    Starch degrading enzymes, viz., beta-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. beta-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.

  20. A neglected modulator of insulin-degrading enzyme activity and conformation: The pH.

    Science.gov (United States)

    Grasso, Giuseppe; Satriano, Cristina; Milardi, Danilo

    2015-01-01

    Insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease, has multiple activities in addition to insulin degradation and its malfunction is believed to connect type 2 diabetes with Alzheimer's disease. IDE has been found in many different cellular compartments, where it may experience significant physio-pathological pH variations. However, the exact role of pH variations on the interplay between enzyme conformations, stability, oligomerization state and catalysis is not understood. Here, we use ESI mass spectrometry, atomic force microscopy, surface plasmon resonance and circular dichroism to investigate the structure-activity relationship of IDE at different pH values. We show that acidic pH affects the ability of the enzyme to bind the substrate and decrease the stability of the protein by inducing an α-helical bundle conformation with a concomitant dissociation of multi-subunit IDE assemblies into monomeric units and loss of activity. These effects suggest a major role played by electrostatic forces in regulating multi-subunit enzyme assembly and function. Our results clearly indicate a pH dependent coupling among enzyme conformation, assembly and stability and suggest that cellular acidosis can have a large effect on IDE oligomerization state, inducing an enzyme inactivation and an altered insulin degradation that could have an impact on insulin signaling.

  1. Changes in the activities of enzymes involved in the degradation of butylbenzyl phthalate by Pleurotus ostreatus.

    Science.gov (United States)

    Hwang, Soon-Seok; Kim, Hyoun-Young; Ka, Jong-Ok; Song, Hong-Gyu

    2012-02-01

    Degradation of butylbenzyl phthalate (BBP) by the white rot fungus Pleurotus ostreatus and the activities of some degrading enzymes were examined in two different media containing 100 mg/l of the compound. P. ostreatus pregrown for 7 days in complex YMG medium was able to completely degrade BBP within an additional 24 h but degraded only 35 mg/l of BBP in 5 days of incubation in minimal medium. Fungal cell mass in the culture in YMG medium was higher in the presence than in the absence of BBP. The esterase activity of the fungal culture in YMG medium was higher than that in minimal medium and increased with the addition of BBP. On the contrary, laccase activity was higher in minimal medium and it did not increase upon the addition of BBP. General peroxidase activity increased for a few days after the addition of BBP to both media. The degradation of BBP and its metabolites by P. ostreatus thus may be attributed mostly to esterase rather than lignin-degrading laccase. In addition, the activities of the enzymes involved in BBP degradation and their changes varied significantly in the different media and culture conditions.

  2. Enzyme activities during degradation of polycyclic aromatic hydrocarbons by white rot fungus Phanerochaete chrysosporium in soils.

    Science.gov (United States)

    Wang, Cuiping; Sun, Hongwen; Li, Jieming; Li, Yimeng; Zhang, Qingmin

    2009-10-01

    The degradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, pyrene and benzo[a]pyrene in soils by Phanerochaete chrysosporium, and the enzyme activities of lignin peroxidase (LiP) and manganese peroxidase (MnP) produced during degradation, were analyzed. The results showed that the 19-d percentage degradation ranged from 72.77+/-1.39% to 25.50+/-3.41% for the three compounds, and the maximum LiP and MnP activities ranged from 0.16+/-0.005 to 0.05+/-0.002 U g(-1) and from 1.92+/-0.03 to 0.54+/-0.03 U g(-1), respectively. Degradation percentage and enzyme activities both exhibited inverse relationships with the octanol/water partition coefficient (K(ow)) of the compounds, indicating that LiP and MnP from P. chrysosporium may be the primary enzymes responsible for PAH degradation in soil. As the soil organic matter (SOM) content increased from 0.3% for Soil 1 to 19% for Soil 4, the 19-d degradation percentage of pyrene decreased from 66.20+/-2.72% to 32.42+/-1.05%, and correspondingly, the maximum of LiP and MnP activities increased from 0.05+/-0.002 to 1.78+/-0.15 U g(-1) and from 0.34+/-0.03 to 1.78+/-0.15 U g(-1), respectively. Hence, it is plausible to conclude that the P. chrysosporium appeared to degrade not only the PAHs with small molecular size but also the macromolecular SOM. When SOM differences are large, as in this study, SOM has greater influence on enzyme activity than low-level exotic pollutants.

  3. Insulin-degrading enzyme: a link between Alzheimer's and type 2 diabetes mellitus.

    Science.gov (United States)

    Haque, Rizwanul; Nazir, Aamir

    2014-03-01

    Enzymes play a very vital role in maintaining the homeostasis inside the body. Improper functioning of enzymes is associated with many diseases. Insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease, is believed to act as a junction point of Type 2 Diabetes and Alzheimer's disease. Recent studies provide inkling for the use of IDE as a potential target hence the design of its regulators would be a viable approach towards treatment of these diseases. This review provides an overview of the IDE structure and function; a relationship is drawn between IDE, Type 2 Diabetes mellitus and Alzheimer's disease and the approaches that make IDE a potential target, are discussed.

  4. Plant Cell Wall Degradation by Saprophytic Bacillus subtilis Strains: Gene Clusters Responsible for Rhamnogalacturonan Depolymerization▿

    Science.gov (United States)

    Ochiai, Akihito; Itoh, Takafumi; Kawamata, Akiko; Hashimoto, Wataru; Murata, Kousaku

    2007-01-01

    Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation. PMID:17449691

  5. Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization.

    Science.gov (United States)

    Ochiai, Akihito; Itoh, Takafumi; Kawamata, Akiko; Hashimoto, Wataru; Murata, Kousaku

    2007-06-01

    Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.

  6. Insulin-degrading enzyme inhibition, a novel therapy for type 2 diabetes?

    Science.gov (United States)

    Costes, Safia; Butler, Peter C

    2014-08-01

    The insulin-degrading enzyme (IDE) has been identified as a type 2 diabetes and Alzheimer's disease susceptibility gene, though its physiological function remains unclear. Maianti et al. (2014) now propose that an IDE inhibitor may be a promising therapeutic strategy for type 2 diabetes.

  7. BACILLUS SUBTILIS SJ01 PRODUCES HEMICELLULOSE DEGRADING MULTI-ENZYME COMPLEXES

    Directory of Open Access Journals (Sweden)

    Brett Ivan Pletschke

    2012-01-01

    Full Text Available Cellulose and hemicellulose account for a large portion of the world’s plant biomass. In nature, these polysaccharides are intertwined, forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The aim of this study was to isolate and characterise a (hemi- cellulolytic MEC from the aerobic bacterium, Bacillus subtilis SJ01, using ultrafiltration followed by size-exclusion chromatography on a Sephacryl S-400 column. Two MECs, C1 and C2 of 371 and 267 kDa, respectively, were purified, consisting of 16 and 18 subunits, respectively, five of which degraded birchwood and oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan with higher efficiency than amorphous cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg carboxymethyl cellulose - CMC. Low or no binding to insoluble substrates indicated that the MECs lacked some of the features characteristic of cellulosomes. The significance of this study lies in the discovery of MECs that differ structurally from cellulosomes that can hydrolyse substrates with high hemicellulose content.

  8. Targeted discovery and functional characterisation of complex-xylan degrading enzymes

    NARCIS (Netherlands)

    Gool, van M.P.

    2012-01-01

    This thesis describes the development of a screening method to discover efficient hemicellulase producers in a wide range of fungi. The method is based on the potential of soil fungi to degrade soluble and insoluble xylan-rich substrates, by assigning various individual enzyme activities. Released m

  9. Enzyme kinetics and identification of the rate-limiting step of enzymatic arabinoxylan degradation

    DEFF Research Database (Denmark)

    Rasmussen, Louise Enggaard; Xu, Cheng; Sørensen, Jens

    2012-01-01

    This study investigated the kinetics of multi-enzymatic degradation of soluble wheat arabinoxylan by monitoring the release of xylose and arabinose during designed treatments with mono-component enzymes at different substrate concentrations. The results of different combinations of α-l-arabinofur...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  12. Degradation study of XVIII century graffiti on the walls of Chiaramonte Palace (Palermo, Italy)

    Science.gov (United States)

    Alberghina, M. F.; Barraco, R.; Brai, M.; Casaletto, M. P.; Ingo, G. M.; Marrale, M.; Policarpo, D.; Schillaci, T.; Tranchina, L.

    2010-09-01

    A systematic investigation of the original materials and the degradation phenomena induced by soluble salts on the wall matrix and on the graffiti of the Inquisition jails of Chiaramonte Palace in Palermo (Italy) was carried out. Built in the XIV century, Chiaramonte Palace was used as Inquisition court during the XV-XVI centuries. The ancient graffiti, recently discovered, represent a unique historical witness of the prisoners that lived during that terrible period. In order to study the nature, the amount and the distribution of the salts in the masonry, stone materials sampled at different depth from the wall matrix and saline efflorescences were analysed. Different physical techniques were used, such as X-ray Photoelectron Spectroscopy, X-ray Fluorescence, Laser Induced Breakdown Spectroscopy, X-ray Diffraction and Ionic Chromatography. Results of the chemical and physical characterisation showed that the main cause of the degradation of graffiti and wall paintings was the presence of soluble salts, such as nitrates, chlorides and sulphates. Traces of oxalates, coming from a previous conservation treatment, were also found. The results obtained by the stratigraphical characterisation of soluble salts in the wall matrix can be used to recommend a procedure based on air humidification at high relative humidity values in order to avoid salt crystallisation and to prevent the crumbling process of the graffiti.

  13. Insulin-degrading enzyme: new therapeutic target for diabetes and Alzheimer's disease?

    Science.gov (United States)

    Pivovarova, Olga; Höhn, Annika; Grune, Tilman; Pfeiffer, Andreas F H; Rudovich, Natalia

    2016-12-01

    Insulin-degrading enzyme (IDE) is a major enzyme responsible for insulin degradation. In addition to insulin, IDE degrades many targets including glucagon, atrial natriuretic peptide, and beta-amyloid peptide, regulates proteasomal degradation and other cell functions. IDE represents a pathophysiological link between type 2 diabetes (T2DM) and late onset Alzheimer's disease (AD). Potent and selective modulators of IDE activity are potential drugs for therapies of both diseases. Acute treatment with a novel IDE inhibitor was recently tested in a mouse study as a therapeutic approach for the treatment of T2DM. In contrast, effective IDE activators can be used for the AD treatment. However, because of the pleiotropic IDE action, the sustained treatment with systemic IDE modulators should be carefully tested in animal studies. Development of substrate-selective IDE modulators could overcome possible adverse effects of IDE modulators associated with multiplicity of IDE targets. KEY MESSAGES Insulin-degrading enzyme (IDE) represents a pathophysiological link between type 2 diabetes (T2DM) and Alzheimer's disease (AD). Selective modulators of IDE activity are potential drugs for both T2DM and AD treatment. Development of substrate-selective IDE modulators could overcome possible adverse effects of IDE modulators associated with multiplicity of IDE targets.

  14. Understanding lignin-degrading reactions of ligninolytic enzymes: binding affinity and interactional profile.

    Science.gov (United States)

    Chen, Ming; Zeng, Guangming; Tan, Zhongyang; Jiang, Min; Li, Hui; Liu, Lifeng; Zhu, Yi; Yu, Zhen; Wei, Zhen; Liu, Yuanyuan; Xie, Gengxin

    2011-01-01

    Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity.

  15. Understanding lignin-degrading reactions of ligninolytic enzymes: binding affinity and interactional profile.

    Directory of Open Access Journals (Sweden)

    Ming Chen

    Full Text Available Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac, lignin peroxidase (LiP and manganese peroxidase (MnP. In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity.

  16. Cysteine 904 is required for maximal insulin degrading enzyme activity and polyanion activation.

    Directory of Open Access Journals (Sweden)

    Eun Suk Song

    Full Text Available Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent. Based on the structure of IDE, Asn 575 was identified as a potential hydrogen bond partner for Cys904 and mutation of this residue also reduced activity and decreased polyanion activation. The oligomerization state of IDE did not correlate with its activity, with the dimer being the predominant form in all the samples examined. These data suggest that there are several conformational states of the dimer that affect activity and polyanion activation.

  17. Preparation of supramolecular hydrogel-enzyme hybrids exhibiting biomolecule-responsive gel degradation.

    Science.gov (United States)

    Shigemitsu, Hajime; Fujisaku, Takahiro; Onogi, Shoji; Yoshii, Tatsuyuki; Ikeda, Masato; Hamachi, Itaru

    2016-09-01

    Hydrogelators are small, self-assembling molecules that form supramolecular nanofiber networks that exhibit unique dynamic properties. Development of supramolecular hydrogels that degrade in response to various biomolecules could potentially be used for applications in areas such as drug delivery and diagnostics. Here we provide a synthetic procedure for preparing redox-responsive supramolecular hydrogelators that are used to create hydrogels that degrade in response to oxidizing or reducing conditions. The synthesis takes ∼2-4 d, and it can potentially be carried out in parallel to prepare multiple hydrogelator candidates. This described solid-phase peptide synthesis protocol can be used to produce previously described hydrogelators or to construct a focused molecular library to efficiently discover and optimize new hydrogelators. In addition, we describe the preparation of redox-responsive supramolecular hydrogel-enzyme hybrids that are created by mixing aqueous solutions of hydrogelators and enzymes, which requires 2 h for completion. The resultant supramolecular hydrogel-enzyme hybrids exhibit gel degradation in response to various biomolecules, and can be rationally designed by connecting the chemical reactions of the hydrogelators with enzymatic reactions. Gel degradation in response to biomolecules as triggers occurs within a few hours. We also describe the preparation of hydrogel-enzyme hybrids arrayed on flat glass slides, enabling high-throughput analysis of biomolecules such as glucose, uric acid, lactate and so on by gel degradation, which is detectable by the naked eye. The protocol requires ∼6 h to prepare the hydrogel-enzyme hybrid array and to complete the biomolecule assay.

  18. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

    Science.gov (United States)

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community. PMID:26441929

  19. Degradation of pheromone and plant volatile components by a same odorant-degrading enzyme in the cotton leafworm, Spodoptera littoralis.

    Directory of Open Access Journals (Sweden)

    Nicolas Durand

    Full Text Available BACKGROUND: Odorant-Degrading Enzymes (ODEs are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. METHODOLOGY: Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis, a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. CONCLUSION: SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

  20. A Cell Wall-degrading Endopolygalacturonase Secreted by Colletotrichum lindemuthianum1

    Science.gov (United States)

    English, Patricia D.; Maglothin, Austin; Keegstra, Kenneth; Albersheim, Peter

    1972-01-01

    Cultures of Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner have been induced to secrete an endopolygalacturonase (polygalacturonide glycanohydrolase EC3.2. 1.15). This enzyme has been brought to a high state of purity by ion exchange, gel filtration, and agarose affinity chromatography. The enzyme has optimal activity at pH 5, has an apparent molecular weight as determined by gel filtration of about 70,000, and prefers polygalacturonic acid to pectin as its substrate. The enzyme, while hydrolyzing only 1% of the glycosidic bonds, reduces the viscosity of a polygalacturonic solution by 50%. Nevertheless, the initial as well as the final products of polygalacturonic acid hydrolysis are predominantly tri- and digalacturonic acid and, to a lesser extent, monogalacturonic acid. The purified enzyme catalyzes the removal of about 80% of the galacturonic acid residues of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus) as well as from the walls isolated from 8-day-old Red Kidney bean (Phaseolus vulgaris) hypocotyls. PMID:16657947

  1. Potential Degradation of Swainsonine by Intracellular Enzymes of Arthrobacter sp. HW08

    Directory of Open Access Journals (Sweden)

    Haili Li

    2013-11-01

    Full Text Available Swainsonine (SW is a toxin produced by locoweeds and harmful to the livestock industry. Degrading SW by Arthrobacter sp. HW08 was demonstrated as a promising way to deal with SW poisoning. However, it is unknown which part of the subcellular enzymes in Arthrobacter sp. HW08 is responsible for biodegrading SW and whether the metabolites are atoxic. In this study, intracellular and extracellular enzymes of Arthrobacter sp. HW08 were isolated and their enzyme activity was evaluated. The metabolites were fed to mice, and physiological and histological properties of the treated mice were investigated. The results showed that only intracellular enzyme of Arthrobacter sp. HW08 (IEHW08 could degrade SW efficiently. Compared with mice in SW treatment group, mice in SW + IEHW08 treatment group (1 increased their body weights; (2 showed higher number of platelets and lower number of white blood cells; (3 decreased the levels of creatinine, urea nitrogen, alanine transaminase and aspartate aminotransferase in serum; (4 reduced the number of vacuolated cells in cerebellum, liver and kidney. All these data demonstrate that IEHW08 was potentially safe for mice, while keeping the capacity of degrading SW. This study indicates a possible application of IEHW08 as an additive in the livestock industry to protect animals from SW poisoning.

  2. Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

    Science.gov (United States)

    Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

    2015-03-18

    Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

  3. Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

    Indian Academy of Sciences (India)

    Dhirendra K Simanshu; Sagar Chittori; H S Savithri; M R N Murthy

    2007-09-01

    In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

  4. Fracturing Fluid (Guar Polymer Gel Degradation Study by using Oxidative and Enzyme Breaker

    Directory of Open Access Journals (Sweden)

    Aung Kyaw

    2012-06-01

    Full Text Available Oxidative and enzyme breakers are used in this research project with the main objective to study on the degradation pattern of fracturing fluid (i.e., guar polymer gel as a function of time, temperature and breaker concentration itself. The fracturing fluid used in hydraulic fracturing or frac pack contain a chemical breakers to reduce the viscosity of the fluid intermingled with the proppant. Chemical breakers reduce viscosity of the guar polymer by cleaving the polymer into small-molecular-weight fragments. The reduction of viscosity will facilitate the flow-back of residual polymer providing rapid recovery of polymer from proppant pack. Ineffective breakers or misapplication of breakers can result in screen-outs or flow-back of viscous fluids both of which can significantly decrease the well productivity. Breaker activity of low to medium temperature range oxidative and enzyme breaker systems was evaluated. ViCon NF an oxidative breaker (Halliburton product and GBW 12- CD an enzyme breaker (BJ Services product were used in this research project with the main objective to study on the degradation pattern of fracturing fluid (guar polymer gel as a function of (time, temperature and breaker concentration itself. This study provides focuses on the way to mix the fracturing fluid, compositions of the fracturing fluid and how to conduct the crosslink and break test. Crosslink test indicate the optimum cross-linker concentration to produce good crosslink gel and the break test gave the characteristic of the gel during degradation process and also the break time. Besides relying on the laboratory experiment, information obtained from research on SPE and US Pattern papers were used to make a comparison study on oxidative and enzyme breakers properties. Degradation pattern observed from the break test showed that reduction in gel viscosity depends on time, temperature and breaker concentration. Observations from experiment also revealed that small

  5. Effect of venous wall immobilization on the thermal degradation of collagen

    Science.gov (United States)

    Ignat'eva, N. Yu.; Zakharkina, O. L.; Lunin, V. V.; Sergeeva, E. A.; Mazaishvili, K. V.; Maksimov, S. V.

    2013-11-01

    The results from a comparative study of the thermal denaturation of collagen in the venous walls of reference samples and samples with varicose disease are presented. Changes in the organization of collagen network of the tissue matrix are detected via thermal analysis and multiphoton microscopy with recording of the second harmonic generation (SHG). It is established that the collagen network of venous walls degrades in varicose disease. It is shown that the disordering of the tertiary structure of collagen molecules is reflected in a 40% drop in the enthalpy of protein denaturation compared to reference (Δ H D = 12.4 ± 4.9 J/g dry residue). The disorganization of fiber structures is recorded on SHG images. It is shown that upon the hydrothermal heating of sequestered samples of venous walls, the complete degradation of the tissue network occurs at 75°C. However, it is noted that upon the mechanical immobilization of samples of both types, the stability of collagen increases and complete denaturation is observed at temperatures above 84°C. It is suggested that the number of available conformations of polypeptide chains in the random coil state falls under tension, lowering Δ S D and raising the temperature of the denaturation of protein.

  6. New approaches to identification and activity estimation of glyphosate degradation enzymes.

    Science.gov (United States)

    Sviridov, A V; Zelenkova, N F; Vinokurova, N G; Ermakova, I T; Leontievsky, A A

    2011-06-01

    We propose a new set of approaches, which allow identifying the primary enzymes of glyphosate (N-phosphonomethyl-glycine) attack, measuring their activities, and quantitative analysis of glyphosate degradation in vivo and in vitro. Using the developed approach we show that glyphosate degradation can follow different pathways depending on physiological characteristics of metabolizing strains: in Ochrobactrum anthropi GPK3 the initial cleavage reaction is catalyzed by glyphosate-oxidoreductase with the formation of aminomethylphosphonic acid and glyoxylate, whereas Achromobacter sp. MPS12 utilize C-P lyase, forming sarcosine. The proposed methodology has several advantages as compared to others described in the literature.

  7. Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states.

    Science.gov (United States)

    Hubin, Ellen; Cioffi, Federica; Rozenski, Jef; van Nuland, Nico A J; Broersen, Kerensa

    2016-06-01

    To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.

  8. Metabolism of cryptic peptides derived from neuropeptide FF precursors: the involvement of insulin-degrading enzyme.

    Science.gov (United States)

    Grasso, Giuseppe; Mielczarek, Przemyslaw; Niedziolka, Magdalena; Silberring, Jerzy

    2014-09-22

    The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

  9. Production of keratinolytic enzyme by an indigenous feather-degrading strain Bacillus cereus Wu2.

    Science.gov (United States)

    Lo, Wei-Hsun; Too, Jui-Rze; Wu, Jane-Yii

    2012-12-01

    A novel feather-degrading microorganism was isolated from a poultry farm in Taiwan, and was identified Bacillus cereus Wu2 according to 16S rRNA sequencing. The isolated strain produces keratinolytic enzyme using chicken feather as the sole carbon and nitrogen source. The experimental results indicated that the extra carbon sources (glucose, fructose, starch, sucrose, or lactose) could act as a catabolite repressor to the enzyme secretion or keratinolytic activity when keratinous substrates were employed as protein sources. However, addition of 2 g/L of NH(4)Cl to the feather medium increased the enzyme production. The optimum temperature and initial pH for enzyme production were 30°C and 7.0, respectively. The maximum yield of the enzyme was 1.75 kU/mL in the optimal chicken feather medium; this value was about 17-fold higher than the yield in the basal hair medium. The B. cereus Wu2 possessed disulfide reductase activity along with keratinolytic activity. The amino acid contents of feathers degradated by B. cereus Wu2 were higher, especially for lysine, methionine and threonine which were nutritionally essential amino acids and usually deficient in the feather meal. Thus, B. cereus Wu2 could be not only used to enhance the nutritional value of feather meal but is also a potential bioinoculant in agricultural environments.

  10. Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization.

    Science.gov (United States)

    Shinozaki, Yukiko; Morita, Tomotake; Cao, Xiao-hong; Yoshida, Shigenobu; Koitabashi, Motoo; Watanabe, Takashi; Suzuki, Ken; Sameshima-Yamashita, Yuka; Nakajima-Kambe, Toshiaki; Fujii, Takeshi; Kitamoto, Hiroko K

    2013-04-01

    Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61-68%); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8±6.3 U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(ε-caprolactone), and poly(lactic acid).

  11. Polyphenols as enzyme inhibitors in different degraded peat soils: Implication for microbial metabolism in rewetted peatlands

    Science.gov (United States)

    Zak, Dominik; Roth, Cyril; Gelbrecht, Jörg; Fenner, Nathalie; Reuter, Hendrik

    2015-04-01

    Recently, more than 30,000 ha of drained minerotrophic peatlands (= fens) in NE Germany were rewetted to restore their ecological functions. Due to an extended drainage history, a re-establishment of their original state is not expected in the short-term. Elevated concentrations of dissolved organic carbon, ammonium and phosphate have been measured in the soil porewater of the upper degraded peat layers of rewetted fens at levels of one to three orders higher than the values in pristine systems; an indicator of increased microbial activity in the upper degraded soil layers. On the other hand there is evidence that the substrate availability within the degraded peat layer is lowered since the organic matter has formerly been subject to intense decomposition over the decades of drainage and intense agricultural use of the areas. Previously however, it was suggested that inhibition of hydrolytic enzymes by polyphenolic substances is suspended during aeration of peat soils mainly due to the decomposition of the inhibiting polyphenols by oxidising enzymes such as phenol oxidase. Accordingly we hypothesised a lack of enzyme inhibiting polyphenols in degraded peat soils of rewetted fens compared to less decomposed peat of more natural fens. We collected both peat samples at the soil surface (0-20 cm) and fresh roots of dominating vascular plants and mosses (as peat parent material) from five formerly drained rewetted sites and five more natural sites of NE Germany and NW Poland. Less decomposed peat and living roots were used to obtain an internal standard for polyphenol analysis and to run enzyme inhibition tests. For all samples we determined the total phenolic contents and in addition we distinguished between the contents of hydrolysable and condensed tannic substances. From a methodical perspective the advantage of internal standards compared to the commercially available standards cyanidin chloride and tannic acid became apparent. Quantification with cyanidin or

  12. Partial purification of chlorophyll degrading enzymes from cavendish banana (Musa Cavendishi).

    Science.gov (United States)

    Janave, Machhindra T; Sharma, Arun

    2004-08-01

    Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30 degrees C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from 'green-ripe' bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from 'green-unripe' and 'yellow-ripe' bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a

  13. Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium.

    OpenAIRE

    Orth, A B; Denny, M.; Tien, M

    1991-01-01

    Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. PSBL-1 is a mutant of this organism that generates the ligninolytic system under nonlimiting conditions during primary metabolism. Lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities for PSBL-1 under nonlimiting conditions were 4- to 10-fold higher than those of the wild type (WT) under nitrogen-limiting conditions. PSBL-1 was still in the log ph...

  14. Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2012-06-01

    Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

  15. Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots.

    Science.gov (United States)

    Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

    2013-05-01

    Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g⁻¹ of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l⁻¹ EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation.

  16. Effects of Lactobacillus plantarum and hydrolytic enzymes on fermentation and ruminal degradability of orange pulp silage

    Directory of Open Access Journals (Sweden)

    HAMID PAYA

    2015-12-01

    Full Text Available The current study was carried out to examine the effect of inoculants, enzymes and mixtures of them on the fermentation, degradability and nutrient value of orange pulp silage. Orange pulp was treated with water (control, inoculant (Lactobacillus plantarum, enzymes (multiple enzyme or inoculants + enzymes prior to ensiling (denoted C, I, E and I+E. For ensiled orange pulp, 84 kg of orange pulp were mixed with 16 kg of wheat straw as an absorbent. Three mini-silos were prepared for each treatment and ensiled for 90 days. Data of each silo within each silage treatment was averaged and used as an experimental unit in a completely random design. Silage pH, total fatty acid and ammonia nitrogen were determined. Silage pH and lactic acid concentration were lowest and highest respectively for I and I+E (p<0.01, while the lowest (p <0.01 NH3N concentration (49.8 g/kg total N was observed in I compared to the control. The lowest acetic and butyric acid concentrations were observed in I and I+E compared with the control (p <0.01. The highest metabolizable energy (ME, net energy lactation (NEl, digestible organic matter in dry matter (DOMD, short chain fatty acid (SCFA and microbial protein (MP values were observed for I+E (p <0.01. The in vitro degradability of dry matter (IVDMD was highest (P<0.01 in I+E, while the highest (P<0.01 effective degradability of DM (EDDM was observed for E and I+E treatments. These results indicated that the bacterial inoculants and combination of enzyme and bacterial inoculants clearly improved silage fermentation characteristic. In addition, the ME, DOM, MP and IVDMD of I+E were significantly improved.

  17. Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

    Science.gov (United States)

    Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

    2017-02-01

    The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

  18. Lytic polysaccharide monooxygenases: a crystallographer's view on a new class of biomass-degrading enzymes

    Directory of Open Access Journals (Sweden)

    Kristian E. H. Frandsen

    2016-11-01

    Full Text Available Lytic polysaccharide monooxygenases (LPMOs are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future.

  19. Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi

    Directory of Open Access Journals (Sweden)

    Boomsma Jacobus J

    2010-12-01

    Full Text Available Abstract Background Leaf-cutting (attine ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth. Results We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21% of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade. Conclusions Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to

  20. Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level

    Directory of Open Access Journals (Sweden)

    Henrissat Bernard

    2011-01-01

    Full Text Available Abstract Background Rhizopus oryzae is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses. Results Carbohydrate Active enzyme (CAZy annotation of the R. oryzae identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs and a high number of glycosyl transferases (GTs and carbohydrate esterases (CEs. A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars, chitin, chitosan, β-1,3-glucan and fungal cell wall fractions suggest specific adaptations of R. oryzae to its environment. Conclusions CAZy analyses of the genome of the zygomycete fungus R. oryzae and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.

  1. Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

    Directory of Open Access Journals (Sweden)

    Anna P Lucarelli

    Full Text Available The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB and extensively drug-resistant strains (XDR-TB is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+ or Mn(2+ and inhibited by Ca(2+ and Cu(2+ but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+ and Cu(2+ ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.

  2. Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq).

    Science.gov (United States)

    Graham, Carly F; Glenn, Travis C; McArthur, Andrew G; Boreham, Douglas R; Kieran, Troy; Lance, Stacey; Manzon, Richard G; Martino, Jessica A; Pierson, Todd; Rogers, Sean M; Wilson, Joanna Y; Somers, Christopher M

    2015-11-01

    Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

  3. Atrazine degradation by fungal co-culture enzyme extracts under different soil conditions.

    Science.gov (United States)

    Chan-Cupul, Wilberth; Heredia-Abarca, Gabriela; Rodríguez-Vázquez, Refugio

    2016-01-01

    This investigation was undertaken to determine the atrazine degradation by fungal enzyme extracts (FEEs) in a clay-loam soil microcosm contaminated at field application rate (5 μg g(-1)) and to study the influence of different soil microcosm conditions, including the effect of soil sterilization, water holding capacity, soil pH and type of FEEs used in atrazine degradation through a 2(4) factorial experimental design. The Trametes maxima-Paecilomyces carneus co-culture extract contained more laccase activity and hydrogen peroxide (H2O2) content (laccase = 18956.0 U mg protein(-1), H2O2 = 6.2 mg L(-1)) than the T. maxima monoculture extract (laccase = 12866.7 U mg protein(-1), H2O2 = 4.0 mg L(-1)). Both extracts were able to degrade atrazine at 100%; however, the T. maxima monoculture extract (0.32 h) achieved a lower half-degradation time than its co-culture with P. carneus (1.2 h). The FEE type (p = 0.03) and soil pH (p = 0.01) significantly affected atrazine degradation. The best degradation rate was achieved by the T. maxima monoculture extract in an acid soil (pH = 4.86). This study demonstrated that both the monoculture extracts of the native strain T. maxima and its co-culture with P. carneus can efficiently and quickly degrade atrazine in clay-loam soils.

  4. A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

    DEFF Research Database (Denmark)

    Kracun, Stjepan Kresimir; Schückel, Julia; Westereng, Bjørge;

    2015-01-01

    Background: Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers...... of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. Results: We have developed a new generation of multi...... carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems...

  5. Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

    Directory of Open Access Journals (Sweden)

    Blewett Ann

    2008-12-01

    Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

  6. Divergent selection for ester-linked diferulates in maize pith stalk tissues. Effects on cell wall composition and degradability.

    Science.gov (United States)

    Barros-Rios, Jaime; Malvar, Rosa A; Jung, Hans-Joachim G; Bunzel, Mirko; Santiago, Rogelio

    2012-11-01

    Cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall properties. However, results of genetic selection for DFA concentration have not been reported for any grass species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith tissues and the associated changes in cell wall composition and biodegradability. After two cycles of divergent selection, maize populations selected for higher total DFA (DFAT) content (CHs) had 16% higher DFAT concentrations than populations selected for lower DFAT content (CLs). These significant DFA concentration gains suggest that DFA deposition in maize pith parenchyma cell walls is a highly heritable trait that is genetically regulated and can be modified trough conventional breeding. Maize populations selected for higher DFAT had 13% less glucose and 10% lower total cell wall concentration than CLs, suggesting that increased cross-linking of feruloylated arabinoxylans results in repacking of the matrix and possibly in thinner and firmer cell walls. Divergent selection affected esterified DFAT and monomeric ferulate ether cross link concentrations differently, supporting the hypothesis that the biosynthesis of these cell wall components are separately regulated. As expected, a more higher DFA ester cross-coupled arabinoxylan network had an effect on rumen cell wall degradability (CLs showed 12% higher 24-h total polysaccharide degradability than CHs). Interestingly, 8-8-coupled DFAs, previously associated with cell wall strength, were the best predictors of pith cell wall degradability (negative impact). Thus, further research on the involvement of these specific DFA regioisomers in limiting cell wall biodegradability is encouraged.

  7. Preparation and properties of bacteriophage-borne enzyme degrading bacterial exopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Mou Haijin; Wang Jingxue; Jiang Xiaolu; Liu Zhihong

    2008-01-01

    Bacteriophages infected different serotypes of Klebsiella were isolated from sewage. Among them, a heat-stable polysaccharide depolymerase enzyme which could degrade bacterial exopolysaccharide effectively was prepared from the phage infecting Klebsiella K13. Treatment at 60 ℃ for 30 min could inactivate most of the K13 phage, with the titration decreasing from 6.4×108 PFU/mL to 1.6×106 PFU/mL. However, no obvious loss of phage enzyme activity was found after this treatment. The optimum hydrolytic temperature of phage enzyme was 60 ℃, with an activity 57% higher than that at 30 ℃. The addition of phage enzyme could result in a rapid decrease of viscosity of exopolysaccharide (EPS) solution within minutes, indicating that K13 phage polysaccharide depolymerase acts as a kind of endo-glycanohydrolase. HPLC and reducing sugar analysis showed that the hydrolysis of EPS approached approximately the maximum at 4h when the final concentration of phage was 6.0 × 108 PFU/mL. The results showed that Klebsiella K13 phage depolymerase enzyme could be used as a good tool for the preparation of EPS oligosaccharide.

  8. Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes.

    Science.gov (United States)

    Nimchua, Thidarat; Thongaram, Taksawan; Uengwetwanit, Tanaporn; Pongpattanakitshote, Somchai; Eurwilaichitr, Lily

    2012-04-01

    A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from 50 degrees C to 55 degrees C. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

  9. Disruption of cell walls for enhanced lipid recovery

    Science.gov (United States)

    Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

    2015-03-24

    Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

  10. Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme.

    Directory of Open Access Journals (Sweden)

    Bilal Çakir

    Full Text Available BACKGROUND: Insulin-degrading enzyme (IDE is an allosteric Zn(+2 metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer's disease (AD and type 2 diabetes mellitus (T2DM, respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only. CONCLUSION/SIGNIFICANCE: This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.

  11. Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme.

    Science.gov (United States)

    Çakir, Bilal; Dağliyan, Onur; Dağyildiz, Ezgi; Bariş, İbrahim; Kavakli, Ibrahim Halil; Kizilel, Seda; Türkay, Metin

    2012-01-01

    Insulin-degrading enzyme (IDE) is an allosteric Zn(+2) metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates. In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only. This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.

  12. Endophytic actinomycetes: a novel source of potential acyl homoserine lactone degrading enzymes.

    Science.gov (United States)

    Chankhamhaengdecha, Surang; Hongvijit, Suphatra; Srichaisupakit, Akkaraphol; Charnchai, Pattra; Panbangred, Watanalai

    2013-01-01

    Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

  13. Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

    Directory of Open Access Journals (Sweden)

    Surang Chankhamhaengdecha

    2013-01-01

    Full Text Available Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL quorum sensing (QS system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9% and 68 (51.5% of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

  14. Synergistic Degradation of a Hyperuricemia-Causing Metabolite Using One-Pot Enzyme-Nanozyme Cascade Reactions

    Science.gov (United States)

    Jung, Secheon; Kwon, Inchan

    2017-01-01

    Multi-enzyme cascade reactions are frequently found in living organisms, in particular when an intermediate should be eliminated. Recently, enzyme-mimic nanomaterials (nanozymes) received much attention for various applications, because they are usually more stable and cost-effective than enzymes. However, enzyme-nanozyme cascade reations have not been yet extensively exploited. Therefore, in this study, we investigated one-pot enzyme-nanozyme cascade reactions using urate oxidase (UOX) and catalase-mimic gold nanoparticle nanozyme (AuNP) with the ultimate goal of treatment of hyperuricemia. UOX degrades hyperuricemia-causing uric acid, but also generates hydrogen peroxide raising several health concerns. We successfully demonstrated that one-pot UOX-AuNP cascade systems degrade uric acid more than five times faster than UOX alone, by eliminating potentially cytotoxic hydrogen peroxide, similar to enzyme-enzyme reactions. PMID:28287162

  15. A monomeric variant of insulin degrading enzyme (IDE loses its regulatory properties.

    Directory of Open Access Journals (Sweden)

    Eun Suk Song

    Full Text Available BACKGROUND: Insulin degrading enzyme (IDE is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structure. METHODOLOGY/PRINCIPAL FINDINGS: IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained approximately 25% of the wild type activity. In contrast with the larger peptide substrates beta-endorphin and amyloid beta peptide 1-40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold. CONCLUSIONS/SIGNIFICANCE: These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.

  16. Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: Key events in the evolution of herbivory in beetles.

    Science.gov (United States)

    Kirsch, Roy; Gramzow, Lydia; Theißen, Günter; Siegfried, Blair D; Ffrench-Constant, Richard H; Heckel, David G; Pauchet, Yannick

    2014-09-01

    Plant cell walls are the largest reservoir of organic carbon on earth. To breach and utilize this carbohydrate-rich protective barrier, microbes secrete plant cell wall degrading enzymes (PCWDEs) targeting pectin, cellulose and hemicelluloses. There is a growing body of evidence that genomes of some herbivorous insects also encode PCWDEs, raising questions about their evolutionary origins and functions. Among herbivorous beetles, pectin-degrading polygalacturonases (PGs) are found in the diverse superfamilies Chrysomeloidea (leaf beetles, long-horn beetles) and Curculionoidea (weevils). Here our aim was to test whether these arose from a common ancestor of beetles or via horizontal gene transfer (HGT), and whether PGs kept their ancestral function in degrading pectin or evolved novel functions. Transcriptome data derived from 10 beetle species were screened for PG-encoding sequences and used for phylogenetic comparisons with their bacterial, fungal and plant counterparts. These analyses revealed a large family of PG-encoding genes of Chrysomeloidea and Curculionoidea sharing a common ancestor, most similar to PG genes of ascomycete fungi. In addition, 50 PGs from beetle digestive systems were heterologously expressed and functionally characterized, showing a set of lineage-specific consecutively pectin-degrading enzymes, as well as conserved but enzymatically inactive PG proteins. The evidence indicates that a PG gene was horizontally transferred ∼200 million years ago from an ascomycete fungus to a common ancestor of Chrysomeloidea and Curculionoidea. This has been followed by independent duplications in these two lineages, as well as independent replacement in two sublineages of Chrysomeloidea by two other subsequent HGTs. This origin, leading to subsequent functional diversification of the PG gene family within its new hosts, was a key event promoting the evolution of herbivory in these beetles. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Effect of insulin and glucose on the activity of insulin-degrading enzymes in rat liver.

    Science.gov (United States)

    Jurcovicová, J; Németh, S; Vigas, M

    1977-09-01

    The degradation of insulin by insulin protease and glutathion-insulin transhydrogenase (glutathioneproteindisulphide oxidoreductase--EC 1.8.4.2, GIT) was measured in rat liver either after replacing food and water by 15% glucose solution, or after daily insulin administration 8 U daily for 3 days or after fasting. The breakdown of radioiodinated insulin was followed by measuring the increase of TCA soluble radioactivity during incubation of cell fractions with 125I insulin at 37 degrees C. The highest GIT activity was observed in liver microsomes of rats after glucose feeding and after insulin administration, whereas enzyme activity of fasted animals did not essentially differ from corresponding values of normally fed controls. The insulin protease in cytosol of liver cells remained unchanged after these procedures. The important role of GIT in insulin degradation seems to be conclusively demonstrated.

  18. Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme

    Directory of Open Access Journals (Sweden)

    Giuseppe Grasso

    2014-09-01

    Full Text Available The term “cryptome” refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor, generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

  19. Targeting Insulin-Degrading Enzyme to Treat Type 2 Diabetes Mellitus.

    Science.gov (United States)

    Tang, Wei-Jen

    2016-01-01

    Insulin-degrading enzyme (IDE) selectively degrades peptides, such as insulin, amylin, and amyloid β (Aβ) that form toxic aggregates, to maintain proteostasis. IDE defects are linked to the development of type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD). Structural and biochemical analyses revealed the molecular basis for IDE-mediated destruction of amyloidogenic peptides and this information has been exploited to develop promising inhibitors of IDE to improve glucose homeostasis. However, the inhibition of IDE can also lead to glucose intolerance. In this review, I focus on recent advances regarding our understanding of the structure and function of IDE and the discovery of IDE inhibitors, as well as challenges in developing IDE-based therapy for human diseases, particularly T2DM.

  20. An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

    Science.gov (United States)

    Lounsbury, Jenny A; Coult, Natalie; Miranian, Daniel C; Cronk, Stephen M; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2012-09-01

    Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

  1. Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface.

    Science.gov (United States)

    Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.

  2. Structure Based Discovery of Small Molecules to Regulate the Activity of Human Insulin Degrading Enzyme

    OpenAIRE

    Bilal Çakir; Onur Dağliyan; Ezgi Dağyildiz; İbrahim Bariş; Ibrahim Halil Kavakli; Seda Kizilel; Metin Türkay

    2012-01-01

    Structure Based Discovery of Small Molecules to Regulate the Activity of Human Insulin Degrading Enzyme Bilal C¸ akir1, Onur Dag˘ liyan1, Ezgi Dag˘ yildiz1, I˙brahim Baris¸1, Ibrahim Halil Kavakli1,2*, Seda Kizilel1*, Metin Tu¨ rkay3* 1 Department of Chemical and Biological Engineering, Koc¸ University, Sariyer, Istanbul, Turkey, 2 Department of Molecular Biology and Genetics, Koc¸ University, Sariyer, Istanbul, Turkey, 3 Department of Industrial Engineering, Koc¸ University...

  3. Protein capsules with cross-linked, semipermeable, and enzyme-degradable surface barriers for controlled release.

    Science.gov (United States)

    Zhou, Jianhua; Hyun, Dong Choon; Liu, Hang; Wu, Hongkai; Xia, Younan

    2014-08-01

    This paper describes a method for fabricating protein-based capsules with semipermeable and enzyme-degradable surface barriers. It involves the use of a simple fluidic device to generate water-in-oil emulsion droplets, followed by cross-linking of proteins at the water-oil interface to generate a semipermeable surface barrier. The capsules can be readily fabricated with uniform and controllable sizes and, more importantly, show selective permeability toward molecules with different molecular weights: small molecules like fluorescein sodium salt can freely diffuse through the surface barrier while macromolecules such as proteins can not. The proteins, however, can be released by digesting the surface barrier with an enzyme such as pepsin. Taken together, the capsules hold great potential for applications in controlled release, in particular, for the delivery of protein drugs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate

    National Research Council Canada - National Science Library

    Greiner, Ralf; Carlsson, Nils-Gunnar

    2006-01-01

    .... The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2...

  5. Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.

    Science.gov (United States)

    Maeda, Hiroshi; Yamagata, Youhei; Abe, Keietsu; Hasegawa, Fumihiko; Machida, Masayuki; Ishioka, Ryoji; Gomi, Katsuya; Nakajima, Tasuku

    2005-06-01

    We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

  6. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    Science.gov (United States)

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schürer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A

    2015-12-18

    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

  7. Degradation kinetics of forchlorfenuron in typical grapevine soils of India and its influence on specific soil enzyme activities.

    Science.gov (United States)

    Banerjee, Kaushik; Dasgupta, Soma; Oulkar, Dasharath P; Patil, Sangram H; Adsule, Pandurang G

    2008-05-01

    The rate of degradation of forchlorfenuron, a cytokinin-based plant growth regulator (PGR) was explored in typical grapevine soils of India with simultaneous evaluation of its effect on biochemical attributes of the test soils in terms of the activities of specific soil microbial enzymes. In all the test soils, namely clay, sandy-loam and silty-clay, the dissipation rate was faster at the beginning, which slowed down with time, indicating a non-linear pattern of degradation. Degradation in soils could best be explained by two-compartment 1st+1st order kinetics with half-life ranging between 4-10 days. The results suggest that organic matter might be playing a major role in influencing the rate of degradation of forchlorfenuron in soil. The rate of degradation in sandy-loam soil was fastest followed by clay and silty-clay soils, respectively. Comparison of the rate of degradation in natural against sterilized soils suggests that microbial degradation might be the major pathway of residue dissipation. Changes in soil enzyme activities as a consequence of forchlorfenuron treatment were studied for extra-cellular enzymes namely acid phosphatase, alkaline phosphatase and beta -glucosidase and intracellular enzyme-dehydrogenase. Although small changes in enzyme activities were observed, forchlorfenuron did not have any significant deleterious effect on the enzymatic activity of the test soils. Simple correlation studies between degradation percentage and individual enzyme activities did not establish any significant relationships. The pattern and change of enzyme activity was primarily the effect of the incubation period rather than the effect of forchlorfenuron itself.

  8. A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jin; Jaroch, David; Rickus, Jenna L; Marshall Porterfield, D [Weldon School of Biomedical Engineering, Purdue University (United States); Claussen, Jonathan C; Ul Haque, Aeraj; Diggs, Alfred R [Physiological Sensing Facility, Bindley Bioscience Center and Birck Nanotechnology Center, Purdue University (United States); McLamore, Eric S [Department of Agricultural and Biological Engineering, University of Florida (United States); Calvo-Marzal, Percy, E-mail: porterf@purdue.edu [Department of Chemistry, Purdue University (United States)

    2011-09-02

    This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 {+-} 0.5 {mu}A mM{sup -1} cm{sup -2}), linear range (0.0037-12 mM), detection limit (3.7 {mu}M), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H{sub 2}O{sub 2} response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

  9. COMPONENTS OF CELL WALL, ENZYME ACTIVITY IN PEDICEL AND SUSCEPTIBILITY OF BANANAS TO FINGER DROP

    Directory of Open Access Journals (Sweden)

    GLORIA ANNABELL COBEÑA RUIZ

    2016-01-01

    Full Text Available ABSTRACT A major problem in post-harvest handling of bananas is the individual detachment of the fruit from the hands. This study aimed to establishing the relationship between carbohydrate concentration and enzyme activity in the pedicel region of three cultivars of bananas, resistant and susceptible to natural dropping, during post-harvest ripening, and the susceptibility of bananas to finger dropping. Cultivars ‘Terra’ (plantain, AAB group and ‘Prata’ (banana, AAB group triploids and the ‘Prata Graúda’ (banana, AAAB group tetraploid were used. The experiment was distributed in split plots, with three plots (cultivars and five subplots (peel color stages in a completely randomized design with three replications and three fruits per sample unit. ‘Terra’ showed resistance to dropping, even though the fruit were ripe, unlike ‘Prata Graúda’, which, starting from the fifth stage (yellow fruit with green tips, exhibited high susceptibility to dropping. At all ripening stages, the ‘Terra’ had the highest dry mass levels. In turn, the ‘Prata Graúda’ always maintained the lowest levels. The ‘Terra’ showed decreasing levels of pectins during ripening, whereas starch remained high even in the ripe fruit. About the enzymes studied, the results confirmed the increased resistance of the ‘Terra’ to dropping, allowing to conclude that polygalacturonase (PG and pectinametylesterase (PME are the key enzymes for the solubilization of the cell wall that accompanies ripening, thus playing a critical role in inducing natural dropping. The high susceptibility of the ’Prata Graúda’ to dropping is associated with the high activity of PG and PME and the low levels of dry mass; the greater resistance of the ‘Terra’ to dropping is related to higher accumulation of dry mass and starch in the pedicel.

  10. No major role for insulin-degrading enzyme in antigen presentation by MHC molecules.

    Directory of Open Access Journals (Sweden)

    Slobodan Culina

    Full Text Available Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.

  11. Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo.

    Science.gov (United States)

    Kim, In Hwang; Kim, Ik-Jung; Wen, Yancheng; Park, Na-Young; Park, Jinyoung; Lee, Keun-Woo; Koh, Ara; Lee, Ji-Hyun; Koo, Seung-Hoi; Kim, Kun-Soo

    2015-07-24

    We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.

  12. No major role for insulin-degrading enzyme in antigen presentation by MHC molecules.

    Science.gov (United States)

    Culina, Slobodan; Mauvais, François-Xavier; Hsu, Hsiang-Ting; Burgevin, Anne; Guénette, Suzanne; Moser, Anna; van Endert, Peter

    2014-01-01

    Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.

  13. Disorder-specific effects of polymorphisms at opposing ends of the Insulin Degrading Enzyme gene

    Directory of Open Access Journals (Sweden)

    Bartl Jasmin

    2011-11-01

    Full Text Available Abstract Background Insulin-degrading enzyme (IDE is the ubiquitously expressed enzyme responsible for insulin and amyloid beta (Aβ degradation. IDE gene is located on chromosome region 10q23-q25 and exhibits a well-replicated peak of linkage with Type 2 diabetes mellitus (T2DM. Several genetic association studies examined IDE gene as a susceptibility gene for Alzheimer's disease (AD, however with controversial results. Methods We examined associations of three IDE polymorphisms (IDE2, rs4646953; IDE7, rs2251101 and IDE9, rs1887922 with AD, Aβ42 plasma level and T2DM risk in the longitudinal Vienna Transdanube Aging (VITA study cohort. Results The upstream polymorphism IDE2 was found to influence AD risk and to trigger the Aβ42 plasma level, whereas the downstream polymorphism IDE7 modified the T2DM risk; no associations were found for the intronic variant IDE9. Conclusions Based on our SNP and haplotype results, we delineate the model that IDE promoter and 3' untranslated region/downstream variation may have different effects on IDE expression, presumably a relevant endophenotype with disorder-specific effects on AD and T2DM susceptibility.

  14. Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum FLU100

    Science.gov (United States)

    Dobslaw, Daniel; Engesser, Karl-Heinrich

    2015-01-01

    Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100. PMID:25130674

  15. ApuA, a multifunctional alpha-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus.

    Science.gov (United States)

    Ferrando, Maria Laura; Fuentes, Susana; de Greeff, Astrid; Smith, Hilde; Wells, Jerry M

    2010-09-01

    We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved alpha-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal alpha-amylase domain of ApuA was shown to have alpha-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase alpha-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host.

  16. Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

    DEFF Research Database (Denmark)

    Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

    2017-01-01

    Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production...... processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase...... at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co2+ and inhibited by Mg2+, K+ and Ca2+ followed by Zn2+, Na+, Mn2+, Al3+, and Cu2+. The Km and kcat values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides...

  17. Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation

    Directory of Open Access Journals (Sweden)

    Henrissat Bernard

    2011-09-01

    Full Text Available Abstract Background Spore-forming Bacilli are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI-tract of animals. Isolates of various Bacillus species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms. Results We report the annotation of carbohydrate active enzymes (CAZymes of two pigmented Bacilli isolated from the human GI-tract and belonging to the Bacillus indicus and B. firmus species. A high number of glycoside hydrolases (GHs and carbohydrate binding modules (CBMs were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both Bacilli. Experimental results on biofilm formation confirmed genomic data on the potentials of B. indicus HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both Bacilli to degrade mammalian glycans. Conclusions CAZy analyses of the genomes of the two pigmented Bacilli, compared to other Bacillus species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented Bacilli are adapted to the intestinal environment and are suited to grow in and colonize the human gut.

  18. Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation

    Science.gov (United States)

    2011-01-01

    Background Spore-forming Bacilli are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI)-tract of animals. Isolates of various Bacillus species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms. Results We report the annotation of carbohydrate active enzymes (CAZymes) of two pigmented Bacilli isolated from the human GI-tract and belonging to the Bacillus indicus and B. firmus species. A high number of glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both Bacilli. Experimental results on biofilm formation confirmed genomic data on the potentials of B. indicus HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both Bacilli to degrade mammalian glycans. Conclusions CAZy analyses of the genomes of the two pigmented Bacilli, compared to other Bacillus species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented Bacilli are adapted to the intestinal environment and are suited to grow in and colonize the human gut. PMID:21892951

  19. Carbohydrate-active enzymes from pigmented Bacilli: a genomic approach to assess carbohydrate utilization and degradation.

    Science.gov (United States)

    Manzo, Nicola; D'Apuzzo, Enrica; Coutinho, Pedro M; Cutting, Simon M; Henrissat, Bernard; Ricca, Ezio

    2011-09-05

    Spore-forming Bacilli are gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI)-tract of animals. Isolates of various Bacillus species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms. We report the annotation of carbohydrate active enzymes (CAZymes) of two pigmented Bacilli isolated from the human GI-tract and belonging to the Bacillus indicus and B. firmus species. A high number of glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both Bacilli. Experimental results on biofilm formation confirmed genomic data on the potentials of B. indicus HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both Bacilli to degrade mammalian glycans. CAZy analyses of the genomes of the two pigmented Bacilli, compared to other Bacillus species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented Bacilli are adapted to the intestinal environment and are suited to grow in and colonize the human gut.

  20. Degradation of Diuron by Phanerochaete chrysosporium: Role of Ligninolytic Enzymes and Cytochrome P450

    Directory of Open Access Journals (Sweden)

    Jaqueline da Silva Coelho-Moreira

    2013-01-01

    Full Text Available The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 μg/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl-3-methylurea] and DCPU [(3,4-dichlorophenylurea], were detected in the culture medium at the concentrations of 0.74 μg/mL and 0.06 μg/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT, a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products.

  1. Atrazine degradation and enzyme activities in an agricultural soil under two tillage systems.

    Science.gov (United States)

    Mahía, Jorge; Martín, Angela; Carballas, Tarsy; Díaz-Raviña, Montserrat

    2007-05-25

    The content of atrazine and its metabolites (hydroxyatrazine, deethylatrazine and deisopropylatrazine) as well as the activities of two soil enzymes (urease and beta-glucosidase) were evaluated in an acid agricultural soil, located in a temperate humid zone (Galicia, NW Spain), with an annual ryegrass-maize rotation under conventional tillage (CT) and no tillage (NT). Samples were collected during two consecutive years from the arable layer at two depths (0-5 cm and 5-20 cm) and different times after atrazine application. Hydroxyatrazine and deisopropylatrazine were the main metabolites resulting from atrazine degradation in the acid soil studied, the highest levels being detected in the surface layer of the NT treatment. A residual effect of atrazine was observed since hydroxyatrazine was detected in the arable layer (0-5 cm, 5-20 cm) even one year after the herbicide application. Soil enzyme activities in the upper 5 cm layer under NT were consistently higher than those in the same layer under CT. Urease and beta-glucosidase activities decreased with depth in the profile under NT but they did not show any differences between the two depths for the plots under CT. For both tillage systems enzyme activities also reflected temporal changes during the maize cultivation; however, no consistent effect of the herbicide application was observed.

  2. Activities of adenine nucleotide and nucleoside degradation enzymes in platelets of rats infected by Trypanosoma evansi.

    Science.gov (United States)

    Oliveira, Camila B; Da Silva, Aleksandro S; Vargas, Lara B; Bitencourt, Paula E R; Souza, Viviane C G; Costa, Marcio M; Leal, Claudio A M; Moretto, Maria B; Leal, Daniela B R; Lopes, Sonia T A; Monteiro, Silvia G

    2011-05-31

    Nucleotide and nucleoside-degrading enzymes, such as nucleoside triphosphate diphosphohydrose (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are present in the surface membranes of platelets, involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activities of these enzymes in platelets of rats experimentally infected with T. evansi. Animals were divided into four groups, according to the level of parasitemia. Blood samples were collected on days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection), post-infection. Group D (control group) was composed of non-infected animals for platelet count, separation and enzymatic assays. Animals from groups A and B showed marked thrombocytopenia, but platelet count was not affected in chronically infected rats. NTPDase, 5'-nucleotidase and ADA activities decreased (pplatelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5'-nucleoside activities decreased (pplatelet count and nucleotide/nucleoside hydrolysis were positive and statistically significant (pPlatelet aggregation was decreased in all infected groups, in comparison to the control group (pplatelets of T. evansi-infected animals might be related to thrombocytopenia, that by reducing the number of platelets, there was less release of ATP and ADP. Another possibility being suggested is that changes have occurred in the membrane of these cells, decreasing the expression of these enzymes in the cell membrane.

  3. Antioxidant and lipoxygenase activities of polyphenol extracts from oat brans treated with polysaccharide degrading enzymes

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    Nisita Ratnasari

    2017-07-01

    Full Text Available This study used polysaccharide degrading enzymes and protein precipitation to extract polyphenols from oats and to determine their bioactivity. Duplicate oat brans were treated with viscozyme (Vis, cellulase (Cel or no enzyme (control, CTL then, proteins were removed in one set (Vis1, Cel1, CTL1 and not in the other (Vis2, Cel2, CTL2. HPLC analyses showed that for cellulase treated brans, precipitation of proteins increased phenolic acids and avenanthramides by 14%. Meanwhile, a decreased of 67% and 20% respectively was found for viscozyme and control brans. The effect of protein precipitation on soluble polyphenols is therefore dependent of the carbohydrase, as proteins with different compositions will interact differently with other molecules. Radical scavenging data showed that Cel1 and Vis1 had higher quenching effects on ROO• radicals with activities of 22.1 ± 0.8 and 23.5 ± 1.2 μM Trolox Equivalents/g defatted brans. Meanwhile, CTL2 had the highest HO• radicals inhibition (49.4 ± 2.8% compared to 10.8–32.3% for others. Samples that highly inhibited lipoxygenase (LOX, an enzyme involved in lipid oxidation were Cel1 (23.4 ± 2.3% and CTL1 (18 ± 0.4%.

  4. PhaC Synthases and PHA Depolymerases: The Enzymes that Produce and Degrade Plastic

    Directory of Open Access Journals (Sweden)

    Amro A. Amara

    2011-12-01

    Full Text Available PHAs are a group of intracellular biodegradable polymer produced by (most bacteria under unbalanced growth conditions. A series of enzymes are involved in different PHAs synthesis, however PhaC synthases are responsible for the polymerization step. PHAs are accumulated in bacterial cells from soluble to insoluble form as storage materials inside the inclusion bodies during unbalanced nutrition or to save organisms from reduces equivalents. PHAs are converted again to soluble components by another pathways and enzymes for the degradation process. PHAs depolymerases are the responsible enzymes. This review is designed to give the non-specialists a condense background about PHAs especially for researcher and students in medicinal and pharmaceutical filled. ABSTRAK: PHAs (polyhydroxyalkanoate merupakan sekumpulan polimer terbiodegradasikan intrasel yang dihasilkan oleh (kebanyakan bakteria di bawah keadaan tumbesaran tak seimbang. Satu rangkaian enzim terlibat dalam sistesis PHAs yang berbeza, namun sintesis PhaC bertanggungjawab dalam peringkat pempolimeran. PHAs dikumpulkan dalam sel bakteria dari bentuk larut dan tak larut sebagai bahan simpan di dalam jasad terangkum semasa nutrisi tak seimbang atau untuk menyelamatkan organisma daripada pengurangan tak keseimbangan. PHAs ditukarkan sekali lagi kepada komponen larut dengan cara lain dan enzim lain untuk proses degradasi. PHAs depoly-merases (enzim yang memangkin penguraian makro molekul kepada molekul yang lebih mudah merupakan enzim yang bertanggunjawab. Kajian semula ini direka untuk memberi mereka yang bukan pakar, satu ringkasan tentang PHAs terutamanya penyelidik dan penuntut dalam bidang peubatan dan farmaseutikal.

  5. Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium

    Energy Technology Data Exchange (ETDEWEB)

    Orth, A.B.; Denny, M.; Ming Tien (Pennsylvania State Univ., University Park (United States))

    1991-09-01

    Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. PSBL-1 is a mutant of this organism that generates the ligninolytic system under nonlimiting conditions during primary metabolism. Lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities for PSBL-1 under nonlimiting conditions were 4- to 10-fold higher than those of the wild type (WT) under nitrogen-limiting conditions. PSBL-1 was still in the log phase of growth while secreting the enzymes, whereas the WT had ceased to grow by this time. As in the WT, manganese(II) increased manganese peroxidase activity in the mutant. However, manganese also caused in increase in lignin peroxidase and glyoxal oxidase activities in PSBL-1. Addition of veratryl alcohol to the culture medium stimulated lignin peroxidase activity, inhibited glyoxal oxidase activity, and had little effect on manganese peroxidase activity in PSBL-1, as in the WT. Fast protein liquid chromatography (FPLC) analysis shows production of larger amounts of isozyme H2 in PSBL-1 than in the WT. These properties make PSBL-1 very useful for isolation of large amounts of all ligninolytic enzymes for biochemical study, and they open the possibility of scale-up production for practical use.

  6. Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing

    Directory of Open Access Journals (Sweden)

    Vasudeo Zambare, Archana Zambare, Kasiviswanath Muthukumarappan, Lew P. Christopher

    2011-01-01

    Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.

  7. Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme.

    Science.gov (United States)

    McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan; Kalas, Vasilios; Hoey, Robert J; Koide, Akiko; Koide, Shohei; Tang, Wei-Jen

    2013-08-20

    Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

  8. Analysis of bulk and inorganic degradation products of stones, mortars and wall paintings by portable Raman microprobe spectroscopy.

    Science.gov (United States)

    Pérez-Alonso, M; Castro, K; Martinez-Arkarazo, I; Angulo, M; Olazabal, M A; Madariaga, J M

    2004-05-01

    This work reports the use of a portable Raman microprobe spectrometer for the analysis of bulk and decaying compounds in carbonaceous materials such as stones, mortars and wall paintings. The analysed stones include limestone, dolomite and carbonaceous sandstone, gypsum and calcium oxalate, both mono- and dihydrated, being the main inorganic degradation products detected. Mortars include bulk phases with pure gypsum, calcite and mixtures of both or with sand, soluble salts being the most important degradation products. The pigments detected in several wall paintings include Prussian blue, iron oxide red, iron oxide yellow, vermilion, carbon black and lead white. Three different decaying processes have been characterised in the mortars of the wall paintings: (a) a massive absorption of nitrates that reacted with calcium carbonate and promoted the unbinding of pigment grains, (b) the formation of black crusts in the vault of the presbytery and (c) the thermodecomposition of pigments due to a fire.

  9. Degradation of wheat straw cell wall by white rot fungi Phanerochaete chrysosporium

    Science.gov (United States)

    Zeng, Jijiao

    The main aim of this dissertation research was to understand the natural microbial degradation process of lignocellulosic materials in order to develop a new, green and more effective pretreatment technology for bio-fuel production. The biodegradation of wheat straw by white rot fungi Phanerochaete chrysosporium was investigated. The addition of nutrients significantly improved the performance of P.chrysosporium on wheat straw degradation. The proteomic analysis indicated that this fungus produced various pepetides related to cellulose and lignin degradation while grown on the biomass. The structural analysis of lignin further showed that P.chrysosporium preferentially degraded hydroxycinnamtes in order to access cellulose. In details, the effects of carbon resource and metabolic pathway regulating compounds on manganeses peroxidase (MnP) were studied. The results indicated that MnP activity of 4.7 +/- 0.31 U mL-1 was obtained using mannose as a carbon source. The enzyme productivity further reached 7.36 +/- 0.05 U mL-1 and 8.77 +/- 0.23 U mL -1 when the mannose medium was supplemented with cyclic adenosine monophosphate (cAMP) and S-adenosylmethionine (SAM) respectively, revealing highest MnP productivity obtained by optimizing the carbon sources and supplementation with small molecules. In addition, the effects of nutrient additives for improving biological pretreatment of lignocellulosic biomass were studied. The pretreatment of wheat straw supplemented with inorganic salts (salts group) and tween 80 was examined. The extra nutrient significantly improved the ligninase expression leading to improve digestibility of lignocellulosic biomass. Among the solid state fermentation groups, salts group resulted in a substantial degradation of wheat straw within one week, along with the highest lignin loss (25 %) and ˜ 250% higher efficiency for the total sugar release through enzymatic hydrolysis. The results were correlated with pyrolysis GC-MS (Py

  10. Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases.

    Science.gov (United States)

    Huang, Yuhong; Busk, Peter Kamp; Lange, Lene

    2015-06-01

    Specific enzymes from plant-pathogenic microbes demonstrate high effectiveness for natural lignocellulosic biomass degradation and utilization. The secreted lignocellulolytic enzymes of Fusarium species have not been investigated comprehensively, however. In this study we compared cellulose and hemicellulose-degrading enzymes of classical fungal enzyme producers with those of Fusarium species. The results indicated that Fusarium species are robust cellulose and hemicellulose degraders. Wheat bran, carboxymethylcellulose and xylan-based growth media induced a broad spectrum of lignocellulolytic enzymes in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose decomposing enzymes (GH3, GH5, GH6, GH7, GH9, GH45 and AA9), and abundant hemicellulases. We further applied peptide pattern recognition to reveal nine and seven subfamilies of GH10 and GH11 family enzymes, respectively. The uncharacterized XYL10A, XYL10B and XYL11 enzymes of F. commune were classified, respectively, into GH10 subfamily 1, subfamily 3 and GH11 subfamily 1. These xylanases were successfully expressed in the PichiaPink™ system with the following properties: the purified recombinant XYL10A had interesting high specific activity; XYL10B was active at alkaline conditions with both endo-1,4-β-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass.

  11. Degradation of wall paints due to sodium sulphate and sodium chloride crystallization

    Directory of Open Access Journals (Sweden)

    Díaz Gonçalves, T.

    2003-03-01

    Full Text Available A test method for evaluating wall paints behaviour to soluble salts crystallization was developed at LNEC. in the present paper, a recent set of tests is described and discussed. The major objectives were: analysing and comparing the behaviour of a common emulsion {"plastic" paint and a silicate-based paint; observing and comparing the effect of sodium sulphate, sodium chloride and distilled water on the paints and on a non-painted stone; evaluating this test method adequacy and effectiveness. The silicate-based paint showed a resistance to soluble salts crystallization greater than the one of the plastic paint. However, the degradation pattern of the silicate-based paint (blistering of a filmic layer was similar to the one of organic paints and distinct from the one of pure mineral paints. The amount of damage that a saline solution can cause to wall paints cannot be inferred from the amount of damage it can cause to stone. Sodium chloride seems to be able to cause more severe degradation to wall paints than sodium sulphate. To the unpainted stone, sodium sulphate seems to be more damaging than sodium chloride. The test method seems adequate to observe and compare the behaviour of wall paints under soluble salts action. However, lower (around 0.5% concentrations for both sodium sulphate and sodium chloride should be tested in the future.

    RESUMEN En el LNEC se desarrolló una metodología de ensayo para evaluar la respuesta de pinturas aplicadas sobre paredes, frente a la cristalización de sales solubles. En este trabajo, se describen y discuten un conjunto de ensayos recientes. Los principales objetivos fueron: el análisis y la comparación del comportamiento de una pintura de emulsión común {''pintura plástica" y la de una pintura de silicato; la observación y la comparación de los efectos del sulfato de sodio, del cloruro de sodio y del agua destilada sobre las pinturas y sobre piedra no pintada; la evaluación de la adecuaci

  12. High-throughput screening system based on phenolics-responsive transcription activator for directed evolution of organophosphate-degrading enzymes.

    Science.gov (United States)

    Jeong, Young-Su; Choi, Su-Lim; Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Eui-Joong; Pan, Jae-Gu; Rha, Eugene; Song, Jae Jun; Lee, Seung-Goo; Kim, Hak-Sung

    2012-11-01

    Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher k(cat)/K(m) than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential.

  13. Production of lignocellulose-degrading enzymes employing Fusarium solani F-552.

    Science.gov (United States)

    Obruca, Stanislav; Marova, Ivana; Matouskova, Petra; Haronikova, Andrea; Lichnova, Andrea

    2012-05-01

    In this work, capability of Fusarium solani F-552 of producing lignocellulose-degrading enzymes in submerged fermentation was investigated. The enzyme cocktail includes hydrolases (cellulases, xylanases, and proteinases) as well as ligninolytic enzymes: manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac). To our knowledge, this is the first report on production of MnP, LiP, and Lac together by one F. solani strain. The enzyme productions were significantly influenced by application of either lignocellulosic material or chemical inducers into the fermentation medium. Among them, corn bran significantly enhanced especially productions of cellulases and xylanases (248 and 170 U/mL, respectively) as compared to control culture (11.7 and 29.2 U/mL, respectively). High MnP activity (9.43 U/mL, control 0.45 U/mL) was observed when (+)-catechin was applied into the medium, the yield of LiP was maximal (33.06 U/mL, control 2.69 U/mL) in gallic acid, and Lac was efficiently induced by, 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] (6.74 U/mL, not detected in control). Finally, in order to maximize the ligninolytic enzymes yields, a novel strategy of introduction of mild oxidative stress conditions caused by hydrogen peroxide into the fermentation broth was tested. Hydrogen peroxide significantly increased activities of MnP, LiP, and Lac which may indicate that these enzymes could be partially involved in stress response against H(2)O(2). The concentration of H(2)O(2) and the time of the stress application were optimized; hence, when 10 mmol/L H(2)O(2) was applied at the second and sixth day of cultivation, the MnP, LiP, and Lac yields reached 21.67, 77.42, and 12.04 U/mL, respectively.

  14. Somatostatin modulates insulin-degrading-enzyme metabolism: implications for the regulation of microglia activity in AD.

    Directory of Open Access Journals (Sweden)

    Grazia Tundo

    Full Text Available The deposition of β-amyloid (Aβ into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer's disease (AD. Insulin-degrading-enzyme (IDE is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity.

  15. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

    Science.gov (United States)

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

  16. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

    Directory of Open Access Journals (Sweden)

    Diego Sbardella

    Full Text Available Insulin-Degrading-Enzyme (IDE is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

  17. Surface binding sites (SBSs), mechanism and regulation of enzymes degrading amylopectin and α-limit dextrins

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Cockburn, Darrell; Nielsen, Jonas W.;

    2013-01-01

    Certain enzymes interact with polysaccharides at surface binding sites (SBSs) situated outside of their active sites. SBSs are not easily identified and their function has been discerned in relatively few cases. Starch degradation is a concerted action involving GH13 hydrolases. New insight...... into barley seed α-amylase 1 (AMY1) and limit dextrinase (LD) includes i. kinetics of bi-exponential amylopectin hydrolysis by AMY1, one reaction having low Km (8 μg/mL) and high kcat (57 s-1) and the other high Km (97 μg/mL) and low kcat (23 s-1). β-Cyclodextrin (β-CD) inhibits the first reaction by binding...

  18. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    Energy Technology Data Exchange (ETDEWEB)

    Radulescu, Razvan T., E-mail: ratura@gmx.net [Molecular Concepts Research (MCR), Muenster (Germany); Duckworth, William C. [Department of Medicine, Phoenix VA Health Care System, Phoenix, AZ (United States); Levy, Jennifer L. [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States); Fawcett, Janet, E-mail: janet.fawcett@va.gov [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States)

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  19. TISSUE REGENERATION. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration.

    Science.gov (United States)

    Zhang, Yongyou; Desai, Amar; Yang, Sung Yeun; Bae, Ki Beom; Antczak, Monika I; Fink, Stephen P; Tiwari, Shruti; Willis, Joseph E; Williams, Noelle S; Dawson, Dawn M; Wald, David; Chen, Wei-Dong; Wang, Zhenghe; Kasturi, Lakshmi; Larusch, Gretchen A; He, Lucy; Cominelli, Fabio; Di Martino, Luca; Djuric, Zora; Milne, Ginger L; Chance, Mark; Sanabria, Juan; Dealwis, Chris; Mikkola, Debra; Naidoo, Jacinth; Wei, Shuguang; Tai, Hsin-Hsiung; Gerson, Stanton L; Ready, Joseph M; Posner, Bruce; Willson, James K V; Markowitz, Sanford D

    2015-06-12

    Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

  20. Gibberellic-acid-induced synthesis and release of cell-wall-degrading endoxylanase by isolated aleurone layers of barley

    Energy Technology Data Exchange (ETDEWEB)

    Dashek, W.V.; Chrispeels, M.J.

    1977-01-01

    When aleurone layers of barley (Hordeum vulgare L.) are incubated with gibberellic acid (GA/sub 3/), xylose and arabinose, both as free sugars and bound to larger molecules, are released into the medium. Release begins 10 to 12 h after the start of incubation and continues for at least 60 h. At the same time there is a GA/sub 3/-induced breakdown of the cell wall resulting in a loss of /sup 2///sub 3/ of the cell-wall pentose during 60 h of incubation. GA/sub 3/ causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 28 to 32 h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of GA/sub 3/. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis of the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA/sub 3/-induced breakdown of the aleurone cell wall.

  1. Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum FLU100.

    Science.gov (United States)

    Dobslaw, Daniel; Engesser, Karl-Heinrich

    2015-01-01

    Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  2. Effects of microbial enzymes on starch and hemicellulose degradation in total mixed ration silages

    Directory of Open Access Journals (Sweden)

    Tingting Ning

    2017-02-01

    Full Text Available Objective This study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR silage. Methods The TMRs were prepared with soybean curd residue, alfalfa hay (ATMR or Leymus chinensis hay (LTMR, corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties. Results Both ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens (B. amyloliquefaciens, B. cereus, B. licheniformis, and B. subtilis in ATMR silage and B. flexus, B. licheniformis, and Paenibacillus xylanexedens (P. xylanexedens in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens, B. licheniformis, and B. subtilis and B. licheniformis, B. pumilus, and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively. Conclusion The microbial amylase contributes to starch hydrolysis during the

  3. Differential Expression of Antioxidant Enzymes During Degradation of Azo Dye Reactive black 8 in Hairy roots of Physalis minima L.

    Science.gov (United States)

    Jha, Pamela; Modi, Nikita; Jobby, Renitta; Desai, Neetin

    2015-01-01

    The enzymes involved in the protection of plant metabolism in presence of azo dye was characterized by studying activities of the role of antioxidant enzymes in the hairy roots (HRs) of Physalis minima L. during degradation of an azo dye, Reactive Black 8 (RB8). When the HRs were exposed to RB8 (30 mg L(-1)), a  nine fold increase in SOD activity was observed after 24 h, while 22 and 50 fold increase in activity was observed for POX and APX respectively after 72 h, whereas there was no significant change in activity of CAT. The activation of different antioxidant enzymes at different time intervals under dye stress suggests the synchronized functioning of antioxidant machinery to protect the HRs from oxidative damage. FTIR analysis confirmed the degradation of dye and the non-toxic nature of metabolites formed after dye degradation was confirmed by phytotoxicity study.

  4. Insights into seasonal variation of litter decomposition and related soil degradative enzyme activities in subtropical forest in China

    Institute of Scientific and Technical Information of China (English)

    WANG Cong-yan; LÜ Yan-na; WANG Lei; LIU Xue-yan; TIAN Xing-jun

    2013-01-01

    We used a litterbag method to investigate litter decomposition and related soil degradative enzyme activities across four seasons in a broad-leaved forest and a coniferous forest on Zijin Mountain in sub-tropical China. Across four seasons, we quantified litter mass losses, soil pH values, and related soil degradative enzyme activities. Litter decomposition rates differed significantly by season. Litter decomposi-tion rates of broadleaf forest leaves were higher than for coniferous for-ests needles across four seasons, and maximal differences in litter de-composition rates between the two litter types were found in spring. Obvious differences in litter decomposition rates of the two litter types were found in winter, which were similar to rates in spring. Litter de-composition rates of the two litter types in autumn were significantly higher than in spring. Soil degradative enzyme activities were lowest in winter and highest in summer in most cases across four seasons.

  5. Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

    Science.gov (United States)

    Machado, M F M; Cunha, F M; Berti, D A; Heimann, A S; Klitzke, C F; Rioli, V; Oliveira, V; Ferro, E S

    2006-01-13

    Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.

  6. Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Cosgrove, Daniel J.

    2015-11-25

    The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the ‘Young's modulus’ of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.

  7. Suppression of Polyfluorene Photo-Oxidative Degradation via Encapsulation of Single-Walled Carbon Nanotubes.

    Science.gov (United States)

    Luck, Kyle A; Arnold, Heather N; Shastry, Tejas A; Marks, Tobin J; Hersam, Mark C

    2016-10-10

    Polyfluorenes have achieved noteworthy performance in organic electronic devices, but exhibit undesired green band emission under photo-oxidative conditions that have limited their broad utility in optoelectronic applications. In addition, polyfluorenes are well-known dispersants of single-walled carbon nanotubes (SWCNTs), although the influence of SWCNTs on polyfluorene photo-oxidative stability has not yet been defined. Here we quantitatively explore the photophysical properties of poly[(9,9-bis(3/-(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) under photo-oxidative conditions when it is in van der Waals contact with SWCNTs. Photoluminescence spectroscopy tracks the spectral evolution of the polymer emission following ambient ultraviolet (UV) exposure, confirming that PFN exhibits green band emission. In marked contrast, PFN-wrapped SWCNTs possess high spectral stability without green band emission under the same ambient UV exposure conditions. By investigating a series of PFN thin films as a function of SWCNT content, it is shown that SWCNT loadings as low as ~23 wt% suppress photo-oxidative degradation. These findings suggest that PFN-SWCNT composites provide an effective pathway toward utilizing polyfluorenes in organic optoelectronics.

  8. PPAR{gamma} transcriptionally regulates the expression of insulin-degrading enzyme in primary neurons

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jing; Zhang, Lang; Liu, Shubo; Zhang, Chi [Protein Science Key Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, School of Medicine, Tsinghua University, Beijing 100084 (China); Huang, Xiuqing; Li, Jian [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730 (China); Zhao, Nanming [Protein Science Key Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, School of Medicine, Tsinghua University, Beijing 100084 (China); Wang, Zhao, E-mail: zwang@tsinghua.edu.cn [Protein Science Key Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, School of Medicine, Tsinghua University, Beijing 100084 (China)

    2009-06-12

    Insulin-degrading enzyme (IDE) is a protease that has been demonstrated to play a key role in degrading both A{beta} and insulin and deficient in IDE function is associated with Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2) pathology. However, little is known about the cellular and molecular regulation of IDE expression. Here we show IDE levels are markedly decreased in DM2 patients and positively correlated with the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) levels. Further studies show that PPAR{gamma} plays an important role in regulating IDE expression in rat primary neurons through binding to a functional peroxisome proliferator-response element (PPRE) in IDE promoter and promoting IDE gene transcription. Finally, we demonstrate that PPAR{gamma} participates in the insulin-induced IDE expression in neurons. These results suggest that PPAR{gamma} transcriptionally induces IDE expression which provides a novel mechanism for the use of PPAR{gamma} agonists in both DM2 and AD therapies.

  9. Multiple insulin degrading enzyme variants alter in vitro reporter gene expression.

    Directory of Open Access Journals (Sweden)

    Olivia Belbin

    Full Text Available The insulin degrading enzyme (IDE variant, v311 (rs6583817, is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ, decreased risk for Alzheimer's disease (AD and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957, for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2 successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04 but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5' to the promoter (1.18 fold-change, p = 0.006 and 3' to the reporter gene (1.29 fold change, p = 0.003, an effect contributed to by four variants (v10, v315, v154 and v311. Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.

  10. Insulin-degrading enzyme prevents α-synuclein fibril formation in a nonproteolytical manner.

    Science.gov (United States)

    Sharma, Sandeep K; Chorell, Erik; Steneberg, Pär; Vernersson-Lindahl, Emma; Edlund, Helena; Wittung-Stafshede, Pernilla

    2015-07-31

    The insulin-degrading enzyme (IDE) degrades amyloidogenic proteins such as Amyloid β (Αβ) and Islet Amyloid Polypeptide (IAPP), i.e. peptides associated with Alzheimer's disease and type 2 diabetes, respectively. In addition to the protease activity normally associated with IDE function an additional activity involving the formation of stable, irreversible complexes with both Αβ and α-synuclein, an amyloidogenic protein involved in Parkinson's disease, was recently proposed. Here, we have investigated the functional consequences of IDE-α-synuclein interactions in vitro. We demonstrate that IDE in a nonproteolytic manner and at sub-stoichiometric ratios efficiently inhibits α-synuclein fibril formation by binding to α-synuclein oligomers making them inert to amyloid formation. Moreover, we show that, within a defined range of α-synuclein concentrations, interaction with α-synuclein oligomers increases IDE's proteolytic activity on a fluorogenic substrate. We propose that the outcomes of IDE-α-synuclein interactions, i.e. protection against α-synuclein amyloid formation and stimulated IDE protease activity, may be protective in vivo.

  11. Insulin-degrading enzyme is activated by the C-terminus of α-synuclein.

    Science.gov (United States)

    Sharma, Sandeep K; Chorell, Erik; Wittung-Stafshede, Pernilla

    2015-10-16

    The insulin-degrading enzyme (IDE) plays a key role in type-2 diabetes and typically degrades small peptides such as insulin, amyloid β and islet amyloid polypeptide. We recently reported a novel non-proteolytical interaction in vitro between IDE and the Parkinson's disease 140-residue protein α-synuclein that resulted in dual effects: arrested α-synuclein oligomers and, simultaneously, increased IDE proteolysis activity. Here we demonstrate that these outcomes arise due to IDE interactions with the C-terminus of α-synuclein. Whereas a peptide containing the first 97 residues of α-synuclein did not improve IDE activity and its aggregation was not blocked by IDE, a peptide with the C-terminal 44 residues of α-synuclein increased IDE proteolysis to the same degree as full-length α-synuclein. Because the α-synuclein C-terminus is acidic, the interaction appears to involve electrostatic attraction with IDE's basic exosite, known to be involved in activation.

  12. Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix.

    Science.gov (United States)

    Niere, Marc; Kernstock, Stefan; Koch-Nolte, Friedrich; Ziegler, Mathias

    2008-01-01

    Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific "knock-down" of the NAD content which is readily detectable by PAR formation.

  13. Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones.

    Science.gov (United States)

    Maianti, Juan Pablo; McFedries, Amanda; Foda, Zachariah H; Kleiner, Ralph E; Du, Xiu Quan; Leissring, Malcolm A; Tang, Wei-Jen; Charron, Maureen J; Seeliger, Markus A; Saghatelian, Alan; Liu, David R

    2014-07-03

    Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide(-/-) mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE's physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.

  14. Lignin degradation, ligninolytic enzymes activities and exopolysaccharide production by Grifola frondosa strains cultivated on oak sawdust

    Directory of Open Access Journals (Sweden)

    Nona A Mikiashvili

    2011-09-01

    Full Text Available Fourteen strains of Grifola frondosa (Dicks. S. F. Gray, originating from different regions (Asia, Europe and North America were tested for lignin degradation, ligninolytic enzyme activities, protein accumulation and exopolysaccharide production during 55 days of cultivation on oak sawdust. Lignin degradation varied from 2.6 to7.1 % of dry weight of the oak sawdust substrate among tested strains. The loss of dry matter in all screened fungi varied between 11.7 and 33.0%, and the amount of crude protein in the dry substrate varied between 0.94 to 2.55%. The strain, MBFBL 596, had the highest laccase activity (703.3 U/l, and the maximum peroxidase activity of 22.6 U/l was shown by the strain MBFBL 684. Several tested strains (MBFBL 21, 638 and 662 appeared to be good producers of exopolysaccharides (3.5, 3.5 and 3.2 mg/ml respectively.

  15. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

    2014-01-01

    are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas...... only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based......The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence...

  16. Insulin-degrading enzyme is exported via an unconventional protein secretion pathway

    Directory of Open Access Journals (Sweden)

    Leissring Malcolm A

    2009-01-01

    Full Text Available Abstract Insulin-degrading enzyme (IDE is a ubiquitously expressed zinc-metalloprotease that degrades several pathophysiologically significant extracellular substrates, including insulin and the amyloid β-protein (Aβ, and accumulating evidence suggests that IDE dysfunction may be operative in both type 2 diabetes mellitus and Alzheimer disease (AD. Although IDE is well known to be secreted by a variety of cell types, the underlying trafficking pathway(s remain poorly understood. To address this topic, we investigated the effects of known inhibitors or stimulators of protein secretion on the secretion of IDE from murine hepatocytes and HeLa cells. IDE secretion was found to be unaffected by the classical secretion inhibitors brefeldin A (BFA, monensin, or nocodazole, treatments that readily inhibited the secretion of α1-antitrypsin (AAT overexpressed in the same cells. Using a novel cell-based Aβ-degradation assay, we show further that IDE secretion was similarly unaffected by multiple stimulators of protein secretion, including glyburide and 3'-O-(4-benzoylbenzoyl-ATP (Bz-ATP. The calcium ionophore, A23187, increased extracellular IDE activity, but only under conditions that also elicited cytotoxicity. Our results provide the first biochemical evidence that IDE export is not dependent upon the classical secretion pathway, thereby identifying IDE as a novel member of the select class of unconventionally secreted proteins. Further elucidation of the mechanisms underlying IDE secretion, which would be facilitated by the assays described herein, promises to uncover processes that might be defective in disease or manipulated for therapeutic benefit.

  17. Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation.

    Science.gov (United States)

    Ouattara, Honoré G; Reverchon, Sylvie; Niamke, Sébastien L; Nasser, William

    2017-05-01

    Pectin degrading enzymes are essential for quality of product from cocoa fermentation. Previously, we studied purified pectate lyases (Pel) produced by Bacillus strains from fermenting cocoa and characterized the cloned pel genes. This study aims to search for biological signals that modulates Pel production and regulators that influence pel gene expression. Strains were grown to the end of exponential phase in media containing various carbon sources. Pel enzymes production in Bacillus was unaffected by simple sugar content variation up to 2%. Additionally, it appeared that pel gene is not under the control of the most common carbon and pectin catabolism regulators ccpA and kdgR, which could explain the insensitivity of Pel production to carbon source variation. However, a 6-fold decrease in Pel production was observed when bacteria were grown in LB rich medium as opposed to basal mineral medium. Subsequently, bioinformatics analysis of cloned pel gene promoter region revealed the presence of DegU binding site. Furthermore, the deletion of degU gene dramatically reduces the pel gene expression, as revealed by real time quantitative PCR, showing an activation effect of DegU on Pel synthesis in Bacillus strains studied. We assumed that, during the latter stage of cocoa fermentation when simple sugars are depleted, production of Pel in Bacillus is stimulated by DegU to supply microbial cells with carbon source from polymeric pectic compounds.

  18. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Science.gov (United States)

    Munir, Riffat I; Schellenberg, John; Henrissat, Bernard; Verbeke, Tobin J; Sparling, Richard; Levin, David B

    2014-01-01

    Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  19. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Directory of Open Access Journals (Sweden)

    Riffat I Munir

    Full Text Available Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes, sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199 and carbohydrate binding modules (95 were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  20. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation

    DEFF Research Database (Denmark)

    Agger, Jane W.; Isaksen, Trine; Várnai, Anikó

    2014-01-01

    The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity ofwell-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora ...

  1. Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw

    DEFF Research Database (Denmark)

    Thygesen, A.; Thomsen, A.B.; Schmidt, A.S.

    2003-01-01

    The production of cellulose and hemicellulose-degrading enzymes by cultivation of Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, Schizophyllum commune ATCC 38548, and Trichoderma reesei Rut-C30 was studied. Wet-oxidised wheat straw suspension suppleme......The production of cellulose and hemicellulose-degrading enzymes by cultivation of Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, Schizophyllum commune ATCC 38548, and Trichoderma reesei Rut-C30 was studied. Wet-oxidised wheat straw suspension...

  2. Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit.

    Science.gov (United States)

    Brecht, J K; Huber, D J

    1988-12-01

    Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO(2) and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.

  3. Immunogenicity of a Moraxella bovis bacterin containing attachment and cornea-degrading enzyme antigens.

    Science.gov (United States)

    Gerber, J D; Selzer, N L; Sharpee, R L; Beckenhauer, W H

    1988-02-01

    An adjuvanted Moraxella bovis bacterin containing attachment antigens and cornea-degrading enzyme antigens protected cattle from infectious bovine keratoconjunctivitis (IBK) when experimentally challenged with homologous and heterologous challenge cultures of M. bovis. This bacterin also protected cattle against field exposure to M. bovis. Transmission electron microscopy and fluorescein labeled anti-M. bovis pili antiserum showed pili on the M. bovis bacterin strain. Scanning electron microscopy demonstrated a fibrillar glycocalyx. The bacterin strain of M. bovis, but not all strains of M. bovis, destroyed bovine corneal cell monolayers in vitro. Bovine corneal cells began to separate from each other within 5 min after M. bovis organisms were added and adhered to the cell monolayers. Moraxella bovis organisms remained attached to the disintegrating cells as the cell membrane separated and was digested. Vaccination stimulated bacterial agglutination antibodies. However, protection against experimental challenge was more closely related to the cornea-degrading enzyme content of the experimental bacterins. Twenty-two of 29 cattle (76%) vaccinated with bacterins containing a relative enzyme activity (REA) greater than 0.4 were protected in a rigorous challenge of immunity test. Only 1 of 21 non-vaccinated calves (5%) was free of IBK. Ninety-two percent (24/26) of calves vaccinated with a bacterin containing a REA greater than 0.29 remained free of IBK following field exposure, whereas 47% (8/17) non-vaccinated calves developed IBK. Only 8 of 12 calves (67%) vaccinated with a bacterin containing a REA of 0.09 remained free of IBK. In a larger field efficacy test consisting of 32 herds in six states, the incidence of IBK in individual herds ranged from 0% to 55%. The overall rate of infection was 11.2%. Vaccination of calves with an M. bovis bacterin that contained a REA of 0.63 reduced the incidence of IBK from 11.2% (217/1931) in the non-vaccinated controls to 4

  4. From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes.

    Science.gov (United States)

    Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A

    2017-03-01

    The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct.

  5. Unravelling the Interactions between Hydrolytic and Oxidative Enzymes in Degradation of Lignocellulosic Biomass by Sporothrix carnis under Various Fermentation Conditions

    Directory of Open Access Journals (Sweden)

    Olusola A. Ogunyewo

    2016-01-01

    Full Text Available The mechanism underlying the action of lignocellulolytic enzymes in biodegradation of lignocellulosic biomass remains unclear; hence, it is crucial to investigate enzymatic interactions involved in the process. In this study, degradation of corn cob by Sporothrix carnis and involvement of lignocellulolytic enzymes in biodegradation were investigated over 240 h cultivation period. About 60% degradation of corn cob was achieved by S. carnis at the end of fermentation. The yields of hydrolytic enzymes, cellulase and xylanase, were higher than oxidative enzymes, laccase and peroxidase, over 144 h fermentation period. Maximum yields of cellulase (854.4 U/mg and xylanase (789.6 U/mg were at 96 and 144 h, respectively. Laccase and peroxidase were produced cooperatively with maximum yields of 489.06 U/mg and 585.39 U/mg at 144 h. Drastic decline in production of cellulase at 144 h (242.01 U/mg and xylanase at 192 h (192.2 U/mg indicates that they play initial roles in biodegradation of lignocellulosic biomass while laccase and peroxidase play later roles. Optimal degradation of corn cob (76.6% and production of hydrolytic and oxidative enzymes were achieved with 2.5% inoculum at pH 6.0. Results suggest synergy in interactions between the hydrolytic and oxidative enzymes which can be optimized for improved biodegradation.

  6. Engineering enzyme-cleavable hybrid click capsules with a pH-sheddable coating for intracellular degradation.

    Science.gov (United States)

    Gunawan, Sylvia T; Liang, Kang; Such, Georgina K; Johnston, Angus P R; Leung, Melissa K M; Cui, Jiwei; Caruso, Frank

    2014-10-29

    The engineering of layer-by-layer (LbL) hybrid click capsules that are responsive to biological stimuli is reported. The capsules comprise a pH-sheddable, non cross-linked outer coating that protects enzyme-cleavable inner layers. Upon cellular uptake, the outer coating is released and the capsules are enzymatically degraded. In vitro cell degradation results in rapid capsule degradation (10 min) upon cellular internalization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Enzyme

    Science.gov (United States)

    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 ...

  8. Partial purification of chlorophyll degrading enzymes from cavendish banana (Musa Cavendishi)

    National Research Council Canada - National Science Library

    Janave, Machhindra T; Sharma, Arun

    2004-01-01

    ...), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided...

  9. Enzymatic degradation of finely divided wood meal. II. Rollmilling and the subsequent enzymic degradation of Pinus densiflora wood

    Energy Technology Data Exchange (ETDEWEB)

    Muraki, E.; Yaku, F.; Tanaka, R.; Koshijima, T.

    1982-01-01

    The roll-milling of wood (P. densiflora) gave products with particle size less than 10 mu and high rate of saccharification under enzymic hydrolysis. About 90% of the available polysaccharides in wood could be converted into reducing sugars when roll-milled wood was subjected to enzymic hydrolysis using cellulase.

  10. Fabrication of enzyme-based coatings on intact multi-walled carbon nanotubes as highly effective electrodes in biofuel cells

    Science.gov (United States)

    Kim, Byoung Chan; Lee, Inseon; Kwon, Seok-Joon; Wee, Youngho; Kwon, Ki Young; Jeon, Chulmin; An, Hyo Jin; Jung, Hee-Tae; Ha, Su; Dordick, Jonathan S.; Kim, Jungbae

    2017-01-01

    CNTs need to be dispersed in aqueous solution for their successful use, and most methods to disperse CNTs rely on tedious and time-consuming acid-based oxidation. Here, we report the simple dispersion of intact multi-walled carbon nanotubes (CNTs) by adding them directly into an aqueous solution of glucose oxidase (GOx), resulting in simultaneous CNT dispersion and facile enzyme immobilization through sequential enzyme adsorption, precipitation, and crosslinking (EAPC). The EAPC achieved high enzyme loading and stability because of crosslinked enzyme coatings on intact CNTs, while obviating the chemical pretreatment that can seriously damage the electron conductivity of CNTs. EAPC-driven GOx activity was 4.5- and 11-times higher than those of covalently-attached GOx (CA) on acid-treated CNTs and simply-adsorbed GOx (ADS) on intact CNTs, respectively. EAPC showed no decrease of GOx activity for 270 days. EAPC was employed to prepare the enzyme anodes for biofuel cells, and the EAPC anode produced 7.5-times higher power output than the CA anode. Even with a higher amount of bound non-conductive enzymes, the EAPC anode showed 1.7-fold higher electron transfer rate than the CA anode. The EAPC on intact CNTs can improve enzyme loading and stability with key routes of improved electron transfer in various biosensing and bioelectronics devices.

  11. Fabrication of enzyme-based coatings on intact multi-walled carbon nanotubes as highly effective electrodes in biofuel cells

    Science.gov (United States)

    Kim, Byoung Chan; Lee, Inseon; Kwon, Seok-Joon; Wee, Youngho; Kwon, Ki Young; Jeon, Chulmin; An, Hyo Jin; Jung, Hee-Tae; Ha, Su; Dordick, Jonathan S.; Kim, Jungbae

    2017-01-01

    CNTs need to be dispersed in aqueous solution for their successful use, and most methods to disperse CNTs rely on tedious and time-consuming acid-based oxidation. Here, we report the simple dispersion of intact multi-walled carbon nanotubes (CNTs) by adding them directly into an aqueous solution of glucose oxidase (GOx), resulting in simultaneous CNT dispersion and facile enzyme immobilization through sequential enzyme adsorption, precipitation, and crosslinking (EAPC). The EAPC achieved high enzyme loading and stability because of crosslinked enzyme coatings on intact CNTs, while obviating the chemical pretreatment that can seriously damage the electron conductivity of CNTs. EAPC-driven GOx activity was 4.5- and 11-times higher than those of covalently-attached GOx (CA) on acid-treated CNTs and simply-adsorbed GOx (ADS) on intact CNTs, respectively. EAPC showed no decrease of GOx activity for 270 days. EAPC was employed to prepare the enzyme anodes for biofuel cells, and the EAPC anode produced 7.5-times higher power output than the CA anode. Even with a higher amount of bound non-conductive enzymes, the EAPC anode showed 1.7-fold higher electron transfer rate than the CA anode. The EAPC on intact CNTs can improve enzyme loading and stability with key routes of improved electron transfer in various biosensing and bioelectronics devices. PMID:28054656

  12. The bacterial cytoskeleton and its putative role in membrane vesicle formation observed in a Gram-positive bacterium producing starch-degrading enzymes.

    Science.gov (United States)

    Mayer, Frank; Gottschalk, Gerhard

    2003-01-01

    Bacteria may possess various kinds of cytoskeleton. In general, bacterial cytoskeletons may play a role in the control and preservation of the cell shape. Such functions become especially evident when the bacteria do not possess a true wall and are nevertheless elongated (e.g. Mycoplasma spp.) or under extreme cultivation conditions whereby loss of the entire bacterial cell wall takes place. Bacterial cytoskeletons may control and preserve the cell shape only if a number of preconditions are fulfilled. They should be present not only transiently, but permanently, they should be located as a lining close to the inner face of the cytoplasmic membrane, enclosing the entire cytoplasm, and they should comprise structural elements (fibrils) crossing the inner volume of the cell in order to provide the necessary stability for the lining. Complete loss of the cell wall layers had earlier been observed to occur during extensive production of bacterial starch-degrading enzymes in an optimized fermentation process by a Gram-positive bacterium. Even under these conditions, the cells had maintained their elongated shape and full viability. Which of the various kinds of bacterial cytoskeleton might have been responsible for shape preservation? Only one of them, the primary or basic cytoskeleton turns out to fulfil the necessary preconditions listed above. Its structural features now provided a first insight into a possible mechanism of formation of membrane blebs and vesicles as observed in the Gram-positive eubacterium Thermoanaerobacterium thermosulfurogenes EM1, and the putative role of the cytoskeletal web in this process.

  13. Production and partial characterization of arabinoxylan-degrading enzymes by Penicillium brasilianum under solid-state fermentation

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Granouillet, P.; Olsson, Lisbeth

    2006-01-01

    The production of a battery of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum grown on brewer's spent grain (BSG) under solid-state fermentation was investigated. Initial moisture content, initial pH, temperature, and nitrogen source content were optimized to achieve maximum...

  14. 3-Ketosteroid 9 alpha-hydroxylase enzymes : Rieske non-heme monooxygenases essential for bacterial steroid degradation

    NARCIS (Netherlands)

    Petrusma, Mirjan; van der Geize, Robert; Dijkhuizen, Lubbert

    2014-01-01

    Various micro-organisms are able to use sterols/steroids as carbon- and energy sources for growth. 3-Ketosteroid 9 alpha-hydroxylase (KSH), a two component Rieske non-heme monooxygenase comprised of the oxygenase KshA and the reductase KshB, is a key-enzyme in bacterial steroid degradation. It initi

  15. Improved mortality of the Formosan subterranean termite by fungi, when amended with cuticle-degrading enzymes or eicosanoid biosynthesis inhibitors

    Science.gov (United States)

    Formosan subterranean termites (FST) were exposed to spores of the fungus Beauveria pseudobassiana (Bpb) strain 8046 to determine virulence of the fungus. Once Bpb was determined to cause mortality of FST it was combined with enzymes capable of degrading the insect cuticle to measure the potential ...

  16. Screening of SDS-degrading Bacteria from Carwash Wastewater and the Study of the Alkylsulfatase Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Razieh Shahbazi

    2013-06-01

    Full Text Available Background and Objectives: Sodium dodecyl sulfate (SDS is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS–degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene.Materials and Methods: Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS.Results and Conclusion: Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg . The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation.

  17. Small-molecule activators of insulin-degrading enzyme discovered through high-throughput compound screening.

    Directory of Open Access Journals (Sweden)

    Christelle Cabrol

    Full Text Available BACKGROUND: Hypocatabolism of the amyloid beta-protein (Abeta by insulin-degrading enzyme (IDE is implicated in the pathogenesis of Alzheimer disease (AD, making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds. METHODOLOGY/PRINCIPAL FINDINGS: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds--designated Ia1 and Ia2--that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Abeta by approximately 700% and approximately 400%, respectively, albeit only when Abeta was presented in a mixture also containing shorter substrates. CONCLUSIONS/SIGNIFICANCE: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

  18. Cell Signaling Mechanisms by which Geniposide Regulates Insulin- Degrading Enzyme Expression in Primary Cortical Neurons.

    Science.gov (United States)

    Zhang, Yonglan; Xia, Zhining; Liu, Jianhui; Yin, Fei

    2015-01-01

    An increasing number of studies have demonstrated that insulin-degrading enzyme (IDE) plays an essential role in both the degradation and its activity of β-amyloid (Aβ). Therefore, the regulation of IDE expression and/or modification of IDE-dependent actions are two emerging strategies for the treatment of Alzheimer's disease (AD). We previously observed that geniposide, a novel agonist of glucagon-like peptide 1 receptor (GLP-1R), could attenuate Aβ-induced neurotoxicity by regulating the expression of IDE in primary cortical neurons. However, the signal transduction mechanisms underlying this effect were not elucidated. The present study, therefore examined and explored the cell signaling transduction and molecular mechanisms by which geniposide induces the expression of IDE in primary cortical neurons. The current study revealed that LY294002 (an inhibitor for phosphatidyl inositol 3-kinase, PI3K), PP1 (inhibitor for c-Src), GW9662 (antagonist for peroxisome proliferator-activated receptor γ, PPARγ), H89 (an inhibitor for protein kinase A, PKA) and AG1478 (an antagonist for epidermal growth factor receptor, EGFR) prohibited the up-regulation of IDE induced by geniposide in primary cortical neurons. Further, geniposide also enhanced the phosphorylation of PPARγ and accelerated the release of phosphorylated FoxO1 (forkhead box O1) from nuclear fraction to the cytosol. Moreover, geniposide directly activated the activity of IDE promoter in PC12 cells, which confirmed the presence of the GLP-1 receptor. Taken together, our findings reveal for the first time the cell signaling transduction pathway of geniposide regulating the expression of IDE in neurons.

  19. Machine Learning and Molecular Dynamics Based Insights into Mode of Actions of Insulin Degrading Enzyme Modulators.

    Science.gov (United States)

    Jamal, Salma; Goyal, Sukriti; Shanker, Asheesh; Grover, Abhinav

    2017-01-01

    Alzheimer's disease (AD) is one of the most common lethal neurodegenerative disorders having impact on the lives of millions of people worldwide. The disease lacks effective treatment options and the unavailability of the drugs to cure the disease necessitates the development of effectual anti-Alzheimer drugs. Several mechanisms have been reported underlying the association of the two disorders, diabetes and dementia, one among which is the insulin-degrading enzyme (IDE) which is known to degrade insulin as well beta-amyloid peptides. The present study is aimed to generate accurate classification models using machine learning techniques, which could identify IDE modulators from a bioassay dataset consisting of IDE inhibitors as well as non-inhibitors. The identified compounds were subjected to docking and Molecular dynamics (MD) studies for an in-depth analysis of the binding modes along with the complex stability. This study proposes that the identified potential active compounds, STK026154 (PubChem ID: CID2927418) with Glide score of -7.70 kcal/mol and BAS05901102 (PubChem ID: CID3152845) with Glide score of -7.06 kcal/mol, could serve as promising leads for the development of novel drugs against AD. The present study shows that such in silico approaches can be effectively used to discover and select active compounds from unseen data for accelerated drug development process. The machine learning models generated in the present study were used to screen Traditional Chinese Medicine (TCM) database to identify the phytocompounds already been reported to have therapeutic effects against AD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases

    DEFF Research Database (Denmark)

    Yuhong, Huang; Busk, Peter Kamp; Lange, Lene

    2015-01-01

    in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose...... decomposing enzymes (GH3, GH5, GH6, GH7, GH9, GH45 and AA9), and abundant hemicellulases. We further applied peptide pattern recognition to reveal nine and seven subfamilies of GH10 and GH11 family enzymes, respectively. The uncharacterized XYL10A, XYL10B and XYL11 enzymes of F. commune were classified......-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass....

  1. Effect of enzyme additions on methane production and lignin degradation of landfilled sample of municipal solid waste.

    Science.gov (United States)

    Jayasinghe, P A; Hettiaratchi, J P A; Mehrotra, A K; Kumar, Sunil

    2011-04-01

    Operation of waste cells as landfill bioreactors with leachate recirculation is known to accelerate waste degradation and landfill gas generation. However, waste degradation rates in landfill bioreactors decrease with time, with the accumulation of difficult to degrade materials, such as lignin-rich waste. Although, potential exists to modify the leachate quality to promote further degradation of such waste, very little information is available in literature. The objective of this study was to determine the viability of augmenting leachate with enzymes to increase the rate of degradation of lignin-rich waste materials. Among the enzymes evaluated MnP enzyme showed the best performance in terms of methane yield and substrate (lignin) utilization. Methane production of 200 mL CH(4)/g VS was observed for the MnP amended reactor as compared to 5.7 mL CH(4)/g VS for the control reactor. The lignin reduction in the MnP amended reactor and control reactor was 68.4% and 6.2%, respectively.

  2. Electrochemical detection and degradation of ibuprofen from water on multi-walled carbon nanotubes-epoxy composite electrode

    Institute of Scientific and Technical Information of China (English)

    Sorina Motoc; Adriana Remes; Aniela Pop; Florica Manea; Joop Schoonman

    2013-01-01

    This work describes the electrochemical behaviour of ibuprofen on two types of multi-walled carbon nanotubes based composite electrodes,i.e.,multi-walled carbon nanotubes-epoxy (MWCNT) and silver-modified zeolite-multi-walled carbon nanotubes-epoxy (AgZMWCNT) composites electrodes.The composite electrodes were obtained using two-roll mill procedure.SEM images of surfaces of the composites revealed a homogeneous distribution of the composite components within the epoxy matrix.AgZMWCNT composite electrode exhibited the better electrical conductivity and larger electroactive surface area.The electrochemical determination of ibuprofen (IBP) was achieved using AgZMWCNT by cyclic voltammetry,differential-pulsed voltammetry,square-wave voltammetry and chronoamperometry.The IBP degradation occurred on both composite electrodes under controlled electrolysis at 1.2 and 1.75 V vs.Ag/AgCl,and IBP concentration was determined comparatively by differential-pulsed voltammetry,under optimized conditions using AgZMWCNT electrode and UV-Vis spectrophotometry methods to determine the IBP degradation performance for each electrode.AgZMWCNT electrode exhibited a dual character allowing a double application in IBP degradation process and its control.

  3. Isolation and screening of strains producing high amounts of rutin degrading enzymes from Fagopyrum tataricum seeds.

    Science.gov (United States)

    Zheng, Ya-Di; Luo, Qing-Lin; Zhou, Mei-Liang; Wang, De-Zhou; Zhang, Ye-Dong; Shao, Ji-Rong; Zhu, Xue-Mei; Tang, Yu

    2013-02-01

    The rutin degrading enzyme (RDE) was isolated and purified from tartary buckwheat seeds. The RDE was purified about 11.34-fold and its final yield was 3.5%, which was very low, due to our purification strategy of giving priority to purity over yield. The RDE molecular weight was estimated to be about 60 kDa. When rutin was used as substrate, an optimal enzyme activity was seen at around pH 5.0 and 40 °C. Strains isolation strategy characterized by the use of rutin as sole carbon source in enrichment cultures was used to isolate RDE-producing strains. Then the active strains were identified by morphology characterization and 18s rDNA-ITS (Internal Transcribed Spacer) gene sequencing. Three isolates coded as B3, W2, Y2 were successfully isolated from fusty Fagopyrum tataricum flour cultures. Strain B3 possessed the highest unit activity among these three strains, and its total activity reached up to 171.0 Unit. The active isolate (B3) could be assigned to Penicillium farinosum. When the Penicillium farinosum strains were added to tartary buckwheat flour cultures at pH 5.0, 30 °C after 5 days fermentation, the quercetin production raised up to 1.78 mg/l, almost 5.1 times higher than the fermentation without the above active strains. Hence, a new approach was available to utilize microorganism-aided fermentation for effective quercetin extraction from Fagopyrum tataricum seeds. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Cvetić Antić, Tijana; Vučinić, Željko

    2015-01-01

    Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

  5. Degradation of different pectins by fungi: correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin

    Directory of Open Access Journals (Sweden)

    Benoit Isabelle

    2012-07-01

    Full Text Available Abstract Background Pectins are diverse and very complex biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. Results The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. Conclusions This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.

  6. Major amyloid-β-degrading enzymes, endothelin-converting enzyme-2 and neprilysin, are expressed by distinct populations of GABAergic interneurons in hippocampus and neocortex.

    Science.gov (United States)

    Pacheco-Quinto, Javier; Eckman, Christopher B; Eckman, Elizabeth A

    2016-12-01

    Impaired clearance of amyloid-β peptide (Aβ) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aβ in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aβ that may be functionally significant. To better understand the differential regulation of Aβ concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aβ degradation is consistent with the possibility that Aβ may have a physiological function related to the regulation of inhibitory signaling.

  7. Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls.

    Science.gov (United States)

    Nari, J; Noat, G; Diamantidis, G; Woudstra, M; Ricard, J

    1986-02-17

    Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.

  8. Effect of the combined probiotics with aflatoxin B₁-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression.

    Science.gov (United States)

    Zuo, Rui-yu; Chang, Juan; Yin, Qing-qiang; Wang, Ping; Yang, Yu-rong; Wang, Xiao; Wang, Guo-qiang; Zheng, Qiu-hong

    2013-09-01

    In order to degrade aflatoxin B₁ (AFB₁), AFB₁-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB₁-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 μg/kg AFB₁ supplement without feed additive, and 200, 400, 800 μg/kg AFB₁ supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB₁ residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB₁ on chicken's production performance and nutrient metabolic rates (P<0.05). It could also improve AFB₁ metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P<0.05). It could be concluded that the feed additive was able to degrade AFB₁ and improve animal production.

  9. Characterization of cell wall degrading enzymes from Chrysosporium lucknowense C1 and their use to degrade sugar beet pulp

    NARCIS (Netherlands)

    Kühnel, S.

    2011-01-01

    Key words: Pectin, arabinan, biorefinery, mode of action, branched arabinose oligomers, ferulic acid esterase, arabinohydrolase, pretreatment Sugar beet pulp is the cellulose and pectin-rich debris remaining after sugar extraction from sugar beets. In order to use sugar beet pulp for biorefinery

  10. Characterization of cell wall degrading enzymes from Chrysosporium lucknowense C1 and their use to degrade sugar beet pulp

    NARCIS (Netherlands)

    Kühnel, S.

    2011-01-01

    Key words: Pectin, arabinan, biorefinery, mode of action, branched arabinose oligomers, ferulic acid esterase, arabinohydrolase, pretreatment Sugar beet pulp is the cellulose and pectin-rich debris remaining after sugar extraction from sugar beets. In order to use sugar beet pulp for biorefinery pu

  11. Predicting Drug Extraction in the Human Gut Wall: Assessing Contributions from Drug Metabolizing Enzymes and Transporter Proteins using Preclinical Models.

    Science.gov (United States)

    Peters, Sheila Annie; Jones, Christopher R; Ungell, Anna-Lena; Hatley, Oliver J D

    2016-06-01

    Intestinal metabolism can limit oral bioavailability of drugs and increase the risk of drug interactions. It is therefore important to be able to predict and quantify it in drug discovery and early development. In recent years, a plethora of models-in vivo, in situ and in vitro-have been discussed in the literature. The primary objective of this review is to summarize the current knowledge in the quantitative prediction of gut-wall metabolism. As well as discussing the successes of current models for intestinal metabolism, the challenges in the establishment of good preclinical models are highlighted, including species differences in the isoforms; regional abundances and activities of drug metabolizing enzymes; the interplay of enzyme-transporter proteins; and lack of knowledge on enzyme abundances and availability of empirical scaling factors. Due to its broad specificity and high abundance in the intestine, CYP3A is the enzyme that is frequently implicated in human gut metabolism and is therefore the major focus of this review. A strategy to assess the impact of gut wall metabolism on oral bioavailability during drug discovery and early development phases is presented. Current gaps in the mechanistic understanding and the prediction of gut metabolism are highlighted, with suggestions on how they can be overcome in the future.

  12. The role of copper(II) and zinc(II) in the degradation of human and murine IAPP by insulin-degrading enzyme.

    Science.gov (United States)

    Bellia, Francesco; Grasso, Giuseppe

    2014-04-01

    Amylin or islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin-degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper- and zinc-induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion-induced changes in substrate accessibility.

  13. Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme.

    Directory of Open Access Journals (Sweden)

    Daishi Yui

    Full Text Available Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD model mice showed decreased insulin-degrading enzyme (IDE levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/- mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3; Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.

  14. Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme.

    Science.gov (United States)

    Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

    2015-01-01

    Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.

  15. Inhibition of Insulin-Degrading Enzyme Does Not Increase Islet Amyloid Deposition in Vitro.

    Science.gov (United States)

    Hogan, Meghan F; Meier, Daniel T; Zraika, Sakeneh; Templin, Andrew T; Mellati, Mahnaz; Hull, Rebecca L; Leissring, Malcolm A; Kahn, Steven E

    2016-09-01

    Islet amyloid deposition in human type 2 diabetes results in β-cell loss. These amyloid deposits contain the unique amyloidogenic peptide human islet amyloid polypeptide (hIAPP), which is also a known substrate of the protease insulin-degrading enzyme (IDE). Whereas IDE inhibition has recently been demonstrated to improve glucose metabolism in mice, inhibiting it has also been shown to increase cell death when synthetic hIAPP is applied exogenously to a β-cell line. Thus, we wanted to determine whether a similar deleterious effect is observed when hIAPP is endogenously produced and secreted from islets. To address this issue, we cultured hIAPP transgenic mouse islets that have the propensity to form amyloid for 48 and 144 hours in 16.7 mM glucose in the presence and absence of the IDE inhibitor 1. At neither time interval did IDE inhibition increase amyloid formation or β-cell loss. Thus, the inhibition of IDE may represent an approach to improve glucose metabolism in human type 2 diabetes, without inducing amyloid deposition and its deleterious effects.

  16. Association between polymorphisms of the insulin-degrading enzyme gene and late-onset Alzheimer disease.

    Science.gov (United States)

    Wang, Shitao; He, Feiyan; Wang, Ying

    2015-06-01

    The insulin-degrading enzyme (IDE) gene is a strong positional and biological candidate for late-onset Alzheimer disease (LOAD) susceptibility, with recent studies independently demonstrating an association between IDE gene variants and LOAD. However, previous data have been controversial. To investigate the relationship between IDE gene polymorphisms and LOAD risk, a case-control association study of 406 Han Chinese participants in Xinjiang, China, was undertaken. The LOAD and control groups consisted of 202 and 204 participants, respectively. The single-nucleotide polymorphisms rs1887922 and rs1999764 of the IDE gene were linked to LOAD incidence. The presence of the CT+CC genotype of rs1999764 had a protective effect compared to the TT genotype (adjusted P=.0001; odds ratio [OR]=0.226; 95% confidence interval [CI]=0.116-0.441), while the CT+CC genotype of rs1887922 was associated with increased LOAD risk (adjusted P=.0001; OR=3.640; 95% CI=1.889-7.016). Moreover, the effects of rs1887922 and rs1999764 were associated with LOAD risk independent of the apolipoprotein E ∊4 polymorphism and were more significant in men and women, respectively. These results demonstrate that the polymorphisms rs1887922 and rs1999764 of the IDE gene are associated with LOAD susceptibility in the Xinjiang Han population.

  17. Association of insulin degrading enzyme gene polymorphisms with Alzheimer's disease: a meta-analysis.

    Science.gov (United States)

    Cheng, Huawei; Wang, Lin; Shi, Tianlu; Shang, Yuping; Jiang, Ling

    2015-05-01

    Alzheimer's disease (AD) is a chronic degenerative disorder. It is caused by both genetic and environmental factors. The association of Insulin Degrading Enzyme (IDE) genotypes rs4646953, rs2251101 and rs1544210 with AD has been detected, but the findings were conflicted, however, Apolipoprotein-E (APOE)-ε4 allele has been observed as a genetic risk factor for AD. To investigate the issue, a meta-analysis was performed. We searched PubMed, Springer Link, AlzGene and CNKI for relevant literatures published by June 2013. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated to explore the significant association. A total of 11 studies comprising 5771 cases and 5474 controls were considered in final meta-analysis. We found that weak connections existed between rs4646953 (TT vs. CC: z = 2.24, p = 0.025, OR = 1.536) and AD, but no significant associations have been found between other IDE gene single nucleotide polymorphisms of rs4646953, rs2251101 and rs1544210 with AD. We certified that APOE-ε4 allele was still be a suspected factor to AD. There was no evidence for obvious publication bias in overall meta-analysis. Furthermore, larger-scale randomized controlled trials are necessary to validate the association between IDE gene polymorphisms with AD.

  18. Characterization of a trehalose-degrading enzyme from the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Moon, Jeong Hyun; Lee, Whiso; Park, Jihee; Choi, Kyoung-Hwa; Cha, Jaeho

    2016-07-01

    We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion-exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40 kDa. Maximum activity was observed at 85°C and pH 4.5. The half-life of TreH was 53 and 41 min at 90°C and 95°C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a Ki of 0.05 mM. Compared with TreH from other trehalases, TreH from S. acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain.

  19. Development of enzyme immunoassay for captan and its degradation product tetrahydrophthalimide in foods.

    Science.gov (United States)

    Newsome, W H; Yeung, J M; Collins, P G

    1993-01-01

    A simple, sensitive, and precise enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of captan as its degradation product tetrahydrophthalimide (THPI) in foods using polyclonal antibodies. Three hapten analogues of THPI with different alkyl spacer arm lengths were synthesized. Immunogens and coating proteins were prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. A 5-carbon spacer arm appeared to be optimum for the production of antibodies. Heterologous coating proteins did not improve the sensitivity, but reduction of homologous coating protein concentration did improve the sensitivity, resulting in a concentration of test compound required to inhibit binding by 50% of 15.5 ng/mL. The antiserum is specific for captan, captafol, and THPI, but not other structurally related compounds. The minimum detection limit was 1 ng/mL; the linearity was 1-200 ng/mL. The overall recoveries of captan and THPI from 11 commodities spiked at 4 levels were 92 and 100%, respectively. The intra-assay and interassay coefficients of variation were 9.1 and 16.8% for apple blanks and 5.9 and 4.2% for apple spiked with 3 ppm THPI, respectively. The ELISA described is suitable for measuring captan and THPI at levels comparable to those typically found in fruit.

  20. Deubiquitinating enzymes Ubp2 and Ubp15 regulate endocytosis by limiting ubiquitination and degradation of ARTs

    Science.gov (United States)

    Ho, Hsuan-Chung; MacGurn, Jason A.; Emr, Scott D.

    2017-01-01

    Endocytic down-regulation of cell-surface proteins is a fundamental cellular process for cell survival and adaptation to environmental stimuli. Ubiquitination of cargo proteins serves as the sorting signal for downstream trafficking and relies on the arrestin-related trafficking adaptor (ART)-Rsp5 ubiquitin ligase adaptor network in yeast. Hence proper regulation of the abundance and activity of these ligase–adaptor complexes is critical for main­tenance of optimal plasma membrane protein composition. Here we report that the stability of ARTs is regulated by the deubiquitinating enzymes (DUBs) Ubp2 and Ubp15. By counteracting the E3 ubiquitin ligase Rsp5, Ubp2 and Ubp15 prevent hyperubiquitination and proteasomal degradation of ARTs. Specifically, we show that loss of both Ubp2 and Ubp15 results in a defect in Hxt6 endocytosis associated with Art4 instability. Our results uncover a novel function for DUBs in the endocytic pathway by which Ubp2 and Ubp15 positively regulate the ART-Rsp5 network. PMID:28298493

  1. Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

    Science.gov (United States)

    Aoyama, Akihisa; Kurane, Ryuichiro; Matsuura, Akira; Nagai, Kazuo

    2015-01-01

    An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

  2. Lignocellulose degradation patterns, structural changes, and enzyme secretion by Inonotus obliquus on straw biomass under submerged fermentation.

    Science.gov (United States)

    Xu, Xiangqun; Xu, Zhiqi; Shi, Song; Lin, Mengmeng

    2017-10-01

    This study examined the white rot fungus I. obliquus on the degradation of three types of straw biomass and the production of extracellular lignocellulolytic enzymes under submerged fermentation. The fungus process resulted in a highest lignin loss of 72%, 39%, and 47% in wheat straw, rice straw, and corn stover within 12days, respectively. In merely two days, the fungus selectively degraded wheat straw lignin by 37%, with only limited cellulose degradation (13%). Fourier transform infrared spectroscopy revealed that the fungus most effectively degraded the wheat straw lignin and rice straw crystalline cellulose. Scanning electronic microscopy showed the most pronounced structural changes in wheat straw. High activities of manganese peroxidase (159.0U/mL) and lignin peroxidase (123.4U/mL) were observed in wheat straw culture on Day 2 and 4, respectively. Rice straw was the best substrate to induce the production of cellulase and xylanase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse.

    Science.gov (United States)

    Mishra, Vartika; Jana, Asim K; Jana, Mithu Maiti; Gupta, Antriksh

    2017-07-01

    The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ∼2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto).

    Science.gov (United States)

    Mitsui, Nobuo; Murasawa, Hisashi; Sekiguchi, Junichi

    2011-01-01

    Poly-γ-glutamic acid (γPGA), a polymer of glutamic acid, is a component of the viscosity substance of natto, a traditional Japanese food made from soybeans fermented with Bacillus subtilis (natto). Here we investigate the effects of the cell wall lytic enzymes belonging to the D,L-endopeptidases (LytE, LytF, CwlO and CwlS) on γPGA production by B. subtilis (natto). γPGA levels in a cwlO disruptant were about twofold higher than that of the wild-type strain, whereas disruption of the lytE, lytF and cwlS genes had little effect on γPGA production. The molecular size of γPGA in the cwlO disruptant was larger than that of the wild-type strain. A complementary strain was constructed by insertion of the entire cwlO gene into the amyE locus of the CwlO mutant genome, and γPGA production was restored to wild-type levels in this complementary strain. These results indicated that the peptidoglycan degradation enzyme, CwlO, plays an important role in γPGA production and affects the molecular size of γPGA.

  5. Binding of human serum albumin to single-walled carbon nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

    Science.gov (United States)

    Lu, Naihao; Li, Jiayu; Tian, Rong; Peng, Yi-Yuan

    2014-06-16

    Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl-) and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase (MPO). However, the importance of protein-SWCNT interactions in the biodegradation of SWCNTs was not stressed. Here, we used both experimental and theoretical approaches to investigate the interactions of SWCNTs with human serum albumin (HSA, one of the most abundant proteins in blood circulation) and found that the binding was involved in the electrostatic interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π-π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Compared with SWCNTs, the binding of HSA could result in a reduced effect for OCl- (or the human MPO system)-induced SWCNTs degradation in vitro. However, the HSA-SWCNT interactions would enhance cellular uptake of nanotubes and stimulate MPO release and OCl- generation in neutrophils, thereby creating the conditions favorable for the degradation of the nanotubes. Upon zymosan stimulation, both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was more for HSA-SWCNTs under these relevant in vivo conditions. Our findings suggest that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in human inflammatory cells and therefore shed light on the biomedical and biotechnological applications of safe carbon nanotubes by comprehensive preconsideration of their interactions with human serum proteins.

  6. Structural characterization of the acid-degraded secondary cell wall polymer of Geobacillus stearothermophilus PV72/p2.

    Science.gov (United States)

    Petersen, Bent O; Sára, Margit; Mader, Christoph; Mayer, Harald F; Sleytr, Uwe B; Pabst, Martin; Puchberger, Michael; Krause, Eberhard; Hofinger, Andreas; Duus, Jens Ø; Kosma, Paul

    2008-06-09

    The secondary cell wall polymer (SCWP) from Geobacillus stearothermophilus PV72/p2, which is involved in the anchoring of the surface-layer protein to the bacterial cell wall layer, is composed of 2-amino-2-deoxy- and 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-mannose, and 2-acetamido-2-deoxy-D-mannuronic acid. The primary structure of the acid-degraded polysaccharide--liberated by HF-treatment from the cell wall--was determined by high-field NMR spectroscopy and mass spectrometry using N-acetylated and hydrolyzed polysaccharide derivatives as well as Smith-degradation. The polysaccharide was shown to consist of a tetrasaccharide repeating unit containing a pyruvic acid acetal at a side-chain 2-acetamido-2-deoxy-alpha-D-mannopyranosyl residue. Substoichiometric substitutions of the repeating unit were observed concerning the degree of N-acetylation of glucosamine residues and the presence of side-chain linked 2-acetamido-2-deoxy-beta-D-glucopyranosyl units: [Formula: see text].

  7. Insulin-degrading enzyme: structure-function relationship and its possible roles in health and disease.

    Science.gov (United States)

    Fernández-Gamba, A; Leal, M C; Morelli, L; Castaño, E M

    2009-01-01

    Insulin-degrading enzyme (IDE) or insulysin is a highly conserved Zn(2+) -dependent endopeptidase with an "inverted" HxxEH motif. In vivo, IDE contributes to regulate the steady state levels of peripheral insulin and cerebral amyloid beta peptide (Abeta) of Alzheimer's disease. In vitro, substrates of IDE include a broad spectrum of peptides with relevant physiological functions such as atrial natriuretic factor, insulin-like growth factor-II, transforming growth factor-alpha, beta-endorphin, amylin or glucagon. The recently solved crystal structures of an inactive IDE mutant bound to four different substrates indicate, in accordance with previous compelling biochemical data, that peptide backbone conformation and size are major determinants of IDE recognition and substrate selectivity. IDE-N and IDE-C halves contribute to substrate binding and may rotate away from each other leading to open and closed conformers that permit or preclude the entry of substrates. Noteworthy, stabilization of substrate beta strands in their IDE-bound form may explain the preference of IDE for peptides with a high tendency to self-assembly as amyloid fibrils. These structural requirements may underlie the capability of some amyloid peptides of forming extremely stable complexes with IDE and raise the possibility of a dead-end chaperone-like function of IDE independent of catalysis. Furthermore, the recent recognition of IDE as a varicella zoster virus receptor and its putative involvement in muscle cell differentiation, steroid receptor signaling or proteasome modulation suggest that IDE is a multi-functional protein with broad and relevant roles in several basic cellular processes. Accordingly, IDE functions, regulation or trafficking may partake in the molecular pathogenesis of major human diseases and become potential targets for therapeutic intervention.

  8. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    Science.gov (United States)

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.

    2010-01-01

    Background Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. Methodology/Principal Findings We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are ∼106 times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's “closed,” inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes. PMID:20498699

  9. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    Energy Technology Data Exchange (ETDEWEB)

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J. (Harvard-Med); (BWH); (Yale-MED); (Scripps); (UC); (Mayo)

    2010-09-20

    Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are {approx} 10{sup 6} times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-a-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's 'closed,' inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

  10. Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin.

    Directory of Open Access Journals (Sweden)

    Malcolm A Leissring

    Full Text Available BACKGROUND: Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE, a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. METHODOLOGY/PRINCIPAL FINDINGS: We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are approximately 10(6 times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's "closed," inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. CONCLUSIONS/SIGNIFICANCE: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

  11. Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

    Directory of Open Access Journals (Sweden)

    Dickson Dennis W

    2009-10-01

    Full Text Available Abstract Background Insulin-degrading enzyme (IDE is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs targeting natively folded human and rodent IDE. Results Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. Conclusion We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

  12. Enzymes for Degradation of Energetic Materials and Demilitarization of Explosives Stockpiles - SERDP Annual (Interim) Report, 12/98

    Energy Technology Data Exchange (ETDEWEB)

    Shah, M.M.

    1999-01-18

    The current stockpile of energetic materials requiring disposal contains about half a million tons. Through 2001, over 2.1 million tons are expected to pass through the stockpile for disposal. Safe and environmentally acceptable methods for disposing of these materials are needed. This project is developing safe, economical, and environmentally sound processes using biocatalyst (enzymes) to degrade energetic materials and to convert them into economically valuable products. Alternative methods for destroying these materials are hazardous, environmentally unacceptable, and expensive. These methods include burning, detonation, land and sea burial, treatment at high temperature and pressure, and treatment with harsh chemicals. Enzyme treatment operates at room temperature and atmospheric pressure in a water solution.

  13. Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases

    DEFF Research Database (Denmark)

    Yuhong, Huang; Busk, Peter Kamp; Lange, Lene

    2015-01-01

    in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose......-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass....

  14. Expression and post-translational processing of a broad-spectrum organophosphorus-neurotoxin-degrading enzyme in insect tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Dave, K.I.; Phillips, L.; Luckow, V.A.; Wild, J.R.

    1994-12-31

    A recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been utilized to express the opd (organophosphate-degrading) gene from Pseudomonas diminuta in insect tissue-culture cells (Sf9) of the fall armyworm (Spodoptera frugiperda). The broad-spectrum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a member of a general class of enzymes (organophosphate (OP) anhyorolases) that include parathion hydrolases, di-isopropyl-fluorophosphatases (DFpases), somanases, and OP phosphotrlesterases. This particular enzyme possesses the ability to hydrolyse paraoxon (P-O bond), DFP, sarin (P-F bond), VX (P-S bond) and tabun (P-CN bond), as well as a number of other extensively used organophosphorus pesticides. The enzyme produced in infected Sf9 cells is post-translationally processed and resembles the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots. N-terminal sequence analysis of enzyme expressed in insect cells revealed Gly-29 as the terminal residue, whereas expression in Escherichia coli removes this residue, exposing Ser-30 at the N-terminus. Conditions for optimal expression of the enzyme in this system are described. Furthermore, hydrolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminuta MG cells.

  15. Peroxidase-induced degradation of single-walled carbon nanotubes: hypochlorite is a major oxidant capable of in vivo degradation of carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Vlasova, I I; Vakhrusheva, T V; Sokolov, A V; Kostevich, V A [Research Institute for Physico-Chemical Medicine, FMBA, M. Pirogovskaya Str. 1a, Moscow (Russian Federation); Ragimov, A A, E-mail: irina.vlasova@yahoo.com [National Research Centre of Surgery, RAMS, Abrikosovskiy per. 2, Moscow (Russian Federation)

    2011-04-01

    Due to their extraordinary properties, single-walled carbon nanotubes (SWNTs) have a tremendous potential for medical applications such as clinical diagnostics, targeted drug (or gene) delivery and cancer therapy. Hence, effects of SWNTs on living systems as well as mechanisms for biodegradation of SWTNs are of great importance and must be studied before starting to explore SWNTs for medical use. This study was undertaken to compare the potential of different peroxidases in degrading carboxylated SWNT (c-SWNT) and to elucidate the role of peroxidase-generated reactive products in this process. A detailed study showed that neither reactive intermediate products nor free radicals generated via peroxidase cycle can considerably oxidize c-SWNT. Biodegradation of c-SWNT in model system can be induced by free radicals generated as a result of heme degradation. The latter explains why hemoglobin, which is a pseudo-peroxidase possessing low peroxidase activity, is able to oxidize carbon nanotubes with a higher efficiency than horseradish peroxidase. However, c-SWNT in the presence of blood plasma (15 vol %) demonstrated no degradation even at high concentrations of hemoglobin and H{sub 2}O{sub 2}. The comparison of the ability of various peroxidases to degrade SWNTs in vitro revealed that MPO, due to its ability to produce hypochlorite, and lactoperoxidase, due to its ability to produce hypobromite, are extremely efficient in degrading carbon nanotubes. Since neutrophils are a main source of human MPO, we tested the effect of SWNTs on these cells. SWNTs were unable to stimulate neutrophils. On the other hand, they dose-dependently enhanced opsonized zymosan-induced cell stimulation as detected by measuring the amount of hypochlorite produced. This finding may be relevant to the in vivo situation, for example, at inflammatory sites. In order to imitate conditions characteristic of phagosomes and inflammatory sites, we titrated the suspension of c-SWNT in the presence of

  16. Extractability and digestibility of plant cell wall polysaccharides during hydrothermal and enzymatic degradation of wheat straw (Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Hansen, Mads A.T.; Ahl, Louise I.; Pedersen, Henriette L.

    2014-01-01

    , regardless their extractability in water or only alkali. Based on the results, AX and MLG appear to be loosely bound in the cell wall matrix while the other polysaccharides are bound more tightly and shielded from enzymatic attack by AX and MLG until pretreatment. The gradual solubilisation and digestion...... and by comprehensive microarray polymer profiling (CoMPP). This way, the effects of each degradation step to the intermolecular organisation of specific polysaccharides in the cell walls were elucidated. After pretreatment, the degree of polymerisation (DP) of released xylo-oligosaccharides in both samples was up...... to about 20, but mostly around 3-8, and notably more acetylated in stems. Arabinoxylan (AX) and mixed-linkage glucan (MLG) became water-extractable while xylan, xyloglucan (XG), mannan and glucan remained only alkali-extractable. All polysaccharides became partly digestible after pretreatment however...

  17. Leucoagaricus gongylophorus produces diverse enzymes for the degradation of recalcitrant plant polymers in leaf-cutter ant fungus gardens.

    Science.gov (United States)

    Aylward, Frank O; Burnum-Johnson, Kristin E; Tringe, Susannah G; Teiling, Clotilde; Tremmel, Daniel M; Moeller, Joseph A; Scott, Jarrod J; Barry, Kerrie W; Piehowski, Paul D; Nicora, Carrie D; Malfatti, Stephanie A; Monroe, Matthew E; Purvine, Samuel O; Goodwin, Lynne A; Smith, Richard D; Weinstock, George M; Gerardo, Nicole M; Suen, Garret; Lipton, Mary S; Currie, Cameron R

    2013-06-01

    Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.

  18. Biochemical characterisation of an allantoate-degrading enzyme from French bean (Phaseolus vulgaris): the requirement of phenylhydrazine.

    Science.gov (United States)

    Raso, María José; Muñoz, Alfonso; Pineda, Manuel; Piedras, Pedro

    2007-10-01

    In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined "in vivo" using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37 degrees C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.

  19. Determination of extra and intracellular content from some lytic enzymes related with carnation (Dianthus caryophyllus L. root cell wall

    Directory of Open Access Journals (Sweden)

    Sixta Tulia Martínez Peralta

    2016-11-01

    Full Text Available The presence of some enzymes related to cell wall (polygalacturonase, the pectate lyase, protease and xylanase in carnation (Dianthus caryophyllus L. roots as well as the activity levels were determined. These levels were analyzed in different cellular places: the intercellular fluid that is part of apoplast, the symplast, and the total level (apoplast and symplast in carnation roots. Two methods were tested to extract the intercellular fluid. To obtain the intracellular content (symplast and total extract (apoplast+symplast, three methods were tested, using as extracting solution  i phosphate buffer, ii phosphate buffer + PVPP,  iii before the extraction with phosphate buffer, the carnation roots were washed with acetone.  The results showed the effect of different extracting solutions in the enzymatic activities and in the protein content. A new only one step method is proposed to extract the four enzymes and make the comparative analysis of enzymatic activity.

  20. The Cell Wall Teichuronic Acid Synthetase (TUAS Is an Enzyme Complex Located in the Cytoplasmic Membrane of Micrococcus luteus

    Directory of Open Access Journals (Sweden)

    Lingyi Lynn Deng

    2010-01-01

    composed of disaccharide repeating units [-4-β-D-ManNAcAp-(1→6α-D-Glcp−1-]n, which is covalently anchored to the peptidoglycan on the inner cell wall and extended to the outer surface of the cell envelope. An enzyme complex responsible for the TUA chain biosynthesis was purified and characterized. The 440 kDa enzyme complex, named teichuronic acid synthetase (TUAS, is an octomer composed of two kinds of glycosyltransferases, Glucosyltransferase, and ManNAcA-transferase, which is capable of catalyzing the transfer of disaccharide glycosyl residues containing both glucose and the N-acetylmannosaminuronic acid residues. TUAS displays hydrophobic properties and is found primarily associated with the cytoplasmic membrane. The purified TUAS contains carotinoids and lipids. TUAS activity is diminished by phospholipase digestion. We propose that TUAS serves as a multitasking polysaccharide assembling station on the bacterial membrane.

  1. Potential efficacy of Lactobacillus casei IBRC_M10711 on expression and activity of insulin degrading enzyme but not insulin degradation.

    Science.gov (United States)

    Neyazi, Nadia; Mohammadi Farsani, Taiebeh; Nouri, Zahra; Ghahremani, Mohammad Hossein; Khorramizadeh, Mohammad Reza; Tajerian, Roksana; Motevaseli, Elahe

    2017-01-01

    Type 2 diabetes (T2D) is a condition with insufficient insulin production or in the setting of insulin resistance with many origins including intestinal microbiota-related molecular mechanism. Insulin-degrading enzyme (IDE) is responsible for insulin breakdown in various tissues and is known as a potential drug target for T2D. Here, we assessed the effects of cell-free supernatant (CFS) and UV-killed Lactobacillus casei IBRC_M10711 on IDE expression, IDE activity, and insulin degradation in Caco-2 cell line. It was found that CFS and UV-killed L. casei IBRC_M10711 led to lower expression of IDE. UV-killed L. casei IBRC_M10711 significantly inhibited IDE activity but CFS did not. Insulin degradation was affected with none of them. In conclusion, L. casei IBRC_M10711 is effective on IDE expression and its activity, but not on insulin degradation. Future studies are recommended to explore the effect of this probiotic on other substrates of IDE.

  2. Indirect resistance to several classes of antibiotics in cocultures with resistant bacteria expressing antibiotic-modifying or -degrading enzymes.

    Science.gov (United States)

    Nicoloff, Hervé; Andersson, Dan I

    2016-01-01

    Indirect resistance (IR), the ability of an antibiotic-resistant population of bacteria to protect a susceptible population, has been previously observed for β-lactamase-producing bacteria and associated with antimicrobial treatment failures. Here, we determined whether other resistance determinants could cause IR in the presence of five other classes of antibiotics. A test was designed to detect IR and 14 antibiotic resistance genes were tested in the presence of 13 antibiotics from six classes. A bioassay was used to measure the ability of resistance-causing enzymes to decrease the concentration of active antibiotics in the medium. We confirmed IR in the presence of β-lactam antibiotics (ampicillin and mecillinam) when TEM-1A was expressed. We found that bacteria expressing antibiotic-modifying or -degrading enzymes Ere(A), Tet(X2) or CatA1 caused IR in the presence of macrolides (erythromycin and clarithromycin), tetracyclines (tetracycline and tigecycline) and chloramphenicol, respectively. IR was not observed with resistance determinants that did not modify or destroy antibiotics or with enzymes modifying aminoglycosides or degrading fosfomycin. IR was dependent on the resistance enzymes decreasing the concentration of active antibiotics in the medium, hence allowing nearby susceptible bacteria to resume growth once the antibiotic concentration fell below their MIC. IR was not limited to β-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    Science.gov (United States)

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms.

  4. Potential of semiarid soil from Caatinga biome as a novel source for mining lignocellulose-degrading enzymes.

    Science.gov (United States)

    Lacerda Júnior, Gileno V; Noronha, Melline F; de Sousa, Sanderson Tarciso P; Cabral, Lucélia; Domingos, Daniela F; Sáber, Mírian L; de Melo, Itamar S; Oliveira, Valéria M

    2017-02-01

    The litterfall is the major organic material deposited in soil of Brazilian Caatinga biome, thus providing the ideal conditions for plant biomass-degrading microorganisms to thrive. Herein, the phylogenetic composition and lignocellulose-degrading capacity have been explored for the first time from a fosmid library dataset of Caatinga soil by sequence-based screening. A complex bacterial community dominated by Proteobacteria and Actinobacteria was unraveled. SEED subsystems-based annotations revealed a broad range of genes assigned to carbohydrate and aromatic compounds metabolism, indicating microbial ability to utilize plant-derived material. CAZy-based annotation identified 7275 genes encoding 37 glycoside hydrolases (GHs) families related to hydrolysis of cellulose, hemicellulose, oligosaccharides and other lignin-modifying enzymes. Taxonomic affiliation of genes showed high genetic potential of the phylum Acidobacteria for hemicellulose degradation, whereas Actinobacteria members appear to play an important role in celullose hydrolysis. Additionally, comparative analyses revealed greater GHs profile similarity among soils as compared to the digestive tract of animals capable of digesting plant biomass, particularly in the hemicellulases content. Combined results suggest a complex synergistic interaction of community members required for biomass degradation into fermentable sugars. This large repertoire of lignocellulolytic enzymes opens perspectives for mining potential candidates of biochemical catalysts for biofuels production from renewable resources and other environmental applications. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Xylose induces the phyllosphere yeast Pseudozyma antarctica to produce a cutinase-like enzyme which efficiently degrades biodegradable plastics.

    Science.gov (United States)

    Watanabe, Takashi; Shinozaki, Yukiko; Yoshida, Shigenobu; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Fujii, Takeshi; Fukuoka, Tokuma; Kitamoto, Hiroko Kuze

    2014-03-01

    There is a need to speed up the degradation of used agricultural mulch films that are made of biodegradable plastics (BPs) in the field. Treating them with BP-degrading enzymes could be a solution to this problem. A cutinase-like enzyme of yeast Pseudozyma antarctica (PaE) has wide specificity of BPs degradation, but needs to be produced efficiently. Here we report that the production of PaE by P. antarctica can be increased by using xylose as carbon source. BP-degradation activity was analyzed using a polybutylene succinate-co-adipate (PBSA) emulsion as the substrate. Strain P. antarctica GB-4(1)W was found to be the best PaE producer among the tested strains. Using a 5-L jar fermentor with xylose fed-batch cultivation, high PaE productivity could be maintained and about 21 U/ml of PaE was obtained in 120 h. This amount was 100 times higher than the amount that we obtained previously (0.21 U/ml by flask cultivation using glycerol as carbon source). Under repeated xylose fed-batch cultivation with 24 h intervals, the maximum PaE production rate (0.34 U/ml/h) was maintained and the highest PaE productivity (28,000 U/2 L/d) was repeatedly obtained for 7 intervals. The activity of filtered jar-culture (crude PaE) was stable over 12 weeks at 4°C. Commercially available BP mulch films (20 μm thickness, cut into 1-cm-squares) were completely degraded by submerging them in crude PaE (2 U/ml) at 30°C in 24 h. These results indicated that concentrated PaE can rapidly degrade the strength of BP mulch films in the field so that they do not interfere with plowing.

  6. Neprilysin and insulin-degrading enzyme levels are increased in Alzheimer disease in relation to disease severity.

    Science.gov (United States)

    Miners, James Scott; Baig, Shabnam; Tayler, Hannah; Kehoe, Patrick Gavin; Love, Seth

    2009-08-01

    Experimental reduction of neprilysin (NEP) or insulin-degrading enzyme (IDE) in vivo exacerbates beta-amyloid accumulation in the brain. The level of these enzymes is reportedly reduced during aging and in postmortem brains of patients with sporadic Alzheimer disease (AD). To distinguish between primary decreases in NEP and IDE activity that might contribute to beta-amyloid accumulation and decreases secondary to neurodegenerative changes in AD, we measured NEP and IDE levels by indirect sandwich ELISA and enzyme activities by immunocapture-based fluorogenic assays in postmortem frontal cortex from patients of different ages and at different pathological stages of AD, as indicated by Braak tangle stage. The ELISA measurements of neuron-specific enolase were used to adjust for neuronal loss. Both unadjusted and neuron-specific enolase-adjusted NEP levels and activity were significantly increased in AD and positively correlated with Braak stage but negatively with age in AD patients. Insulin-degrading enzyme activity was higher in AD than controls; this was significant after adjustment for neuron-specific enolase level; unadjusted IDE protein level was decreased in AD but not after adjustment. Our findings suggest that reduction in NEP and IDE activity is not the primary cause of beta-amyloid accumulation in AD, but rather a late-stage phenomenon secondary to neurodegeneration.

  7. Development of Quantum Dot Probes for Studies of Synergy Between Components of the Wood-Degrading Fungal Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Haw; Nixon, B. Tracy; Tien, Ming

    2011-09-01

    “Development of Quantum Dot Probes for Studies of Synergy Between Components of the Wood-Degrading Fungal Enzymes,” aims to develop quantum dot-based tagging and imaging technologies tailored for simultaneously monitoring, in real time and in the natural fungal / lignocellulose environment, the mode of action of several lignocellulosic enzymes at the single-molecule level. With a three-year research scope, it is designed to be the first project of a long-term research program for which the overarching goal is to bridge the aforementioned knowledge gap by a quantitative determination of the biochemical and biophysical properties of these fungal enzymes in realistic plant biomass-microbe milieus.

  8. Lytic polysaccharide monooxygenases: a crystallographer’s view on a new class of biomass-degrading enzymes

    Science.gov (United States)

    Frandsen, Kristian E. H.; Lo Leggio, Leila

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future. PMID:27840684

  9. Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

    Science.gov (United States)

    Liu, Yu-Huan; Chung, Ying-Cheng; Xiong, Ya

    2001-01-01

    A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (Km) and Vmax for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively. PMID:11472959

  10. Interaction of Carthamus tinctorius lignan arctigenin with the binding site of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase☆

    Science.gov (United States)

    Temml, Veronika; Kuehnl, Susanne; Schuster, Daniela; Schwaiger, Stefan; Stuppner, Hermann; Fuchs, Dietmar

    2013-01-01

    Mediterranean Carthamus tinctorius (Safflower) is used for treatment of inflammatory conditions and neuropsychiatric disorders. Recently C. tinctorius lignans arctigenin and trachelogenin but not matairesinol were described to interfere with the activity of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in peripheral blood mononuclear cells in vitro. We examined a potential direct influence of compounds on IDO enzyme activity applying computational calculations based on 3D geometry of the compounds. The interaction pattern analysis and force field-based minimization was performed within LigandScout 3.03, the docking simulation with MOE 2011.10 using the X-ray crystal structure of IDO. Results confirm the possibility of an intense interaction of arctigenin and trachelogenin with the binding site of the enzyme, while matairesinol had no such effect. PMID:24251110

  11. myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate.

    Science.gov (United States)

    Greiner, Ralf; Carlsson, Nils-Gunnar

    2006-08-01

    For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.

  12. Polymorphisms within insulin-degrading enzyme (IDE) gene determine insulin metabolism and risk of type 2 diabetes.

    Science.gov (United States)

    Rudovich, Natalia; Pivovarova, Olga; Fisher, Eva; Fischer-Rosinsky, Antje; Spranger, Joachim; Möhlig, Matthias; Schulze, Matthias B; Boeing, Heiner; Pfeiffer, Andreas F H

    2009-11-01

    Insulin-degrading enzyme (IDE) is the ubiquitously expressed major enzyme responsible for insulin degradation. Insulin-degrading enzyme gene is located on chromosome region 10q23-q25 and exhibits a well-replicated peak of linkage with type 2 diabetes (T2DM). Several genetic association studies examined IDE gene as a susceptibility gene for T2DM with controversial results. However, pathophysiological mechanisms involved have remained elusive. We verified associations of two IDE polymorphisms (rs1887922 and rs2149632) with T2DM risk in two independent German cohorts and evaluated in detail the association of common variants with insulin metabolism and glycemic traits. We confirmed previously published findings for diabetes-associated rs1887922 and rs2149632 in the European Prospective Investigation into Cancer and Nutrition-Potsdam cohort (n = 3049; RR 1.26, p = 0.003 and RR 1.33, p < 0.0001 for additive model). Haplotypes which carried one risk allele of rs2149632 or two risk alleles of both studied IDE SNPs also demonstrated a strong association with increased T2DM risk in this cohort (p = 0.001 and p < 0.0001, respectively). However, we found no significant T2DM association in the cross-sectional metabolic syndrome Berlin-Potsdam cohort (n = 1026). In nondiabetic subjects (NGT+IFG/IGT; n = 739), we found an association of rs2149632 with impaired glucose-derived insulin secretion and a trend to decreased insulin sensitivity for rs1887922. In the NGT subjects (n = 440), the association with decreased insulin secretion for rs2149632 remain significant, and the association with decreased hepatic insulin degradation for rs1887922 were observed additionally. This study validates and confirms the association of IDE polymorphisms with T2DM risk in the prospective German cohort and provides novel evidence of influences of IDE genetic variants on insulin metabolism.

  13. [Studies on lignocellulolytic enzymes production and biomass degradation of Pleurotus sp2 and Trametes gallica in wheat straw cultures].

    Science.gov (United States)

    Xie, J; Sun, X; Ren, L; Zhang, Y Z

    2001-09-01

    Pleurotus sp2 and Trametes gallica were selected in this assay because of their high activities of lignocellulolytic enzymes and the enzyme peaks appeared at the early stage of liquid state fermentation. Solid state fermentation was also investigated for their abilities and behaviors of enzyme-production. The capabilities and characteristics of the two strains in degrading biomass were studied. When Pleurotus sp2 was incubated in wheat straw powder containing the liquid medium of low-nitrogen, no-carbon and high inorganic salt, the activities of MnP and Lac reached the peaks on the tenth day, but the activities of hemicellulases reached the peak on the 40th day. Pleurotus sp2 caused 17.6% of biomass loss. When T. gallica was incubated in wheat straw powder containing the liquid medium of hlig-nitrogen, or low-nitrogen, no-carbon and high inorganic salt, the activities of MnP reached the peaks on the tenth day, the lac and hemicelluloses on the 40th day, and the lignin peroxidases reached the peaks on the 50th day, and it caused more than 64% of biomass loss. Among them the hemicellulose was degraded by 71.96%, and the cellulose 66.21%. T. gallica was very capable of degrading lignin of wheat straw and caused 34.37% loss during 20 days, 46. 71% loss during 30 days and 70.14% loss during 60 days. It was interesting that T. gallica degraded lignin preferentially with respect to cellulose, which was very beneficial to biopulping of paper industry.

  14. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

    Science.gov (United States)

    Vlasova, Irina I; Vakhrusheva, Tatyana V; Sokolov, Alexey V; Kostevich, Valeria A; Gusev, Alexandr A; Gusev, Sergey A; Melnikova, Viktoriya I; Lobach, Anatolii S

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H(2)O(2) system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes.

  15. Purification and characterization of a hexanol-degrading enzyme extracted from apple

    Science.gov (United States)

    An enzyme having activity towards n-hexanol was purified from apple and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 column. The obtained enzyme had a yi...

  16. Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans.

    NARCIS (Netherlands)

    Vis, van de J.W.

    1994-01-01

    Agricultural biomass consisting mainly of cellulose, hemicellulose and lignin, is a renewable source of fuels and chemicals. An interesting option is enzymic conversion of biomass to readily usable material. To improve the overall economics of enzymic conversion of biomass not only cellulose but als

  17. Sonochemical Degradation of Reactive Black 5 with a Composite Catalyst of TiO2/Single-Walled Carbon Nanotubes

    Science.gov (United States)

    Cho, Eunju; Choi, Jongbok; Lee, Yonghyeon; Park, Jeong Min; Khim, Jeehyeong

    2013-07-01

    In the sonocatalytic process, composites of TiO2-carbon were used because carbon provides more adsorption sites and acts like an electron sink to prevent the recombination of an electron/hole. Therefore, in the present study, the characteristics of a TiO2/single-walled carbon nanotubes catalyst (TiO2/SWCNTs) have been investigated, and the optimal weight ratio of SWCNTs and the dose for degradation of reactive black 5 (RB5) were also evaluated. TiO2/SWCNT composite was characterized using Brunauer-Emmett-Teller analysis, scanning electron microscopy, energy-dispersive X-ray diffraction microanalysis and spectra, and X-ray diffraction patterns. The degradation rate constants of RB5 with the ratio of SWCNTs were found to depend on the adsorption phenomenon of a surface catalyst, light absorbance, and the recombination of electrons and holes. As a result, the optimal ratio of carbon in the sono-TiO2/SWCNTs process for degradation of RB5 was TiO2:SWCNTs= 200:1. Additionally, the optimal dose of the catalyst was 0.5 g/L.

  18. Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp. : a comparative study of wild-type and genetically manipulated strains

    Energy Technology Data Exchange (ETDEWEB)

    Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

    1987-12-01

    The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward ..cap alpha..-naphthyl acetate and ..cap alpha..-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.

  19. wall

    Directory of Open Access Journals (Sweden)

    Irshad Kashif

    2016-01-01

    Full Text Available Maintaining indoor climatic conditions of buildings compatible with the occupant comfort by consuming minimum energy, especially in a tropical climate becomes a challenging problem for researchers. This paper aims to investigate this problem by evaluating the effect of different kind of Photovoltaic Trombe wall system (PV-TW on thermal comfort, energy consumption and CO2 emission. A detailed simulation model of a single room building integrated with PV-TW was modelled using TRNSYS software. Results show that 14-35% PMV index and 26-38% PPD index reduces as system shifted from SPV-TW to DGPV-TW as compared to normal buildings. Thermal comfort indexes (PMV and PPD lie in the recommended range of ASHARE for both DPV-TW and DGPV-TW except for the few months when RH%, solar radiation intensity and ambient temperature were high. Moreover PVTW system significantly reduces energy consumption and CO2 emission of the building and also 2-4.8 °C of temperature differences between indoor and outdoor climate of building was examined.

  20. COMPONENTS OF CELL WALL, ENZYME ACTIVITY IN PEDICEL AND SUSCEPTIBILITY OF BANANAS TO FINGER DROP

    OpenAIRE

    GLORIA ANNABELL COBEÑA RUIZ; LUIZ CARLOS CHAMHUM SALOMÃO; DALMO LOPES DE SIQUEIRA; SEBASTIÃO TAVARES DE REZENDE; LEILA CRISTINA ROSA DE LINS

    2016-01-01

    ABSTRACT A major problem in post-harvest handling of bananas is the individual detachment of the fruit from the hands. This study aimed to establishing the relationship between carbohydrate concentration and enzyme activity in the pedicel region of three cultivars of bananas, resistant and susceptible to natural dropping, during post-harvest ripening, and the susceptibility of bananas to finger dropping. Cultivars ‘Terra’ (plantain, AAB group) and ‘Prata’ (banana, AAB group) triploids and th...

  1. Enzymatic degradation of plant cell wall polysaccharides: the kinetic effect of competitive adsorption

    DEFF Research Database (Denmark)

    Bergsøe, Merete Norsker; Bloch, Line; Adler-Nissen, Jens

    1999-01-01

    Insoluble potato dietary fibre, isolated from potato pulp, can be enzymatically hydrolysed with the pectolytic enzyme preparation Pectinex Ultra SP from Novo Nordisk A/S, in order to produce soluble fibre. The soluble fibre has valuable functional properties for the food industry. Cloned monocomp...

  2. Enhancement of Biodegradable Plastic-degrading Enzyme Production from Paraphoma-like Fungus, Strain B47-9.

    Science.gov (United States)

    Sameshima-Yamashita, Yuka; Koitabashi, Motoo; Tsuchiya, Wataru; Suzuki, Ken; Watanabe, Takashi; Shinozaki, Yukiko; Yamamoto-Tamura, Kimiko; Yamazaki, Toshimasa; Kitamoto, Hiroko

    2016-01-01

    To improve the productivity of Paraphoma-like fungal strain B47-9 for biodegradable plastic (BP)-degrading enzyme (PCLE), the optimal concentration of emulsified poly(butylene succinate-co-adipate) (PBSA) in the medium was determined. Emulsified PBSA was consumed as a sole carbon source and an inducer of PCLE production by strain B47-9. Among the various concentrations of emulsified PBSA [0.09-0.9% (w/v)] used in flask cultivation, 0.27% yielded the maximum enzyme activity within a short cultivation period. To evaluate the residual concentration of emulsified PBSA in culture, emulsified PBSA in aliquots of culture supernatant was digested in vitro, and the concentration of released monomerised succinic acid was determined. Regardless of the initial concentration of emulsified PBSA in medium, PCLE activity was detected after residual succinic acid decreased below 0.04 mg/mL in culture broth. Jarfermentation was performed at a 0.27% PBSA concentration. Among the various airflow rates tested, 1 LPM resulted in a PCLE production rate of 1.0 U/mL/day. The enzyme activity in the resulting culture filtrate (4.2 U/2 mL) was shown to degrade commercial BP films (1 × 1 cm, 20 µm thickness) within 8 hours.

  3. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    Science.gov (United States)

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  4. Host cell capable of producing enzymes useful for degradation of lignocellulosic material

    Energy Technology Data Exchange (ETDEWEB)

    Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schoonneveld-Bergmans, Margot Elisabeth Francoise; Damveld, Robbertus Antonius

    2017-08-22

    The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

  5. Enzyme-catalyzed degradation of biodegradable polymers derived from trimethylene carbonate and glycolide by lipases from Candida antarctica and Hog pancreas.

    Science.gov (United States)

    Liu, Feng; Yang, Jian; Fan, Zhongyong; Li, Suming; Kasperczyk, Janusz; Dobrzynski, Piotr

    2012-01-01

    Enzyme-catalyzed degradation of poly(trimethylene carbonate) homo-polymer (PTMC) and poly(trimethylene carbonate-co-glycolide) co-polymer (PTGA) was investigated in the presence of lipases from Candida antarctica and Hog pancreas. Degradation was monitored by gravimetry, size-exclusion chromatography (SEC), nuclear magnetic resonance (NMR), tensiometry and environmental scanning electron microscopy (ESEM). PTMC can be rapidly degraded by Candida antarctica lipase with 98% mass loss after 9 days, while degradation by Hog pancreas lipase leads to 27% mass loss. Introduction of 16% glycolide units in PTMC chains strongly affects the enzymatic degradation. Hog pancreas lipase becomes more effective to PTGA co-polymer with a mass loss of 58% after 9 days, while Candida antarctica lipase seems not able to degrade PTGA. Bimodal molecular weight distributions are observed during enzymatic degradation of both PTMC and PTGA, which can be assigned to the fact that the surface is largely degraded while the internal part remains intact. The composition of the PTGA co-polymer remains constant, and ESEM shows that the polymers are homogeneously eroded during enzymatic degradation. Contact angle measurements confirm the enzymatic degradation mechanism, i.e., enzyme adsorption on the polymer surface followed by enzyme-catalyzed chain cleavage.

  6. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason...... kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both...

  7. Toxicity of textile dyes and their degradation by the enzyme horseradish peroxidase (HRP).

    Science.gov (United States)

    Ulson de Souza, Selene Maria Arruda Guelli; Forgiarini, Eliane; Ulson de Souza, Antônio Augusto

    2007-08-25

    The enzyme peroxidase is known for its capacity to remove phenolic compounds and aromatic amines from aqueous solutions and also to decolorize textile effluents. This study evaluates the potential of the enzyme horseradish peroxidase (HRP) in the decolorization of textile dyes and effluents. Some factors such as pH and the amount of H(2)O(2) and the enzyme were evaluated in order to determine the optimum conditions for the enzyme performance. For the dyes tested, the results indicated that the decolorization of the dye Remazol Turquoise Blue G 133% was approximately 59%, and 94% for the Lanaset Blue 2R; for the textile effluent, the decolorization was 52%. The tests for toxicity towards Daphnia magna showed that there was a reduction in toxicity after the enzymatic treatment. However, the toxicity of the textile effluent showed no change towards Artemia salina after the enzyme treatment. This study verifies the viability of the use of the enzyme horseradish peroxidase in the biodegradation of textile dyes.

  8. Examinations of a new long-term degradable electrospun polycaprolactone scaffold in three rat abdominal wall models.

    Science.gov (United States)

    Jangö, Hanna; Gräs, Søren; Christensen, Lise; Lose, Gunnar

    2017-02-01

    Alternative approaches to reinforce native tissue in reconstructive surgery for pelvic organ prolapse are warranted. Tissue engineering combines the use of a scaffold with the regenerative potential of stem cells and is a promising new concept in urogynecology. Our objective was to evaluate whether a newly developed long-term degradable polycaprolactone scaffold could provide biomechanical reinforcement and function as a scaffold for autologous muscle fiber fragments. We performed a study with three different rat abdominal wall models where the scaffold with or without muscle fiber fragments was placed (1) subcutaneously (minimal load), (2) in a partial defect (partial load), and (3) in a full-thickness defect (heavy load). After 8 weeks, no animals had developed hernia, and the scaffold provided biomechanical reinforcement, even in the models where it was subjected to heavy load. The scaffold was not yet degraded but showed increased thickness in all groups. Histologically, we found a massive foreign body response with numerous large giant cells intermingled with the fibers of the scaffold. Cells from added muscle fiber fragments could not be traced by PKH26 fluorescence or desmin staining. Taken together, the long-term degradable polycaprolactone scaffold provided biomechanical reinforcement by inducing a marked foreign-body response and attracting numerous inflammatory cells to form a strong neo-tissue construct. However, cells from the muscle fiber fragments did not survive in this milieu. Properties of the new neo-tissue construct must be evaluated at the time of full degradation of the scaffold before its possible clinical value in pelvic organ prolapse surgery can be evaluated.

  9. Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice.

    Science.gov (United States)

    Deprez-Poulain, Rebecca; Hennuyer, Nathalie; Bosc, Damien; Liang, Wenguang G; Enée, Emmanuelle; Marechal, Xavier; Charton, Julie; Totobenazara, Jane; Berte, Gonzague; Jahklal, Jouda; Verdelet, Tristan; Dumont, Julie; Dassonneville, Sandrine; Woitrain, Eloise; Gauriot, Marion; Paquet, Charlotte; Duplan, Isabelle; Hermant, Paul; Cantrelle, François-Xavier; Sevin, Emmanuel; Culot, Maxime; Landry, Valerie; Herledan, Adrien; Piveteau, Catherine; Lippens, Guy; Leroux, Florence; Tang, Wei-Jen; van Endert, Peter; Staels, Bart; Deprez, Benoit

    2015-09-23

    Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.

  10. Decomposition of insoluble and hard-to-degrade animal proteins by enzyme E77 and its potential applications.

    Science.gov (United States)

    Zhao, Hui; Mitsuiki, Shinji; Takasugi, Mikako; Sakai, Masashi; Goto, Masatoshi; Kanouchi, Hiroaki; Oka, Tatsuzo

    2012-04-01

    Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.

  11. Formation of insulin fragments by insulin-degrading enzyme: the role of zinc(II) and cystine bridges.

    Science.gov (United States)

    Bellia, Francesco; Pietropaolo, Adriana; Grasso, Giuseppe

    2013-02-01

    Insulin is the hormone mainly involved in widespread diseases such as diabetes mellitus. It is widely recognized that metal ions such as zinc(II) as well as insulin degradation and insulin fragments are inexplicably linked to the hormone action. Insulin-degrading enzyme (IDE) has been identified as the main factor of insulin degradation, but it is still unknown the exact way and location at which IDE action toward insulin occurs and how metal ions can modulate this interaction. Interestingly, some insulin fragments have different biological activity from the intact hormone, and it is not clear how they can be generated from insulin. In this work, the role of zinc(II) and cystine bridges in the degradation of insulin by IDE are investigated by high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the experimental conditions at which peculiar insulin fragments having biological activity are formed by the action of IDE are found and discussed. Docking simulations of IDE/insulin A and B chains are in good accordance with the insulin fragments detected by HPLC-MS.

  12. Antipathy of Trichoderma against Sclerotium rolfsii Sacc.: Evaluation of Cell Wall-Degrading Enzymatic Activities and Molecular Diversity Analysis of Antagonists.

    Science.gov (United States)

    Hirpara, Darshna G; Gajera, Harsukh P; Hirpara, Hitesh Z; Golakiya, Balubhai A

    2017-01-01

    The fungus Trichoderma is a teleomorph of the Hypocrea genus and associated with biological control of plant diseases. The microscopic, biochemical, and molecular characterization of Trichoderma was carried out and evaluated for in vitro antagonistic activity against the fungal pathogen Sclerotium rolfsii causing stem rot disease in groundnut. In total, 11 isolates of Trichoderma were examined for antagonism at 6 and 12 days after inoculation (DAI). Out of 11, T. virens NBAII Tvs12 evidenced the highest (87.91%) growth inhibition of the test pathogen followed by T. koningii MTCC 796 (67.03%), T. viride NBAII Tv23 (63.74%), and T. harzianum NBAII Th1 (60.44%). Strong mycoparasitism was observed in the best antagonist Tvs12 strain during 6-12 DAI. The specific activity of cell wall-degrading enzymes - chitinase and β-1,3-glucanase - was positively correlated with growth inhibition of the test pathogen. In total, 18 simple sequence repeat (SSR) polymorphisms were reported to amplify 202 alleles across 11 Trichoderma isolates. The average polymorphism information content for SSR markers was found to be 0.80. The best antagonist Tvs 12 was identified with 7 unique SSR alleles amplified by 5 SSR markers. Clustering patterns of 11 Trichoderma strains showed the best antagonist T. virens NBAII Tvs 12 outgrouped with a minimum 3% similarity from the rest of Trichoderma. © 2017 S. Karger AG, Basel.

  13. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Vlasova, Irina I., E-mail: irina.vlasova@yahoo.com [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Vakhrusheva, Tatyana V. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Sokolov, Alexey V.; Kostevich, Valeria A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Research Institute for Experimental Medicine, Russian Academy of Medical Science, Saint Petersburg (Russian Federation); Gusev, Alexandr A.; Gusev, Sergey A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Melnikova, Viktoriya I. [Institute of Developmental Biology, Russian Academy of Science, Moscow (Russian Federation); Lobach, Anatolii S. [Institute of Problems of Chemical Physics, Russian Academy of Science, Chernogolovka (Russian Federation)

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H{sub 2}O{sub 2} system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. -- Highlights: ► Myeloperoxidase (MPO) product hypochlorous acid is able to degrade CNTs. ► PEGylated SWCNTs stimulate isolated neutrophils to produce hypochlorous acid. ► SWCNTs are capable of activating neutrophils in blood samples. ► Activation of

  14. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

    2014-01-01

    . In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important...... feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases...

  15. Effect of subacute benzene exposure on the activity of two neuropeptide-degrading enzymes in the rat brain.

    Science.gov (United States)

    de Gandarias, J M; Casis, O; Irazusta, J; Echevarría, E; Casis, L

    1992-04-01

    Benzene (Bz) is an important industrial chemical, a petroleum by-product, a component of unleaded gas, and thus a ubiquitous environmental pollutant. It is well established that this organic solvent possesses neurotoxic and behavioral effects. However, the neurochemical mechanism of the solvent action remains obscure. The aminopeptidases (AP) are proteolytic enzymes that have been proposed as a candidate regulator of the degradation of several neuropeptides. In this work, changes in Lys- and Leu-aminopeptidase activities in several rat brain regions after benzene administration are described. The AP activity was determined by measuring the rate of hydrolysis of the artificial substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities decrease in the thalamus, hypothalamus, hippocampus, and amygdala after Bz treatment. It is suggested that these aminopeptidase activities play a part in the benzene action mechanism, possibly by regulating the activity of several neuroactive peptides.

  16. Improved production of raw starch degrading enzyme by Aspergillus oryzae F-30 using methyl glucoside sesqui-stearate.

    Science.gov (United States)

    Sun, Hai-Yan; Wang, Lu; Liu, Jian-Wen; Peng, Ming

    2009-10-01

    The effect of methyl glucoside sesqui-stearate (MGS) on the production of raw starch degrading enzyme (RSDE) by Aspergillus oryzae F-30 was studied in this paper. The activity of RSDE formed by Aspergillus oryzae F-30 was enhanced dramatically by the addition of MGS to the medium. As a result, with the addition of 1.5 g MGS in 1 L basal medium, RSDE activity and productivity were 107 U mL(-1) and 1.49 U mL(-1) h(-1), 4.3-fold and 7.1-fold greater than the values obtained in the basal medium, respectively. The effect of MGS on the synthesis of RSDE by Aspergillus oryzae F-30 was also studied on a molecular level. It was observed that the regulation of RSDE synthesis in Aspergillus oryzae F-30 occurs at both transcriptional and translational level and the enzyme synthesis was provoked by the addition of MGS at transcriptional level.

  17. Biochemical characterization and bioinformatic analysis of two large multi-domain enzymes from Microbacterium aurum B8.A involved in native starch degradation

    NARCIS (Netherlands)

    Valk, Vincent

    2017-01-01

    Microbacterium aurum B8.A is a unique bacterium with the ability to degrade starch granules through pore formation. In this study two enzymes (MaAmyA and MaAmyB) which are involved in granular starch degradation and were specific for the M. aurum B8.A strain, have been characterized in detail. Both

  18. Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

    Directory of Open Access Journals (Sweden)

    Bruna Silveira Lamanes dos Santos

    2015-09-01

    Full Text Available Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3, A. sydowii (SBCM7 and A. fumigatus (SBC4. The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g. For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g and 4.0 (573 U/g, respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

  19. An Application of Microcapsules Having Enzyme-degradable Gel Membrane to Cell Culture

    Science.gov (United States)

    Dobashi, Toshiaki; Koike, Michiru; Kobayashi, Kentaro; Maki, Yasuyuki; Yamamoto, Takao; Tanaka, Susumu

    Newly developed microcapsules having gelatin wall membrane was applied as a scaffold for suspension cell culture. The optimum preparation condition was determined, and the stability of the cultured human fibroblast cells using the microcapsules was examined at both protein and gene levels.

  20. Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification.

    Science.gov (United States)

    Robertson, James M; Dineen, Shauna M; Scott, Kristina A; Lucyshyn, Jonathan; Saeed, Maria; Murphy, Devonie L; Schweighardt, Andrew J; Meiklejohn, Kelly A

    2014-09-01

    Forensic scientists have used several approaches to obtain short tandem repeat (STR) profiles from compromised DNA samples, including supplementing the polymerase chain reaction (PCR) with enhancers and using procedures yielding reduced-length amplicons. For degraded DNA, the peak intensities of the alleles separated by electrophoresis generally decrease as the length of the allele increases. When the intensities of the alleles decrease below an established threshold, they are described as drop-outs, thus contributing to a partial STR profile. This work assesses the use of repair enzymes to improve the STR profiles from artificially degraded DNA. The commercial PreCR™ repair kit of DNA repair enzymes was tested on both purified DNA and native DNA in body fluids exposed to oxidizing agents, hydrolytic conditions, ultraviolet (UV) and ionizing radiation, and desiccation. The strategy was to restrict the level of DNA damage to that which yields partial STR profiles in order to test for allele restoration as opposed to simple allele enhancement. Two protocols were investigated for allele restoration: a sequential protocol using the manufacturer's repair procedure and a modified protocol reportedly designed for optimal STR analysis of forensic samples. Allele restoration was obtained with both protocols, but the peak height appeared to be higher for the modified protocol (determined by Mann-Kendall Trend Test). The success of the approach using the PreCR™ repair enzymes was sporadic; it led to allele restoration as well as allele drop-out. Additionally, allele restoration with the PreCR™ enzymes was compared with restoration by alternative, but commonly implemented approaches using Restorase™, PCRBoost™, bovine serum albumin (BSA) and the Minifiler™ STR system. The alternative methods were also successful in improving the STR profile, but their success also depended on the quality of the template encountered. Our results indicate the PreCR™ repair kit may

  1. Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans.

    Science.gov (United States)

    Bezsudnova, Ekaterina Yu; Sorokin, Dimitry Yu; Tikhonova, Tamara V; Popov, Vladimir O

    2007-12-01

    Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the , high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed.

  2. Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase, proteolytic enzymes and enzymes degrading non-starch polysaccharides.

    Science.gov (United States)

    Kłosowski, Grzegorz; Mikulski, Dawid; Czupryński, Bogusław; Kotarska, Katarzyna

    2010-05-01

    The aim of the research was to assess the possibility of the fermentation productivity rising through the increase in corn mashes extract from 16-17 to 20-21 degrees Balling, yet keeping a 3-day fermentation period. The second goal was to obtain the highest possible utilization of starch in the raw material through deep enzymatic degradation and utilization of available sugars and simultaneous maintenance of high quality spirit. It was found that fulfilling the above during the 3-day fermentation period was possible with the application of pullulanase as an additional amylolytic enzyme. Adding pullulanase resulted in the acceleration of the starch hydrolysis degree, which led to lower amounts of unhydrolyzed dextrins and higher ethanol yield. When the supportive enzymes complex (pullulanase, protease and cellulase) was used, the final ethanol concentration reached 10.86+/-0.04% v/v, with ethanol yield at 68.41+/-0.23 dm(3) of absolute ethanol (A(100)) per 100 kg of starch, which was 95.25+/-0.32% at the theoretical value. The acceleration of starch enzymatic degradation and the application of a proteolytic preparation visibly shortened both initial and main fermentation phases. This in turn increased the time of the final fermentation phase and resulted in more extensive utilization of substrates by yeasts with simultaneous reduction of the final concentration of acetaldehyde (26.0+/-0.5 mg/dm(3)A(100)) and diethyl acetal of acetaldehyde (2.5+/-0.5 mg/dm(3)A(100)). The quality of spirit obtained was positively verified also in terms of relatively low concentration of higher alcohol (3912.2+/-9.8 mg/dm(3)A(100)). Preliminary analysis of costs (without raw-material) of 1 l distillate production indicated the possibility to reduce the costs by 18-20%.

  3. A novel analytical method for D-glucosamine quantification and its application in the analysis of chitosan degradation by a minimal enzyme cocktail

    DEFF Research Database (Denmark)

    Mekasha, Sophanit; Toupalová, Hana; Linggadjaja, Eka

    2016-01-01

    action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β...

  4. Indigenous West African plants as novel sources of polysaccharide degrading enzymes: application in the reduction of the viscosity of cereal porridges

    NARCIS (Netherlands)

    Dicko, M.H.; Hilhorst, M.H.; Traore, A.S.

    2005-01-01

    Ethnobotanical and biochemical surveys revealed that some local plants from West Africa are novel sources of polysaccharide degrading enzymes such as amylases and glucanases. The study shows that these enzymes could be used for various biotechnological applications. In a crude extract of Curculigo p

  5. Indigenous West African plants as novel sources of polysaccharide degrading enzymes: application in the reduction of the viscosity of cereal porridges

    NARCIS (Netherlands)

    Dicko, M.H.; Hilhorst, M.H.; Traore, A.S.

    2005-01-01

    Ethnobotanical and biochemical surveys revealed that some local plants from West Africa are novel sources of polysaccharide degrading enzymes such as amylases and glucanases. The study shows that these enzymes could be used for various biotechnological applications. In a crude extract of Curculigo

  6. Pectin modifications and the role of pectin-degrading enzymes during postharvest softening of Jonagold apples.

    Science.gov (United States)

    Gwanpua, Sunny George; Van Buggenhout, Sandy; Verlinden, Bert E; Christiaens, Stefanie; Shpigelman, Avi; Vicent, Victor; Kermani, Zahra Jamsazzadeh; Nicolai, Bart M; Hendrickx, Marc; Geeraerd, Annemie

    2014-09-01

    This study aimed at understanding softening in Jonagold apple (Malus×domestica Borkh.) fruits, by investigating pectin modifications and the evolution of pectin-modifying enzymes during postharvest storage and ripening. Jonagold apples were harvested at commercial maturity and stored at different temperatures and controlled atmosphere conditions for 6 months, followed by exposure to ambient shelf life conditions (20 °C under air) for 2 weeks. The composition of the pectic material was analysed. Furthermore, the firmness and the ethylene production of the apples were assessed. Generally, the main changes in pectin composition associated with the loss of firmness during ripening in Jonagold apples were a loss of side chains neutral sugars, increased water solubility and decreased molar mass. Also, the activities of four important enzymes possibly involved in apple softening, β-galactosidase, α-arabinofuranosidase, polygalacturonase and pectin methylesterase, were measured. Pectin-related enzyme activities highly correlated with ethylene production, but not always with pectin modifications.

  7. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    Science.gov (United States)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2010-12-28

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  8. Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D.

    2016-08-02

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  9. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-07-30

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  10. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-04-23

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  11. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2013-10-15

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  12. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2015-06-02

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  13. The Impact of Enzyme Characteristics on Corn Stover Fiber Degradation and Acid Production During Ensiled Storage

    Science.gov (United States)

    Ren, Haiyu; Richard, Tom L.; Moore, Kenneth J.

    Ensilage can be used to store lignocellulosic biomass before industrial bioprocessing. This study investigated the impacts of seven commerical enzyme mixtures derived from Aspergillus niger, Trichoderma reesei, and T. longibrachiatum. Treatments included three size grades of corn stover, two enzyme levels (1.67 and 5 IU/g dry matter based on hemicellulase), and various ratios of cellulase to hemicellulase (C ∶ H). The highest C ∶ H ratio tested, 2.38, derived from T. reesei, resulted in the most effective fermentation, with lactic acid as the dominant product. Enzymatic activity during storage may complement industrial pretreatment; creating synergies that could reduce total bioconversion costs.

  14. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants.

    Science.gov (United States)

    Aylward, Frank O; Khadempour, Lily; Tremmel, Daniel M; McDonald, Bradon R; Nicora, Carrie D; Wu, Si; Moore, Ronald J; Orton, Daniel J; Monroe, Matthew E; Piehowski, Paul D; Purvine, Samuel O; Smith, Richard D; Lipton, Mary S; Burnum-Johnson, Kristin E; Currie, Cameron R

    2015-01-01

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

  15. Production of a cell wall-associated endopolygalacturonase by Colletotrichum lindemuthianum and pectin degradation during bean infection.

    Science.gov (United States)

    Herbert, Corentin; O'Connell, Richard; Gaulin, Elodie; Salesses, Vincent; Esquerré-Tugayé, Marie Thérèse; Dumas, Bernard

    2004-02-01

    The bean pathogen Colletotrichum lindemuthianum expresses two endopolygalacturonase genes, CLPG1 and CLPG2, during interaction with its host plant. However, only CLPG1 was found to be secreted to the extracellular medium during saprophytic growth of the fungus on pectin. To localize CLPG2, a FLAG epitope sequence was inserted in the C-terminal sequence of CLPG2 and the modified gene was introduced into C. lindemuthianum. Western blot analysis using a FLAG monoclonal antibody allowed the detection of CLPG2 in intracellular protein extracts and in the cell wall fraction, but not in the culture medium. Indirect immunofluorescence microscopy was performed to detect CLPG2 during saprophytic or parasitic growth. According to the expression pattern of CLPG2, it was found that CLPG2 accumulates in the fungal cell wall during growth on pectin medium and during appressorium formation, both in vitro and during interaction with the plant. Pectin degradation was not detected around the infection peg using the monoclonal antibody JIM7, specific for methyl-esterified galacturonan. However, extensive pectin dissolution was observed during the development of secondary hyphae.

  16. Diversity in production of xyaln-degrading enzymes among species belonging to the Trichoderma section Longibrachiatum

    Science.gov (United States)

    Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzy...

  17. Diversity in Production of Xylan-Degrading Enzymes Among Species Belonging to the Trichoderma Section Longibrachiatum

    NARCIS (Netherlands)

    Toth, K.; Gool, van M.P.; Schols, H.A.; Samuels, G.J.; Gruppen, H.; Szakacs, G.

    2013-01-01

    Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase

  18. Computational Enzymology and Organophosphorus Degrading Enzymes: Promising Approaches Toward Remediation Technologies of Warfare Agents and Pesticides.

    Science.gov (United States)

    Ramalho, Teodorico C; de Castro, Alexandre A; Silva, Daniela R; Silva, Maria Cristina; Franca, Tanos C C; Bennion, Brian J; Kuca, Kamil

    2016-01-01

    The re-emergence of chemical weapons as a global threat in hands of terrorist groups, together with an increasing number of pesticides intoxications and environmental contaminations worldwide, has called the attention of the scientific community for the need of improvement in the technologies for detoxification of organophosphorus (OP) compounds. A compelling strategy is the use of bioremediation by enzymes that are able to hydrolyze these molecules to harmless chemical species. Several enzymes have been studied and engineered for this purpose. However, their mechanisms of action are not well understood. Theoretical investigations may help elucidate important aspects of these mechanisms and help in the development of more efficient bio-remediators. In this review, we point out the major contributions of computational methodologies applied to enzyme based detoxification of OPs. Furthermore, we highlight the use of PTE, PON, DFP, and BuChE as enzymes used in OP detoxification process and how computational tools such as molecular docking, molecular dynamics simulations and combined quantum mechanical/molecular mechanics have and will continue to contribute to this very important area of research.

  19. Characterisation of three starch degrading enzymes: thermostable β-amylase, maltotetraogenic and maltogenic α-amylases.

    Science.gov (United States)

    Derde, L J; Gomand, S V; Courtin, C M; Delcour, J A

    2012-11-15

    Maltogenic α-amylase from Bacillus stearothermophilus (BStA) is widely used as bread crumb anti-firming enzyme. A maltotetraose-forming α-amylase from Pseudomonas saccharophila (PSA) was recently proposed as alternative, hence the need to compare both exo-acting enzymes with some endo-action component. A purely exo-acting thermostable β-amylase from Clostridium thermosulfurogenes (CTB) was included for reference purposes. Under the experimental conditions used, temperature optima of the enzymes are rather similar (60-65 °C), but temperature stability decreased in the order BStA, PSA and CTB. The action of the enzymes on different substrates and their impact on the rheological behaviour of maize starch suspensions demonstrated that, while CTB acts exclusively through an exo-action mechanism, BStA displayed limited endo-action which became more pronounced at higher temperatures. PSA has more substantial endo-action than BStA, which is rather temperature independent. This is important for their impact in processes such as breadmaking, where temperature is gradually increased.

  20. Screening for cellulose and hemicellulose degrading enzymes from the fungal genus Ulocladium

    DEFF Research Database (Denmark)

    Pedersen, Mads; Hollensted, Morten; Lange, L.

    2009-01-01

    The fungal genus Ulocladium consists mostly of saprotrophic species and can readily be isolated from dead vegetation, rotten wood. paper, textiles and other cellulose containing materials. Thus, they must produce cellulolytic and hemicellulolytic enzymes. In this study fifty Ulocladium strains from...

  1. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...

  2. Improved mortality of the Formosan subterranean termite by fungi, when amended with cuticle-degrading enzymes or eicosanoid biosynthesis inhibitors.

    Science.gov (United States)

    Wright, Maureen S; Lax, Alan R

    2016-01-01

    Formosan subterranean termites (FST) were exposed to strains of Beauveria pseudobassiana (Bpb) and Isaria fumosorosea (Ifr) to determine virulence of the fungi. Once lethality was determined, sublethal doses of Bpb were combined with enzymes capable of degrading the insect cuticle to measure the potential to enhance fungal infection. Bpb applied to FST in combination with proteinases and a chitinase caused increased mortality over the fungus alone. Mortality was enhanced when Ifr was applied to FST in combination with a chitinase isolated from Serratia marcesans. A lipase isolated from Pseudomonas cepacia, when combined with Ifr, also resulted in greater mortality than all control treatments. FST were also exposed to the eicosanoid biosynthesis inhibitors (EBIs) dexamethasone (DEX), ibuprofen (IBU), and ibuprofen sodium salt (IBUNA), in combination with Ifr. Combining Ifr with IBUNA caused significantly increased mortality on days 6, 7, and 9. Cuticle-degrading enzymes and EBIs may have potential to enhance the pathogenic effect of a fungal control agent against the Formosan subterranean termite.

  3. A novel enzymatic approach in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine degradative enzymes.

    Science.gov (United States)

    Jankowska, Dagmara A; Trautwein-Schult, Anke; Cordes, Arno; Bode, Rüdiger; Baronian, Keith; Kunze, Gotthard

    2015-01-01

    The purine degradation pathway in humans ends with uric acid, which has low water solubility. When the production of uric acid is increased either by elevated purine intake or by impaired kidney function, uric acid will accumulate in the blood (hyperuricemia). This increases the risk of gout, a disease described in humans for at least 1000 years. Many lower organisms, such as the yeast Arxula adeninivorans, possess the enzyme, urate oxidase that converts uric acid to 5-hydroxyisourate, thus preventing uric acid accumulation. We have examined the complete purine degradation pathway in A. adeninivorans and analyzed enzymes involved. Recombinant adenine deaminase, guanine deaminase, urate oxidase and endogenous xanthine oxidoreductase have been investigated as potential additives to degrade purines in the food. Here, we review the current model of the purine degradation pathway of A. adeninivorans and present an overview of proposed enzyme system with perspectives for its further development.

  4. Application of fluorescent antibody and enzyme-linked immunosorbent assays for TCE and PAH degrading bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Brigmon, R.L.; Franck, M.; Brey, J.; Scott, D.; Lanclos, K.; Fliermans, C.

    1996-07-01

    Historically, methods used to identify methanotrophic and polyaromatic hydrocarbon-degrading (PAH) bacteria in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate specific bacteria from other environmental microorganisms. Methanotrophic bacteria have been isolated and characterized from TCE-contaminated soils (Bowman et al. 1993; Fliermans et al., 1988). Fliermans et al., (1988) and others demonstrated that cultures enriched with methane and propane could cometabolically degrade a wide variety of chlorinated aliphatic hydrocarbons including ethylene; 1,2-cisdichloroethylene (c-DCE); 1,2-trans-dichloroethylene (t-DCE); vinyl chloride (VC); toluene; phenol and cresol. Characterization of select microorganisms in the natural setting is important for the evaluation of bioremediation potential and its effectiveness. This realization has necessitated techniques that are selective, sensitive and easily applicable to soils, sediments, and groundwater (Fliermans, et al., 1994). Additionally these techniques can identify and quantify microbial types in situ in real time

  5. Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

    Science.gov (United States)

    de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L.; Jáuregui, Ruy; Vilchez-Vargas, Ramiro

    2015-01-01

    Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. PMID:26475106

  6. Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Min; Guo, Qing; Guo, Liang; Lenz, Martin; Qian, Feng; Koenen, Rory R.; Xu, Hua; Schilling, Alexander B.; Weber, Christian; Ye, Richard D.; Dinner, Aaron R.; Tang, Wei-Jen (IIT); (Aachen); (UC); (UIC)

    2010-12-07

    Macrophage inflammatory protein-1 (MIP-1), MIP-1{alpha} (CCL3) and MIP-1{beta} (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1{alpha} and MIP-1{beta} form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1{alpha} and MIP-1{beta} form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1{alpha}, thus depolymerization mutations enhance MIP-1{alpha} to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1{alpha} ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.

  7. Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: organisms, strategies and key enzymes.

    Science.gov (United States)

    Boll, Matthias; Löffler, Claudia; Morris, Brandon E L; Kung, Johannes W

    2014-03-01

    Next to carbohydrates, aromatic compounds are the second most abundant class of natural organic molecules in living organic matter but also make up a significant proportion of fossil carbon sources. Only microorganisms are capable of fully mineralizing aromatic compounds. While aerobic microbes use well-studied oxygenases for the activation and cleavage of aromatic rings, anaerobic bacteria follow completely different strategies to initiate catabolism. The key enzymes related to aromatic compound degradation in anaerobic bacteria are comprised of metal- and/or flavin-containing cofactors, of which many use unprecedented radical mechanisms for C-H bond cleavage or dearomatization. Over the past decade, the increasing number of completed genomes has helped to reveal a large variety of anaerobic degradation pathways in Proteobacteria, Gram-positive microbes and in one archaeon. This review aims to update our understanding of the occurrence of aromatic degradation capabilities in anaerobic microorganisms and serves to highlight characteristic enzymatic reactions involved in (i) the anoxic oxidation of alkyl side chains attached to aromatic rings, (ii) the carboxylation of aromatic rings and (iii) the reductive dearomatization of central arylcarboxyl-coenzyme A intermediates. Depending on the redox potential of the electron acceptors used and the metabolic efficiency of the cell, different strategies may be employed for identical overall reactions.

  8. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes

    KAUST Repository

    Zhang, Meiling

    2014-08-18

    Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT-04215 and BACOVA-04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xy-lose- configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM fromits homolog in the Prevotella bryantii B 14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. Aminimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

  9. Analysis of Calcium Content,Hormones,and Degrading Enzymes in Tomato Pedicel Explants During Calcium-Inhibited Abscission

    Institute of Scientific and Technical Information of China (English)

    XU Tao; LI Tian-lai; QI Ming-fang

    2009-01-01

    This study was designed to analyze the changes of phytohormone concentrations,calcium fraction,and the activities of degrading enzymes during calcium-inhibited and ethyleneglycol-bis-(β-aminoethyl ether)N,N'-tetraacetic acid (EGTA)induced abscission of tomato pedicel explants.Added calcium caused an increase in the total calcium content within the abscission zone and produced a corresponding reduction (20%) in pedicel explant abscission.As expected,EGTA treatment produced the opposite effect and resulted in a decrease in the total calcium content,while accelerating abscission of pedicel explants.Hormone analysis revealed that indole-3-acetic acid (IAA) concentrations in the abscission zone first decreased and then increased before the occurrence of abscission in all treatments,with the greatest effect produced by addition of EGTA.Similarly,abscisic acid (ABA),and gibberellin (GA1+3) concentrations,and ethylene production were elevated in the abscission zone during the first 16 h before abscission when explants imbibed EGTA.With calcium treatment,the concentrations of ABA,ethylene,and GA1+3 also increased in pedicels throughout the first 16 h following exposure,but the increase was slower and less dramatic than with EGTA.Both cellulase and polygalacturonase were induced in the explants during abscission and the activities were also strengthened by treatment with EGTA.Calciumtreated explants produced lower hydrolysing enzyme activities than controls throughout abscission.Calcium played a role of mediating hormone balance and degrading enzymes activities and affected on abscission.

  10. Pathogenesis of mucosal injury in the blind loop syndrome. Brush border enzyme activity and glycoprotein degradation.

    Science.gov (United States)

    Jonas, A; Flanagan, P R; Forstner, G G

    1977-12-01

    The effect of intestinal bacterial over-growth on brush border hydrolases and brush border glycoproteins was studied in nonoperated control rats, control rats with surgically introduced jejunal self-emptying blind loops, and rats with surgically introduced jejunal self-filling blind loops. Data were analyzed from blind loop segments, segments above and below the blind loops, and three corresponding segments in the nonoperated controls. Rats with self-filling blind loops had significantly greater fat excretion than controls and exhibited significantly lower conjugated:free bile salt ratios in all three segments. Maltase, sucrase, and lactase activities were significantly reduced in homogenates and isolated brush borders from the self-filling blind loop, but alkaline phosphatase was not affected. The relative degradation rate of homogenate and brush border glycoproteins was assessed by a double-isotope technique involving the injection of d-[6-(3)H]glucosamine 3 h and d-[U-(14)C]glucosamine 19 h before sacrifice, and recorded as a (3)H:(14)C ratio. The relative degradation rate in both homogenate and brush border fractions was significantly greater in most segments from rats with self-filling blind loops. In the upper and blind loop segments from rats with self-filling blind loops, the (3)H:(14)C ratios were higher in the brush border membrane than in the corresponding homogenates, indicating that the increased rates of degradation primarily involve membrane glycoproteins. Incorporation of d-[6-(3)H]glucosamine by brush border glycoproteins was not reduced in rats with self-filling blind loops, suggesting that glycoprotein synthesis was not affected. Polyacrylamide gel electrophoresis of brush border glycoproteins from the contaminated segments indicated that the large molecular weight glycoproteins, which include many of the surface hydrolases, were degraded most rapidly. Brush border maltase, isolated by immunoprecipitation, had (3)H:(14)C ratios characteristic of

  11. Growth of bacteria and yeast on enzymically degraded alkali treated rice and wheat straws

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, J.K.; Shirkot, C.K.; Dhawan, S.

    1981-01-01

    An enzyme filtrate of Trichoderma viride QM 9414 was used to saccharify rice and wheat straw. Delignification of the straw by alkali treatment increased the enzymic saccharification of both materials to approximately 70%. The optimum conditions for delignification were autoclaving at 120 degrees for 30 minutes with 2% Sodium Hydroxide. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Lactobacillus acidophilus, Bacillus megaterium, and Saccharomyces cerevisiae grew very well on enriched hydrolyzates of rice and wheat straws. Even nonenriched straw hydrolyzates supported better growth of L. acidophilus, B. megaterium, and E. coli on rice straw than the enriched synthetic medium containing equivalent glucose. S. cerevisiae grown in shake flasks containing 25 mL of enriched rice and wheat straw hydrolyzates yielded 0.595 g and 0.450 g of dry cells, respectively. The corresponding yield was 0.396 g from enriched synthetic medium containing equal amounts of glucose.

  12. Screening Psychrophilic Fungi of Cellulose Degradation and Characteristic of Enzyme Production

    Institute of Scientific and Technical Information of China (English)

    Wang Da-qing; Jin Wen-ran; Sun Tai-peng; Meng Yu-tian; Zhao Wei; Wang Hong-yan

    2016-01-01

    A fungus (WR-C1) decomposed cellulose was isolated from a hypothermal litter layer using Congo red medium as the preliminary screening culture medium and then using a filter as the secondary screening medium at low temperature. The experiment showed that the weight loss rate of filter paper on the 15th days could reach 30.69%. A morphologic and ITS gene sequence analysis suggested that CF-C1 wasCladosporium. We mainly studied the effects of culture time, inoculation amount, initial pH and different sources of carbon, nitrogen and inorganic salt on the cellulase production of strain WR-C1. Under optimum cultural condition, the highest value of WR-C1 enzyme production and filter paper enzyme were 3.27 U• mL-1 and 0.51 U• mL-1.

  13. Biochemical Control of Fungal Biomass and Enzyme Production During Native Hawaiian Litter Degradation

    Science.gov (United States)

    Amatangelo, K. L.; Cordova, T. P.; Vitousek, P. M.

    2007-12-01

    Microbial growth and enzyme production during decomposition is controlled by the availability of carbon substrates, essential elements, and the ratios of these (such as lignin:N). We manipulated carbon:nutrient stoichiometry during decomposition using a natural fertility gradient in Hawaii and litter of varying initial biochemistry. We collected freshly senesced litter of seven biochemically distinct species from three sites offering differing levels of N, P, cations, and 15N , but similar yearly rainfall and temperature patterns. Litter types were decomposed at both the sites they were collected, and at the other site(s) that species was found. Litter was collected at multiple time points, and after one year of decomposition, calculated K constants varied an order of magnitude, from 0.276 to 2.76. Decomposition rates varied significantly with both litter site of origin and deployment, except at the oldest, P-limited site, where litter site of origin was not significantly correlated with decomposition within species. As microbial exocellular enzymes provide the catalyst for the breakdown of organic molecules including phenols, cellulose, and cutin, we assayed polyphenol oxidase, cellobiohydrolase, cutinase, chitinase, and lignin peroxidase to evaluate the breakdown sequence of different litter types. To measure the fungal biomass accumulating during decomposition, we extracted (22E)-Ergosta-5,7,22-trien-3beta- ol (ergosterol) on a subset of samples. The production of particular exocellular enzymes on litter species responded distinctly to origin and decomposition sites: after six months, chitinase and cellobiohydrolase were significantly affected by origin site, whereas polyphenol oxidase activity was controlled by deployment site. We conclude that site characteristics can alter the interaction between litter carbon:nutrient ratios and decomposition rate, mediated through microbial biomass and enzyme production.

  14. The phylogeny and evolution of deoxyribonuclease II: An enzyme essential for lysosomal DNA degradation

    OpenAIRE

    Shpak, Max; Kugelman, Jeffrey R.; Varela-Ramirez, Armando; Aguilera, Renato J.

    2007-01-01

    Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the g...

  15. Isolation and characterization of feather degrading enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site.

    Science.gov (United States)

    Agrahari, S; Wadhwa, N

    2012-01-01

    The SN1 strain of Bacillus megaterium, isolated from soil of Ghazipur poultry waste site (India) produced extracellular caseinolytic and keratinolytic enzymes in basal media at 30 degrees C, 160 rpm in the presence of 10% feather. Feathers were completely degraded after 72 h of incubation. The caesinolytic enzyme was separated from the basal media following ammonium sulphate precipitation and ion exchange chromatography. We report 29.3-fold purification of protease after Q Sepharose chromatography. The molecular weight of this enzyme was estimated to be 30 kDa as shown by SDS-PAGE and zymography studies. Protease activity increased by 2-fold in presence of 10 mM Mn2+ whereas Ba2+ and Hg2+ inhibited it. Ratio of milk clotting activity to caseinolytic was found to be 520.8 activity for the 30-60% ammonium sulphate fraction in presence of Mn2+ ion suggesting potential application in dairy industry. Keratinase was purified to 655.64 fold with specific activity of 544.7 U/mg protein and 12.4% recovery. We adopted the strategy of isolating the keratinolytic and caesinolytic producing microorganism by its selective growing in enriched media and found that feather protein can be metabolized for production of animal feed protein concentrates.

  16. Identification of novel biomass-degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation.

    Science.gov (United States)

    Piao, Hailan; Froula, Jeff; Du, Changbin; Kim, Tae-Wan; Hawley, Erik R; Bauer, Stefan; Wang, Zhong; Ivanova, Nathalia; Clark, Douglas S; Klenk, Hans-Peter; Hess, Matthias

    2014-08-01

    Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this "genomic dark matter." Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as "genomic dark matter" and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications.

  17. Enhanced cellulose degradation by nano-complexed enzymes: Synergism between a scaffold-linked exoglucanase and a free endoglucanase.

    Science.gov (United States)

    Moraïs, Sarah; Heyman, Arnon; Barak, Yoav; Caspi, Jonathan; Wilson, David B; Lamed, Raphael; Shoseyov, Oded; Bayer, Edward A

    2010-06-01

    Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populustremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosomal cohesin) modules. In the present work, we have displayed a potent exoglucanase (Cel6B) from the aerobic cellulolytic bacterium, Thermobifida fusca, on a cohesin-bearing SP1 scaffold. For this purpose, a chimaeric form of the enzyme, fused to a cellulosomal dockerin module, was prepared. Full incorporation of 12 dockerin-bearing exoglucanase molecules onto the cohesin-bearing scaffold was achieved. Cellulase activity was tested on two cellulosic substrates with different levels of crystallinity, and the activity of the scaffold-linked exoglucanase was significantly reduced, compared to the free dockerin-containing enzyme. However, addition of relatively low concentrations of a free wild-type endoglucanase (T. fusca Cel5A) that bears a cellulose-binding module, in combination with the complexed exoglucanase resulted in a marked rise in activity on both cellulosic substrates. The endoglucanase cleaves internal sites of the cellulose chains, and the new chain ends of the substrate were now readily accessible to the scaffold-borne exoglucanase, thereby resulting in highly effective, synergistic degradation of cellulosic substrates.

  18. Plasmid control of 6-aminohexanoic acid cyclic dimer degradation enzymes of Flavobacterium sp. KI72.

    Science.gov (United States)

    Negoro, S; Shinagawa, H; Nakata, A; Kinoshita, S; Hatozaki, T; Okada, H

    1980-07-01

    Flavobacterium sp. K172, which is able to grow on 6-aminohexanoic acid cyclic dimer as the sole source of carbon and nitrogen, and plasmid control of the responsible enzymes, 6-aminohexanoic acid cyclic dimer hydrolase and 6-aminohexanoic acid linear oligomer hydrolase, were studied. The wild strain of K172 harbors three kinds of plasmid, pOAD1 (26.2 megadaltons), pOAD2 (28.8 megadaltons), and pOAD3 (37.2 megadaltons). The wild strain K172 was readily cured of its ability to grow on the cyclic dimer by mitomycin C, and the cyclic dimer hydrolase could not be detected either as catalytic activity or by antibody precipitation. No reversion of the cured strains was detected. pOAD2 was not detected in every cured strain tested but was restored in a transformant. The transformant recovered both of the enzyme activities, and the cyclic dimer hydrolase of the transformant was immunologically identical with that of the wild strain. All of the strains tested, including the wild, cured, and transformant ones, possessed identical pOAD3 irrespective of the metabolizing activity. Some of the cured strains possessed pOAD1 identical with the wild strain, but the others harbored plasmids with partially altered structures which were likely to be derived from pOAD1 by genetic rearrangements such as deletion, insertion, or substitution. These results suggested that the genes of the enzymes were borne on pOAD2.

  19. Genome mining of fungal lipid-degrading enzymes for industrial applications.

    Science.gov (United States)

    Vorapreeda, Tayvich; Thammarongtham, Chinae; Cheevadhanarak, Supapon; Laoteng, Kobkul

    2015-08-01

    Lipases are interesting enzymes, which contribute important roles in maintaining lipid homeostasis and cellular metabolisms. Using available genome data, seven lipase families of oleaginous and non-oleaginous yeast and fungi were categorized based on the similarity of their amino acid sequences and conserved structural domains. Of them, triacylglycerol lipase (patatin-domain-containing protein) and steryl ester hydrolase (abhydro_lipase-domain-containing protein) families were ubiquitous enzymes found in all species studied. The two essential lipases rendered signature characteristics of integral membrane proteins that might be targeted to lipid monolayer particles. At least one of the extracellular lipase families existed in each species of yeast and fungi. We found that the diversity of lipase families and the number of genes in individual families of oleaginous strains were greater than those identified in non-oleaginous species, which might play a role in nutrient acquisition from surrounding hydrophobic substrates and attribute to their obese phenotype. The gene/enzyme catalogue and relevant informative data of the lipases provided by this study are not only valuable toolboxes for investigation of the biological role of these lipases, but also convey potential in various industrial applications.

  20. Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Vassiliadis, Efstathios; Larsen, Lise

    2011-01-01

    Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine....

  1. Developments in Analytical Chemistry: Acoustically Levitated Drop Reactors for Enzyme Reaction Kinetics and Single-Walled Carbon Nanotube-Based Sensors for Detection of Toxic Organic Phosphonates

    Science.gov (United States)

    Field, Christopher Ryan

    2009-01-01

    Developments in analytical chemistry were made using acoustically levitated small volumes of liquid to study enzyme reaction kinetics and by detecting volatile organic compounds in the gas phase using single-walled carbon nanotubes. Experience gained in engineering, electronics, automation, and software development from the design and…

  2. Plasmid dependence of Pseudomonas sp. strain NK87 enzymes that degrade 6-aminohexanoate-cyclic dimer.

    Science.gov (United States)

    Kanagawa, K; Negoro, S; Takada, N; Okada, H

    1989-06-01

    A bacterial strain, Pseudomonas sp. strain NK87, that can use 6-aminohexanoate-cyclic dimer as the sole source of carbon and nitrogen was newly isolated from wastewater of a factory which produces nylon-6. Two responsible enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (P-EI) and 6-aminohexanoate-dimer hydrolase (P-EII), were found in the NK87 strain, as is the case with Flavobacterium sp. strain KI72, another 6-aminohexanoate-cyclic-dimer-metabolizing bacterium (H. Okada, S. Negoro, H. Kimura, and S. Nakamura, Nature [London] 306:203-206, 1983). The P-EI enzyme is immunologically identical to the 6-aminohexanoate-cyclic-dimer hydrolase of KI72 (F-EI). However, antiserum against the 6-aminohexanoate-dimer hydrolase purified from KI72 (F-EII) did not react with cell extracts of NK87, indicating that the F-EII and P-EII enzymes are immunologically different. Restriction endonuclease analyses show that the NK87 strain harbors at least six plasmids ranging in size from 20 to 80 kilobase pairs (kbp). The P-EI and P-EII genes were cloned in Escherichia coli. Both the P-EI and F-EI probes strongly hybridized with a 23-kbp plasmid in Southern hybridization analyses. The P-EII probe hybridized specifically with an 80-kbp plasmid, but the F-EII probe hybridized with none of the plasmids harbored in NK87. These results indicate that the P-EI gene and P-EII gene are encoded on the 23-kbp and 80-kbp plasmids, respectively.

  3. Tannin degradation by a novel tannase enzyme present in some Lactobacillus plantarum strains.

    Science.gov (United States)

    Jiménez, Natalia; Esteban-Torres, María; Mancheño, José Miguel; de Las Rivas, Blanca; Muñoz, Rosario

    2014-05-01

    Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29 L. plantarum strains analyzed in the study possess the tanBLp gene, the gene tanALp was present in only four strains. Upon methyl gallate exposure, the expression of tanBLp was induced, whereas tanALp expression was not affected. TanALp showed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALp was observed at 30°C and pH 6 in the presence of Ca(2+) ions. TanALp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments.

  4. Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Fernanda Cortez Lopes

    2011-01-01

    Full Text Available A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

  5. Production and regulation of lignocellulose-degrading enzymes of Poria-like wood-inhabiting basidiomycetes.

    Science.gov (United States)

    Tomsovský, M; Popelárová, P; Baldrian, P

    2009-01-01

    The wood-decomposing fungal species Antrodia macra, A. pulvinascens, Ceriporiopsis aneirina, C. resinascens and Dichomitus albidofuscus were determined for production of laccase (LAC), Mn peroxidase (MnP), lignin peroxidase (LiP), endo-l,4-P-beta-glucanase, endo-l,4-beta-xylanase, cellobiohydrolase, 1,4-beta-glucosidase and 1,4-beta-xylosidase. The results confirmed the brown-rot mode of Antrodia spp. which did not produce the activity of LAC and MnP. The remaining species performed detectable activity of both enzymes while no strain produced LiP. Significant inhibition of LAC production by high nitrogen was found in all white-rot species while only MnP of D. albidofuscus was regulated in the same way. The endoglucanase and endoxylanase activities of white-rotting species were inhibited by glucose in the medium while those of Antrodia spp. were not influenced by glucose concentration. The regulation of enzyme activity and bio-mass production can vary even within a single fungal genus.

  6. Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

    Science.gov (United States)

    Lopes, Fernanda Cortez; Silva, Lucas André Dedavid e; Tichota, Deise Michele; Daroit, Daniel Joner; Velho, Renata Voltolini; Pereira, Jamile Queiroz; Corrêa, Ana Paula Folmer; Brandelli, Adriano

    2011-01-01

    A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism. PMID:22007293

  7. Single-walled carbon nanotube release affects the microbial enzyme-catalyzed oxidation processes of organic pollutants and lignin model compounds in nature.

    Science.gov (United States)

    Chen, Ming; Qin, Xiaosheng; Zeng, Guangming

    2016-11-01

    The question how microbial enzyme-catalyzed oxidation processes of organic pollutants and lignin model compounds (LMCs) are affected by the release of single-walled carbon nanotube (SWCNT) into the environment remains to be addressed at the molecular level. We have, therefore concentrated the effects of SWCNT on some important properties associated with enzyme activity and function during microbial oxidation of polycyclic aromatic hydrocarbons (benzo(a)pyrene, acenaphthene and anthracene), LMCs (2,6-dimethoxyphenol, guaiacol and veratryl alcohol) and β-hexachlorocyclohexane, including the behaviour of water molecules, hydrogen bonds (HBs) and hydrophobic interactions (HYs) between ligand and the enzyme, and conformational dynamics in N- and C-terminus. Our study revealed that SWCNT significantly affected the behaviour of water molecules within 5 Å of both these substrates and their respective enzymes during oxidation (p microbial enzyme-catalyzed processes of organic pollutants and LMCs in nature.

  8. Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger

    Science.gov (United States)

    van Munster, Jolanda M.; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L.; Gray, Christopher J.; Archer, David B.; Flitsch, Sabine L.

    2017-01-01

    Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry. PMID:28220903

  9. The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes.

    Science.gov (United States)

    Moreira, Leonora Rios de Souza; Ferreira, Gaspar Virgilio; Santos, Sheila Sousa Thurler; Ribeiro, Ana Paula Souza; Siqueira, Félix Gonçalves; Filho, Edivaldo Ximenes Ferreira

    2012-04-01

    Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

  10. The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

    Directory of Open Access Journals (Sweden)

    Leonora Rios de Souza Moreira

    2012-06-01

    Full Text Available Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

  11. Potentiality of yeast Candida sp. SMN04 for degradation of cefdinir, a cephalosporin antibiotic: kinetics, enzyme analysis and biodegradation pathway.

    Science.gov (United States)

    Selvi, A; Das, Devlina; Das, Nilanjana

    2015-01-01

    A new yeast strain isolated from the pharmaceutical wastewater was capable of utilizing cefdinir as a sole carbon source for their growth in mineral medium. The yeast was identified and named as Candida sp. SMN04 based on morphology and 18S-ITS-D1/D2/D3 rRNA sequence analysis. The interaction between factors pH (3.0-9.0), inoculum dosage (1-7%), time (1-11 day) and cefdinir concentration (50-450 mg/L) was studied using a Box-Behnken design. The factors were studied as a result of their effect on cell dry weight (R1; g/L), extended spectrum β-lactamase (ESBL) assay (R2; mm), P450 activity (R3; U/mL) and degradation (R4; %). Maximum values of R1, R2, R3 and R4 were obtained at central values of all the parameters. The isolated yeast strain efficiently degraded 84% of 250 mg L⁻¹ of cefdinir within 6 days with a half-life of 2.97 days and degradation rate constant of 0.2335 per day. Pseudo-first-order model efficiently described the process. Among the various enzymes tested, the order of activity at the end of Day 4 was noted to be: cytochrome P450 (1.76 ± 0.03) > NADPH reductase (1.51 ± 0.20) > manganese peroxidase and amylase (0.66 ± 0.15; 0.66 ± 0.70). Intermediates were successfully characterized by liquid chromatography-mass spectrometry. The opening of the β-lactam ring involving ESBL activity was considered as one of the major steps in the cefdinir degradation process. Fourier transform-infrared spectroscopy analysis showed the absence of spectral vibrations between 1766 and 1519 cm⁻¹ confirming the complete removal of lactam ring during cefdinir degradation. The results of the present study are promising for the use of isolated yeast Candida sp. SMN04 as a potential bioremediation agent.

  12. Comparative study on the rumen microbial populations, hydrolytic enzyme activities and dry matter degradability between different species of ruminant.

    Science.gov (United States)

    Moon, Yea Hwang; Ok, Ji Un; Lee, Shin Ja; Ha, Jong Kyu; Lee, Sung Sill

    2010-12-01

    A comparative study among Korean native cow (Hanwoo), Holstein dairy cow, Korean native goat and crossbred sheep on the population and marker concentration of ruminal microbes, the activities of carboxymethylcellulase (CMCase), xylanase and amylase, and in situ dry matter (DM) degradability were conducted. Twelve ruminally cannulated animals, three of each species, were used. Animals were fed the same diet containing 40% formula feed and 60% rice straw at the level of 2.5% of body weight. Total viable microbial populations in the rumen fluid were significantly (P < 0.01) greater for bacteria and fungi in goat than those of Holstein. The protozoan population among ruminant species was the reverse from that of bacteria. The concentrations of 2,6-diaminopimelic acid and chitin as markers for bacteria and fungi in the rumen fluid, respectively, were highest in goat, which is in accordance with the above population data. The concentration of aminoethylphosphonic acid as marker of protozoa was highest in Hanwoo and lowest in sheep (P < 0.01). Goat had the highest (P < 0.01) activities of all the enzymes investigated among ruminants. In situ effective degradation of the DM of rice straw was approximately 19% higher in the rumen of goat compared with other animals.

  13. Proteolytically inactive insulin-degrading enzyme inhibits amyloid formation yielding non-neurotoxic aβ peptide aggregates.

    Directory of Open Access Journals (Sweden)

    Matias B de Tullio

    Full Text Available Insulin-degrading enzyme (IDE is a neutral Zn(2+ peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid β (Aβ are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aβ in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ was used. IDEQ blocked the amyloidogenic pathway of Aβ yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aβ aggregation by light scattering showed that 1 IDEQ effect was promoted by ATP independent of its hydrolysis, 2 end products of Aβ-IDEQ co-incubation were incapable of "seeding" the assembly of monomeric Aβ and 3 IDEQ was ineffective in reversing Aβ aggregation. Moreover, Aβ aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals.

  14. The deubiquitinating enzyme USP8 promotes trafficking and degradation of the chemokine receptor 4 at the sorting endosome.

    Science.gov (United States)

    Berlin, Ilana; Higginbotham, Katherine M; Dise, Rebecca S; Sierra, Maria I; Nash, Piers D

    2010-11-26

    Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.

  15. The Mechanism of Carotenoid Degradation in Flue-Cured Tobacco and Changes in the Related Enzyme Activities at the Leaf-Drying Stage During the Bulk Curing Process

    Institute of Scientific and Technical Information of China (English)

    SONG Zhao-peng; LI Tong-shuai; ZHANG Yong-gang; CAO Hui-jing; GONG Chang-rong; ZHANG Wei-jian

    2010-01-01

    The mechanism of carotenoid degradation and the changes in the activities of related enzymes in flue-cured tobacco at the leaf-drying stage during the bulk-curing process were studied in order to provide theoretical basis for optimization of curing technology.The effect of different rising speeds of temperature on the carotenoid degradation and the related enzymes activities at the color-fixing stage during the bulk curing process was studied by using the electric-heated fluecuring barn designed by Henan Agricultural University,China,based on curing technology with yellowing at low temperature and moderate humidity and leaf drying at moderate humidity.The results showed that the carotenoid degradation components(β-carotene,lutein,neoxanthin,and violaxthin)decreased gradually at the color-fixing stage during the bulk curing process.The carotenoid degradation components viz.,β-carotene,lutein,neoxanthin,and violaxthin at the slow heating curing(T1)were relatively higher than the rapid heating curing(T2)accounting for 10,2,32 and 32%respectively,but there were no differences among treatments(P>0.05).The effect of different conditions of curing on the activities of enzymes related to carotenoids degradation were significant.The lipoxygenase,phenylalanine ammonialyase,peroxidase,and polyphenol oxidase enzymes had a bidirectional effect on the quality of tobacco leaves and it was beneficial to form more premise matter of aroma based on the higher enzyme activities at the early leaf-drying stage.The slow heating could regulate the change in various enzymes' activities reasonably,making cell redox reaction to reach the dynamic balance and make the degradation of carotenoids adequately.Meanwhile,it could avoid the occurrence of browning reaction and provide foundation for improving the quality of tobacco and optimization of technology for bulk curing and further enhancing aroma.

  16. Respiratory syncytial virus infection down-regulates antioxidant enzyme expression by triggering deacetylation-proteasomal degradation of Nrf2.

    Science.gov (United States)

    Komaravelli, Narayana; Tian, Bing; Ivanciuc, Teodora; Mautemps, Nicholas; Brasier, Allan R; Garofalo, Roberto P; Casola, Antonella

    2015-11-01

    Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or treatment is available. RSV infection in cells, mice, and children leads to rapid generation of reactive oxygen species, which are associated with oxidative stress and lung damage, due to a significant decrease in the expression of airway antioxidant enzymes (AOEs). Oxidative stress plays an important role in the pathogenesis of RSV-induced lung disease, as antioxidants ameliorate clinical disease and inflammation in vivo. The aim of this study is to investigate the unknown mechanism(s) of virus-induced inhibition of AOE expression. RSV infection is shown to induce a progressive reduction in nuclear and total cellular levels of the transcription factor NF-E2-related factor 2 (Nrf2), resulting in decreased binding to endogenous AOE gene promoters and decreased AOE expression. RSV induces Nrf2 deacetylation and degradation via the proteasome pathway in vitro and in vivo. Histone deacetylase and proteasome inhibitors block Nrf2 degradation and increase Nrf2 binding to AOE endogenous promoters, resulting in increased AOE expression. Known inducers of Nrf2 are able to increase Nrf2 activation and subsequent AOE expression during RSV infection in vitro and in vivo, with significant amelioration of oxidative stress. This is the first study to investigate the mechanism(s) of virus-induced inhibition of AOE expression. RSV-induced inhibition of Nrf2 activation, due to deacetylation and proteasomal degradation, could be targeted for therapeutic intervention aimed to increase airway antioxidant capacity during infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.

    Science.gov (United States)

    Ralat, Luis A; Ren, Min; Schilling, Alexander B; Tang, Wei-Jen

    2009-12-01

    Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.

  18. Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells.

    Science.gov (United States)

    Pivovarova, O; Gögebakan, O; Pfeiffer, A F H; Rudovich, N

    2009-08-01

    Hepatic insulin degradation decreases in type 2 diabetes. Insulin-degrading enzyme (IDE) plays a key role in insulin degradation and its gene is located in a diabetes-associated chromosomal region. We hypothesised that IDE may be regulated by insulin and/or glucose in a liver cell model. To validate the observed regulation of IDE in vivo, we analysed biopsies of human adipose tissue during different clamp experiments in men. Human hepatoma HepG2 cells were incubated in normal (1 g/l) or high (4.5 g/l) glucose medium and treated with insulin for 24 h. Catalytic activity, mRNA and protein levels of IDE were assessed. IDE mRNA levels were measured in biopsies of human subcutaneous adipose tissue before and at 240 min of hyperinsulinaemic, euglycaemic and hyperglycaemic clamps. In HepG2 cells, insulin increased IDE activity under normal glucose conditions with no change in IDE mRNA or protein levels. Under conditions of high glucose, insulin increased mRNA levels of IDE without changes in IDE activity. Both in normal and high glucose medium, insulin increased levels of the catalytically more active 15a IDE isoform compared with the 15b isoform. In subcutaneous adipose tissue, IDE mRNA levels were not significantly upregulated after euglycaemic or hyperglycaemic clamps. Insulin increases IDE activity in HepG2 cells in normal but not in high glucose conditions. This disturbance cannot be explained by corresponding alterations in IDE protein levels or IDE splicing. The loss of insulin-induced regulation of IDE activity under hyperglycaemia may contribute to the reduced insulin extraction and peripheral hyperinsulinaemia in type 2 diabetes.

  19. Triesterase and promiscuous diesterase activities of a di-Co(II)-containing organophosphate degrading enzyme reaction mechanisms.

    Science.gov (United States)

    Alberto, Marta E; Pinto, Gaspar; Russo, Nino; Toscano, Marirosa

    2015-02-23

    The reaction mechanism for the hydrolysis of trimethyl phosphate and of the obtained phosphodiester by the di-Co(II) derivative of organophosphate degrading enzyme from Agrobacterium radiobacter P230(OpdA), have been investigated at density functional level of theory in the framework of the cluster model approach. Both mechanisms proceed by a multistep sequence and each catalytic cycle begins with the nucleophilic attack by a metal-bound hydroxide on the phosphorus atom of the substrate, leading to the cleavage of the phosphate-ester bond. Four exchange-correlation functionals were used to derive the potential energy profiles in protein environments. Although the enzyme is confirmed to work better as triesterase, as revealed by the barrier heights in the rate-limiting steps of the catalytic processes, its promiscuous ability to hydrolyze also the product of the reaction has been confirmed. The important role played by water molecules and some residues in the outer coordination sphere has been elucidated, while the binuclear Co(II) center accomplishes both structural and catalytic functions. To correctly describe the electronic configuration of the d shell of the metal ions, high- and low-spin arrangement jointly with the occurrence of antiferromagnetic coupling, have been herein considered.

  20. Statistical Correlation between Ligninolytic Enzymes Secretion and Remazol Brilliant Yellow-3GL Dye Degradation Potential of Trametes versicolor IBL-04.

    Science.gov (United States)

    Asgher, Muhammad; Shah, Syed Agha Hassan; Iqbal, Hafiz Muhammad Nasir

    2016-04-01

    Trametes versicolor IBL-04 was used for biodegradation of Remazol Brilliant Yellow 3-GL (RBY3-GL) reactive textile dye in Kirk's basal salts medium. During the initial screening study, the maximum decolorization (93.5%) of RBY3-GL was achieved in 7 days' shaking incubation period at pH 4 and 30 °C. Different physical and nutritional factors were statistically optimized to enhance the efficiency of T. versicolor IBL-04 for maximum decolorization. Under optimal conditions T. versicolor IBL-04 completely decolorized (100%) the RBY3-GL in 2 days of incubation with negligible adsorption on fungal mycelia. Laccase was the major enzyme (938.3 U/mL) secreted by T. versicolor IBL-04 along with comparatively lower activities of MnP. In this article and for the first time, a statistical correlation has been successfully investigated between the ligninolytic enzymes from an indigenously isolated white rot fungi, T. versicolor IBL-04, and the degradation of RBY3-GL.

  1. Novel Platinum(II) compounds modulate insulin-degrading enzyme activity and induce cell death in neuroblastoma cells.

    Science.gov (United States)

    Tundo, Grazia R; Sbardella, Diego; De Pascali, Sandra A; Ciaccio, Chiara; Coletta, Massimo; Fanizzi, Francesco P; Marini, Stefano

    2015-01-01

    The properties of three novel Platinum(II) compounds toward the insulin-degrading enzyme (IDE) enzymatic activity have been investigated under physiological conditions. The rationale of this study resides on previous observations that these compounds, specifically designed and synthesized by some of us, induce apoptosis in various cancer cell lines, whereas IDE has been proposed as a putative oncogene involved in neuroblastoma onset and progression. Two of these compounds, namely [PtCl(O,O'-acac)(DMSO)] and [Pt(O,O'-acac)(γ-acac)(DMS)], display a modulatory behavior, wherefore activation or inhibition of IDE activity occurs over different concentration ranges (suggesting the existence of two binding sites on the enzyme). On the other hand, [Pt(O,O'-acac)(γ-acac)(DMSO)] shows a typical competitive inhibitory pattern, characterized by a meaningful affinity constant (K i  = 0.95 ± 0.21 μM). Although all three compounds induce cell death in neuroblastoma SHSY5Y cells at concentrations exceeding 2 μM, the two modulators facilitate cells' proliferation at concentrations ≤ 1.5 μM, whereas the competitive inhibitor [Pt(O,O'-acac)(γ-acac)(DMSO)] only shows a pro-apoptotic activity at all investigated concentrations. These features render the [Pt(O,O'-acac)(γ-acac)(DMSO)] a promising "lead compound" for the synthesis of IDE-specific inhibitors (not characterized yet) with therapeutic potentiality.

  2. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

    Science.gov (United States)

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

    2016-06-01

    The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

  3. Expression of a nitric oxide degrading enzyme induces a senescence programme in Arabidopsis.

    Science.gov (United States)

    Mishina, Tatiana E; Lamb, Chris; Zeier, Jürgen

    2007-01-01

    Nitric oxide (NO) has been proposed to act as a factor delaying leaf senescence and fruit maturation in plants. Here we show that expression of a NO degrading dioxygenase (NOD) in Arabidopsis thaliana initiates a senescence-like phenotype, an effect that proved to be more pronounced in older than in younger leaves. This senescence phenotype was preceded by a massive switch in gene expression in which photosynthetic genes were down-regulated, whereas many senescence-associated genes (SAGs) and the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene ACS6 involved in ethylene synthesis were up-regulated. External fumigation of NOD plants with NO as well as environmental conditions known to stimulate endogenous NO production attenuated the induced senescence programme. For instance, both high light conditions and nitrate feeding reduced the senescence phenotype and attenuated the down-regulation of photosynthetic genes as well as the up-regulation of SAGs. Treatment of plants with the cytokinin 6-benzylaminopurin (BAP) reduced the down-regulation of photosynthesis, although it had no consistent effect on SAG expression. Metabolic changes during NOD-induced senescence comprehended increases in salicylic acid (SA) levels, accumulation of the phytoalexin camalexin and elevation of leaf gamma-tocopherol contents, all of which occurred during natural senescence in Arabidopsis leaves as well. Moreover, NO fumigation delayed the senescence process induced by darkening individual Arabidopsis Columbia-0 (Col-0) leaves. Our data thus support the notion that NO acts as a negative regulator of leaf senescence.

  4. Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes.

    Science.gov (United States)

    Aarstad, Olav Andreas; Tøndervik, Anne; Sletta, Håvard; Skjåk-Bræk, Gudmund

    2012-01-09

    Distribution and proportion of β-D-mannuronic and α-L-guluronic acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diguluronic acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with (1)H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (F(G) = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerases act by a multiple attack mechanism. Alternating sequences (MG-blocks) were found to be much shorter than the other block types. In connection with method development, an oligomer library comprising both saturated and unsaturated oligomers of various composition and DP 2-15 was made.

  5. Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme*S⃞

    OpenAIRE

    Manolopoulou, Marika; Guo, Qing; Malito, Enrico; Schilling, Alexander B.; Tang, Wei-Jen

    2009-01-01

    Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentatio...

  6. Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

    Science.gov (United States)

    Suzuki, Ken; Sakamoto, Hironori; Shinozaki, Yukiko; Tabata, Jun; Watanabe, Takashi; Mochizuki, Atsushi; Koitabashi, Motoo; Fujii, Takeshi; Tsushima, Seiya; Kitamoto, Hiroko K

    2013-09-01

    Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

  7. Purification of a new manganese peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the enzyme.

    Science.gov (United States)

    Baborová, Petra; Möder, Monika; Baldrian, Petr; Cajthamlová, Kamila; Cajthaml, Tomás

    2006-04-01

    The white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDS-PAGE showed the purified MnP to be a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0 to 7.0 with a peak at pH 5.5. Kinetic constants K(m) were 8 microM for H(2)O(2) and 12 or 31 microM for Mn(2+) depending on the substrate. The enzyme did not perform oxidation in the absence of H(2)O(2) or Mn(2+). MnP was active at 5-70 degrees C with an optimum between 50-60 degrees C. At temperatures above 65 degrees C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.

  8. Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an enzyme for the degradation of type II arabinogalactan.

    Science.gov (United States)

    Fujita, Kiyotaka; Sakaguchi, Takenori; Sakamoto, Ayami; Shimokawa, Michiko; Kitahara, Kanefumi

    2014-08-01

    Type II arabinogalactan (AG-II) is a suitable carbohydrate source for Bifidobacterium longum subsp. longum, but the degradative enzymes have never been characterized. In this study, we characterized an exo-β-1,3-galactanase, BLLJ_1840, belonging to glycoside hydrolase family 43 from B. longum subsp. longum JCM1217. The recombinant BLLJ_1840 expressed in Escherichia coli hydrolyzed β-1,3-linked galactooligosaccharides but not β-1,4- and β-1,6-linked galactooligosaccharides. The enzyme also hydrolyzed larch wood arabinogalactan (LWAG), which comprises a β-1,3-linked galactan backbone with β-1,6-linked galactan side chains. The kcat/Km ratio of dearabinosylated LWAG was 24-fold higher than that of β-1,3-galactan. BLLJ_1840 is a novel type of exo-β-1,3-galactanase with a higher affinity for the β-1,6-substituted β-1,3-galactan than for nonsubstituted β-1,3-galactan. BLLJ_1840 has 27% to 28% identities with other characterized exo--1,3-galactanases from bacteria and fungi. The homologous genes are conserved in several strains of B. longum subsp. longum and B. longum subsp. infantis but not in other bifidobacteria. Transcriptional analysis revealed that BLLJ_1840 is intensively induced with BLLJ_1841, an endo-β-1,6-galactanase candidate, in the presence of LWAG. This is the first report of exo-β-1,3-galactanase in bifidobacteria, which is an enzyme used for the acquisition of AG-II in B. longum subsp. longum.

  9. Arabinoxylan-degrading enzyme system of the fungus Aspergillus awamori: purification and properties of an alpha-L-arabinofuranosidase.

    Science.gov (United States)

    Wood, T M; McCrae, S I

    1996-05-01

    An alpha-L-arabinofuranosidase produced by the fungus Aspergillus awamori had a molecular mass of approximately 64 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and was optimally active at pH 4.6 and 50 degrees C. The enzyme, which chromatographed as a single component on SDS-PAGE, appeared to consist of two isoenzymes of pI 3.6 and 3.2. Acting in isolation, the alpha-L-arabinofuranosidase had only a very limited capacity to release L-arabinose (less than 11%) directly from arabinoxylans that had been extracted from a number of plant cell wall preparations using 18% alkali, but a much higher proportion of the L-arabinose (46%) was released from a wheat straw arabinoxylan that had been isolated by steam treatment. There was a marked synergistic effect between the alpha-L-arabinofuranosidase and an endo-(1 --> 4)-beta-D-xylanase produced by A. awamori in both the rate and extent of the release of L-arabinose from both oat straw and wheat straw arabinoxylans, suggesting that L-arabinose-substituted oligosaccharides generated by the endoxylanase action were better substrates for enzyme action. A novel property of the alpha-L-arabinofuranosidase was its capacity to release a substantial proportion (42%) of feruloyl L-arabinose from intact wheat straw arabinoxylan. The concerted action of the alpha-L-arabinofuranosidase and endoxylanase released 71% of the feruloyl L-arabinose and 69% of the p-coumaroyl L-arabinose substituents from wheat straw arabinoxylan.

  10. Studies on the mode of action of non-starch-polysaccharides (NSP)-degrading enzymes in vitro. 2. Communication: effects on nutrient release and hydration properties.

    Science.gov (United States)

    Aulrich, K; Flachowsky, G

    2001-01-01

    By use of an in vitro model, the effects of NSP-degrading enzymes on the cage effect and the hydration properties were demonstrated using wheat bran. The in vitro model simulates the conditions (pH, dry matter, temperature and transit time) in the fore sections of the porcine gastro-intestinal tract (GIT) by neglecting endogenous enzyme activities. Enzyme treatment caused a dose-dependent increase in wheat bran solubility and thus resulted in improved protein and mineral release from the insoluble NSP fraction. Up to 17% protein and 40% crude ash from the insoluble NSP-fraction were dissolved after enzyme treatment. Hydrating properties of wheat bran were strongly affected by enzyme treatment and particle size. Water-binding capacity (WBC) and water-holding capacity (WHC) decreased with increasing enzyme dosage in dependence on particle size. The studies confirmed the applicability of the tested in vitro model as a useful tool for preliminary tests to estimate the effects of NSP-degrading enzymes on nutrient release and changes in some physico-chemical properties.

  11. Draft Genome Sequence of the Fungus Paraphoma sp. B47-9, a Producer of a Biodegradable Plastic-Degrading Enzyme.

    Science.gov (United States)

    Sameshima-Yamashita, Yuka; Koike, Hideaki; Koitabashi, Motoo; Saika, Azusa; Morita, Tomotake; Yarimizu, Tohru; Kitamoto, Hiroko

    2016-10-20

    Paraphoma sp. B47-9 is a producer of a biodegradable plastic-degrading enzyme. Here, we report the draft genome sequence of this strain. The draft genome assembly has a size of 39.3 Mb with a GC content of 52.4% and consists of 185 scaffolds.

  12. Purification, characterization, and cloning of the gene for a biodegradable plastic-degrading enzyme from Paraphoma-related fungal strain B47-9.

    Science.gov (United States)

    Suzuki, Ken; Noguchi, Masako Tsujimoto; Shinozaki, Yukiko; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Yoshida, Shigenobu; Fujii, Takeshi; Kitamoto, Hiroko K

    2014-05-01

    Paraphoma-related fungal strain B47-9 secreted a biodegradable plastic (BP)-degrading enzyme which amounted to 68 % (w/w) of the total secreted proteins in a culture medium containing emulsified poly(butylene succinate-co-adipate) (PBSA) as sole carbon source. The gene for this enzyme was found to be composed of an open reading frame consisting of 681 nucleotides encoding 227 amino acids and two introns. Southern blot analysis showed that this gene exists as a single copy. The deduced amino acid sequence suggested that this enzyme belongs to the cutinase (E.C.3.1.1.74) family; thus, it was named P araphoma-related fungus cutinase-like enzyme (PCLE). It degraded various types of BP films, such as poly(butylene succinate), PBSA, poly(butylene adipate-co-terephthalate), poly(ε-caprolactone), and poly(DL-lactic acid). It has a molecular mass of 19.7 kDa, and an optimum pH and temperature for degradation of emulsified PBSA of 7.2 and 45 °C, respectively. Ca(2+) ion at a concentration of about 1.0 mM markedly enhanced the degradation of emulsified PBSA.

  13. Degradation of granular starch by the bacterium Microbacterium aurum B8.A involves a novel modular α-amylase enzyme system with FNIII and CBM25 domains

    NARCIS (Netherlands)

    Valk, Vincent; Eeuwema, Wieger; Sarian, Fean D; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

    2015-01-01

    The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant waste water facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multi-domain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1417 amino

  14. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    Science.gov (United States)

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

  15. Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

    Institute of Scientific and Technical Information of China (English)

    SHEN; Xihui; LIU; Shuangjiang

    2005-01-01

    Although the protocatechuate branch of the β-ketoadipate pathway in Gram bacteria has been well studied, this branch is less understood in Gram+ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C.glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.

  16. Structural and functional characterization of pathogenic non- synonymous genetic mutations of human insulin-degrading enzyme by in silico methods.

    Science.gov (United States)

    Shaik, Noor A; Kaleemuddin, Mohammed; Banaganapalli, Babajan; Khan, Fazal; Shaik, Nazia S; Ajabnoor, Ghada; Al-Harthi, Sameer E; Bondagji, Nabeel; Al-Aama, Jumana Y; Elango, Ramu

    2014-04-01

    Insulin-degrading enzyme (IDE) is a key protease involved in degrading insulin and amyloid peptides in human body. Several non-synonymous genetic mutations of IDE gene have been recently associated with susceptibility to both diabetes and Alzheimer's diseases. However, the consequence of these mutations on the structure of IDE protein and its substrate binding characteristics is not well elucidated. The computational investigation of genetic mutation consequences on structural level of protein is recently found to be an effective alternate to traditional in vivo and in vitro approaches. Hence, by using a combination of empirical rule and support vector machine based in silico algorithms, this study was able to identify that the pathogenic nonsynonymous genetic mutations corresponding to p.I54F, p.P122T, p.T533R, p.P581A and p.Y609A have more potential role in structural and functional deviations of IDE activity. Moreover, molecular modeling and secondary structure analysis have also confirmed their impact on the stability and secondary properties of IDE protein. The molecular docking analysis of IDE with combinational substrates has revealed that peptide inhibitors compared to small non-peptide inhibitor molecules possess good inhibitory activity towards mutant IDE. This finding may pave a way to design novel potential small peptide inhibitors for mutant IDE. Additionally by un-translated region (UTR) scanning analysis, two regulatory pathogenic genetic mutations i.e., rs5786997 (3' UTR) and rs4646954 (5' UTR), which can influence the translation pattern of IDE gene through sequence alteration of upstream-Open Reading Frame and Internal Ribosome Entry Site elements were identified. Our findings are expected to help in narrowing down the number of IDE genetic variants to be screened for disease association studies and also to select better competitive inhibitors for IDE related diseases.

  17. EFFECT OF DIETARY SUPPLEMENTATION OF NON-STARCH POLYSACCHARIDE DEGRADING ENZYMES ON GROWTH PERFORMANCE OF BROILER CHICKS

    Directory of Open Access Journals (Sweden)

    M. A. Nadeem, M. I. Anjum, A. G. Khan and A. Azim

    2005-10-01

    Full Text Available An experiment was conducted to study the performance and carcass parameters of broilers chicks fed diets with and without supplementing non-starch polysaccharide degrading enzymes (NSPDE at the rate of 0.5 g/kg diet. A total of 300 day-old broiler chicks were randomly divided into 12 sets (replicates each comprising 25 chicks and three sets per treatment group, reared on deep litter from 1-42 days post-hatch. Group A was fed diets without NSPDE supplementation, while group B was fed diets supplemented with NSPDE (0.5 g/kg. Group C was fed diets containing 50 kcal/kg less metabolizable energy (ME without NSPDE and group D was fed diets having 50 kcal/kg less ME with NSPDE (0.5 g/kg supplementation. Feed and water were provided ad libitum. Feed intake and feed conversion ratio (FCR from 1-28 days and 1-42 days was significantly (p<0.05 improved in chicks fed NSPDE supplemented diets (groups B and D compared to non-supplemented diets (groups A and C. However, during 29-42 days of growing period enzymes supplementation did not influence feed intake and FCR. Body weight gain, dressing percentage and relative weights of heart, gizzard and shank at 42 days of age was found to be non-significantly different among all groups. However, liver weight reduced significantly (p<0.05 in NSPDE supplemented groups. The study suggested that NSPDE supplementation was beneficial in enhancing feed utilization during the starter phase, while its effects on weight gain, dressing percentage and weights of organs, except liver weight, were found to be non-significant.

  18. Early maternal deprivation induces changes on the expression of 2-AG biosynthesis and degradation enzymes in neonatal rat hippocampus.

    Science.gov (United States)

    Suárez, Juan; Rivera, Patricia; Llorente, Ricardo; Romero-Zerbo, Silvana Y; Bermúdez-Silva, Francisco J; de Fonseca, Fernando Rodríguez; Viveros, María-Paz

    2010-08-19

    Early maternal deprivation (MD) in rats (24h, PND 9-10) is a model for neurodevelopmental stress. Our previous data showed that MD altered the hippocampal levels of the endocannabinoid 2-AG and the expression of hippocampal cannabinoid receptors in 13-day-old rats, with males being more markedly affected. The aim of this study was to analyze the impact of MD on the enzymes involved in 2-AG biosynthesis (DAGLalpha and DAGLbeta) and degradation (MAGL) in relevant areas (DG, CA1, CA3) of the hippocampus in 13-day-old neonatal rats. The expression of the enzymes was evaluated by quantitative RT-PCR, immunohistochemistry, and densitometry. MD induced a significant increase in DAGLalpha immunoreactivity in both males and females, which was mainly associated with fibers in the polymorphic cell layer of the dentate gyrus and in the stratum pyramidale of CA3. In contrast, the molecular layer of the dentate gyrus showed a significant decrease in DAGLalpha immunoreactivity in MD males and females. No changes were observed in DAGLbeta immunoreactivity. MD induced a significant decrease in MAGL immunoreactivity in hippocampal CA3 and CA1 areas, more marked in males than in females, and that was mainly associated with fibers in all strata of CA3 and CA1. The results also showed a significant decrease of MAGL mRNA levels in MD males. These data support a clear association between neurodevelopmental stress and dysregulation of the endocannabinoid system. This association may be relevant for schizophrenia and other neurodevelopmental psychiatric disorders.

  19. Effects of ageing and experimental diabetes on insulin-degrading enzyme expression in male rat tissues.

    Science.gov (United States)

    Kochkina, Ekaterina G; Plesneva, Svetlana A; Vasilev, Dmitrii S; Zhuravin, Igor A; Turner, Anthony J; Nalivaeva, Natalia N

    2015-08-01

    Due to an increasing life expectancy in developing countries, cases of type 2 diabetes and Alzheimer's disease (AD) in the elderly are growing exponentially. Despite a causative link between diabetes and AD, general molecular mechanisms underlying pathogenesis of these disorders are still far from being understood. One of the factors leading to cell death and cognitive impairment characteristic of AD is accumulation in the brain of toxic aggregates of amyloid-β peptide (Aβ). In the normally functioning brain Aβ catabolism is regulated by a cohort of proteolytic enzymes including insulin-degrading enzyme (IDE) and their deficit with ageing can result in Aβ accumulation and increased risk of AD. The aim of this study was a comparative analysis of IDE expression in the brain structures involved in AD, as well as in peripheral organs (the liver and kidney) of rats, during natural ageing and after experimentally-induced diabetes. It was found that ageing is accompanied by a significant decrease of IDE mRNA and protein content in the liver (by 32 and 81%) and brain structures (in the cortex by 58 and 47% and in the striatum by 53 and 68%, respectively). In diabetic animals, IDE protein level was increased in the liver (by 36%) and in the striatum (by 42%) while in the brain cortex and hippocampus it was 20-30% lower than in control animals. No significant IDE protein changes were observed in the kidney of diabetic rats. These data testify that ageing and diabetes are accompanied by a deficit of IDE in the brain structures where accumulation of Aβ was reported in AD patients, which might be one of the factors predisposing to development of the sporadic form of AD in the elderly, and especially in diabetics.

  20. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

    Energy Technology Data Exchange (ETDEWEB)

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel; McDonald, Bradon R.; Nicora, Carrie D.; Wu, Si; Moore, Ronald J.; Orton, Daniel J.; Monroe, Matthew E.; Piehowski, Paul D.; Purvine, Samuel O.; Smith, Richard D.; Lipton, Mary S.; Burnum-Johnson, Kristin E.; Currie, Cameron R.

    2015-08-28

    Leaf-cutter ants are prolific and conspicuous Neotropical herbivores that derive energy from specialized fungus gardens they cultivate using foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain lignocellulases that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as it is foraged by the ants. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous lignocellulases likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three-quarters of all lignocellulases identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 23 lignocellulases enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

  1. Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

    Science.gov (United States)

    Laurenzana, Anna; Biagioni, Alessio; D'Alessio, Silvia; Bianchini, Francesca; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Mazzanti, Benedetta; Pimpinelli, Nicola; Torre, Eugenio; Danese, Silvio; Calorini, Lido; Rosso, Mario Del; Fibbi, Gabriella

    2014-01-01

    The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of “targeted therapies” as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a “personalized therapy”, without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis. PMID:25003596

  2. The Role of Insulin, Insulin Growth Factor, and Insulin-Degrading Enzyme in Brain Aging and Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Claude Messier

    2005-01-01

    Full Text Available Most brain insulin comes from the pancreas and is taken up by the brain by what appears to be a receptor-based carrier. Type 2 diabetes animal models associated with insulin resistance show reduced insulin brain uptake and content. Recent data point to changes in the insulin receptor cascade in obesity-related insulin resistance, suggesting that brain insulin receptors also become less sensitive to insulin, which could reduce synaptic plasticity. Insulin transport to the brain is reduced in aging and in some animal models of type 2 diabetes; brain insulin resistance may be present as well. Studies examining the effect of the hyperinsulinic clamp or intranasal insulin on cognitive function have found a small but consistent improvement in memory and changes in brain neuroelectric parameters in evoked brain potentials consistent with improved attention or memory processing. These effects appear to be due to raised brain insulin levels. Peripheral levels of Insulin Growth Factor-I (IGF-I are associated with glucose regulation and influence glucose disposal. There is some indication that reduced sensitivity to insulin or IGF-I in the brain, as observed in aging, obesity, and diabetes, decreases the clearance of Aβ amyloid. Such a decrease involves the insulin receptor cascade and can also increase amyloid toxicity. Insulin and IGF-I may modulate brain levels of insulin degrading enzyme, which would also lead to an accumulation of Aβ amyloid.

  3. Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease.

    Science.gov (United States)

    Son, Sung Min; Cha, Moon-Yong; Choi, Heesun; Kang, Seokjo; Choi, Hyunjung; Lee, Myung-Shik; Park, Sun Ah; Mook-Jung, Inhee

    2016-05-01

    The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.

  4. Overexpression of Insulin Degrading Enzyme could Greatly Contribute to Insulin Down-regulation Induced by Short-Term Swimming Exercise.

    Science.gov (United States)

    Kim, Min Sun; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Choi, Sun Il; Lee, Hye Ryun; Hwang, In Sik; Shim, Sun Bo; Jee, Seung Wan; Lee, Su Hae; Bae, Chang Joon; Cho, Jung Sik; Cho, Jun Yong; Hwang, Dae Youn

    2011-03-01

    Exercise training is highly correlated with the reduced glucose-stimulated insulin secretion (GSIS), although it enhanced insulin sensitivity, glucose uptake and glucose transporter expression to reduce severity of diabetic symptoms. This study investigated the impact of short-term swimming exercise on insulin regulation in the Goto-Kakizaki (GK) rat as a non-obese model of non-insulin-dependent diabetes mellitus. Wistar (W/S) and GK rats were trained 2 hours daily with the swimming exercise for 4 weeks, and then the changes in the metabolism of insulin and glucose were assessed. Body weight was markedly decreased in the exercised GK rats compare to their non-exercised counterpart, while W/S rats did not show any exercise-related changes. Glucose concentration was not changed by exercise, although impaired glucose tolerance was improved in GK rats 120 min after glucose injection. However, insulin concentration was decreased by swimming exercise as in the decrease of GSIS after running exercise. To identify the other cause for exercise-induced insulin down-regulation, the changes in the levels of key factors involved in insulin production (C-peptide) and clearance (insulin-degrading enzyme; IDE) were measured in W/S and GK rats. The C-peptide level was maintained while IDE expression increased markedly. Therefore, these results showed that insulin down-regulation induced by short-term swimming exercise likely attributes to enhanced insulin clearance via IDE over-expression than by altered insulin production.

  5. Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme.

    Science.gov (United States)

    Laurenzana, Anna; Biagioni, Alessio; D'Alessio, Silvia; Bianchini, Francesca; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Mazzanti, Benedetta; Pimpinelli, Nicola; Torre, Eugenio; Danese, Silvio; Calorini, Lido; Del Rosso, Mario; Fibbi, Gabriella

    2014-06-15

    The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of "targeted therapies" as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a "personalized therapy", without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis.

  6. Capability of a selected bacterial consortium for degrading diesel/biodiesel blends (B20): enzyme and biosurfactant production.

    Science.gov (United States)

    Meyer, Daniel Derrossi; Santestevan, Naiara Aguiar; Bücker, Francielle; Salamoni, Sabrina Pinto; Andreazza, Robson; De Oliveira Camargo, Flávio Anastácio; Bento, Fátima Menezes

    2012-01-01

    The search for alternative sources of energy, such as biodiesel, has been stimulated, since this biofuel is highly susceptible for biodegradation and has low toxicity, thus, reducing the impact in ecosystems. The objective of this study was to select a bacterial consortium with potential for degrading diesel/biodiesel blends (B20) obtained from areas contaminated with hydrocarbons/esters. In order to evaluate the biodegrability of the blend, six enzyme assays were conducted: alkane hydroxylase, Catechol 1,2-dioxygenase, Catechol 2,3-dioxygenase, Protocatechol 3,4-dioxygenase, ρ-NPA hydrolysis (esterase), and release of fatty acids through titration (lipase), with estimative of total protein and biosurfactant production (surface tension measurement and emulsifying index E(24)). The best results obtained allowed the selection of four bacteria isolates (Bacillus megaterium, Bacillus pumilus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia) for compiling a consortium, which will be used for bioaugmentation strategies in soils contaminated with these fuels. This consortium exhibited high potential for biodegradation of biodiesel, and might be an efficient alternative for cleaning up these contaminated environments.

  7. Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic condition: characteristics, degradation products, enzyme analysis and phytotoxicity.

    Science.gov (United States)

    Du, Lin-Na; Wang, Sheng; Li, Gang; Wang, Bing; Jia, Xiao-Ming; Zhao, Yu-Hua; Chen, Yun-Long

    2011-03-01

    Malachite green (MG), a widely-used and recalcitrant dye, has been confirmed to be carcinogenic and mutagenic against many organisms. The main objective of this study is to investigate the capability of Pseudomonas sp. strain DY1 to decolorize MG, and to explore the possible mechanism. The results sho