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Sample records for cellulolytic enzyme complex

  1. Cellulolytic enzyme compositions and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Prashant; Gaspar, Armindo Ribiero; Croonenberghs, James; Binder, Thomas P.

    2017-07-25

    The present invention relates enzyme composition comprising a cellulolytic preparation and an acetylxylan esterase (AXE); and the used of cellulolytic enzyme compositions for hydrolyzing acetylated cellulosic material. Finally the invention also relates to processes of producing fermentation products from acetylated cellulosic materials using a cellulolytic enzyme composition of the invention.

  2. EXTRACELLULAR CELLULOLYTIC COMPLEXES PRODUCTION BY MICROSCOPIC FUNGI

    Directory of Open Access Journals (Sweden)

    S. O. Syrchin

    2015-10-01

    Full Text Available The aim of this work was to screen and to study the effect of inducers on the synthesis of the cellulolytic enzyme complexes by microscopic fungi. Cellulolytic and xylanolytic activities were determined by reducing sugar with DNS reagent, and β-glucosidase activity by pNPG hydrolysis. The enzyme preparations were obtained by ammonium sulphate precipitation. Among 32 studied strains of microscopic fungi 14 produced cellulo- and xylanolytic enzyme complexes. Fusarium sp. 5 and Fennellia sp. 2806 demonstrated the highest levels of all studied enzyme activities. Enzyme preparations with high endo-, exoglucanase, xylanase and β-glucosidase activities were obtained from these strains. Fusarium sp. 5 and Fennellia sp. 2806 were active producers of cellulase enzyme complexes during growth on natural substrates. It was shown that inductors of cellulolytic enzymes in Fusarium sp. 5 and Fennellia sp. 2806 differed from the ones in Trichoderma reesei.

  3. Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

    Science.gov (United States)

    Dojnov, Biljana; Grujić, Marica; Vujčić, Zoran

    2015-08-01

    A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-β-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Production of cellulolytic enzymes from ascomycetes

    DEFF Research Database (Denmark)

    Hansen, Gustav Hammerich; Lübeck, Mette; Frisvad, Jens Christian

    2015-01-01

    Optimizing production of cellulose degrading enzymes is of great interest in order to increase the feasibility of constructing biorefinery facilities for a sustainable supply of energy and chemical products. The ascomycete phylum has a large potential for the production of cellulolytic enzymes....... Although numerous enzymatic profiles have already been unraveled, the research has been covering only a limited number of species and genera, thus leaving many ascomycetes to be analyzed. Such analysis requires choosing appropriate media and cultivation methods that ensure enzyme profiles with high...... specificities and activities. However, the choice of media, cultivation methods and enzyme assays highly affect the enzyme activity profile observed. This review provides an overview of enzymatic profiles for several ascomycetes covering phylogenetically distinct genera and species. The profiles of cellulose...

  5. Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes

    DEFF Research Database (Denmark)

    Krogh, Kristian Bertel Rømer; Mørkeberg, Astrid; Frisvad, Jens Christian

    2004-01-01

    For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commenly used, but, there is a need for more efficient enzyme cocktails. In this study, the production of cellulolytic and xylanolytic enzymes was investigated in 12 filamentous fungi from ...

  6. The significance of cellulolytic enzymes produced by Trichoderma in opportunistic lifestyle of this fungus.

    Science.gov (United States)

    Strakowska, Judyta; Błaszczyk, Lidia; Chełkowski, Jerzy

    2014-07-01

    The degradation of native cellulose to glucose monomers is a complex process, which requires the synergistic action of the extracellular enzymes produced by cellulolytic microorganisms. Among fungi, the enzymatic systems that can degrade native cellulose have been extensively studied for species belonging to the genera of Trichoderma. The majority of the cellulolytic enzymes described so far have been examples of Trichoderma reesei, extremely specialized in the efficient degradation of plant cell wall cellulose. Other Trichoderma species, such as T. harzianum, T. koningii, T. longibrachiatum, and T. viride, known for their capacity to produce cellulolytic enzymes, have been isolated from various ecological niches, where they have proved successful in various heterotrophic interactions. As saprotrophs, these species are considered to make a contribution to the degradation of lignocellulosic plant material. Their cellulolytic potential is also used in interactions with plants, especially in plant root colonization. However, the role of cellulolytic enzymes in species forming endophytic associations with plants or in those existing in the substratum for mushroom cultivation remains unknown. The present review discusses the current state of knowledge about cellulolytic enzymes production by Trichoderma species and the encoding genes, as well as the involvement of these proteins in the lifestyle of Trichoderma. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Biosynthesis of cellulolytic enzymes by Tricothecium roseum with ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-11-05

    Nov 5, 2008 ... good inducer for extracellular cellulolytic enzyme production by the fungus. Key words: Tricothecium ... feasible for the conversion of cellulose into fermentable sugars and fuel ... Biomass of the culture was dried at 70°C in an ...

  8. [Isolation and identification of rumen bacteria for cellulolytic enzyme production].

    Science.gov (United States)

    Aihemaiti, Maierhaba; Zhen, Fan; Li, Yuezhong; Aibaidoula, Gulisimayi; Yimit, Wusiman

    2013-05-04

    We screened aerobic bacteria with cellulolytic activity from ruminal fluid of sheep, cattle and camel in Xinjiang. Fresh ruminal fluid was inoculated on sterilized sodium carboxymethylcellulose agar plates. Highly cellulolytic aerobic bacteria were screened out by using Congo red staining and liquid secondary screening culture media. The combination of morphological and biochemical test with 16SrDNA sequence analysis were used to classify the strains. Enzymatic activities of four strains with strong cellulose-decomposing abilities were studied under different culture conditions. Out 84 isolated cellulolytic strains, 40 exhibited strong abilities in decomposing cellulose. They are including 37 Gram-negative isolates and 3 Gram-positive strains. Identification of these 40 strains shows that they belong to 11 species of 6 genera, 16 strains in Stenotrophomonas maltophilia, 10 Ochrobactrum, 5 Sphingobacterium, 3 Microbacterium, 3 Paracoccus and 2 Pseudomonas. The results of the enzymatic studies of four strains with strong cellulolytic abilities indicates that the strains have the best enzyme producing property when straw powder was chosen as the carbon source; the pH at 5.5 -6.0 and temperature at 37 degrees C. The strains with highly cellulolytic abilities isolated from ruminal fluid show strong abilities in cellulose decomposition.

  9. Production of cellulolytic enzymes by Aspergillus fumigatus

    Energy Technology Data Exchange (ETDEWEB)

    Trivedi, L S; Rao, K K

    1979-01-01

    Production of extracellular cellulase by A. fumigatus was studied in liquid shake culture. The effect of culture conditions such as C and N source and pH on cellulase was examined. Sequential appearance of cellulase components, beta-glucosidase on the 2nd day, followed by endo-beta-glucanase on the 4th day and exobeta-gluconase on the 6th day of growth was observed. Maximum production of all cellulase components was achieved on the 12th day of growth in basal medium containing cellulose as C source and a combination of (NH4)2S04 and NH4H2PO4 as N source. High cellulolytic activities were observed only in the presence of insoluble cellulose as C source, while no significant activities were observed in the presence of simple sugars.

  10. Anaerobic gut fungi: Advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production.

    Science.gov (United States)

    Haitjema, Charles H; Solomon, Kevin V; Henske, John K; Theodorou, Michael K; O'Malley, Michelle A

    2014-08-01

    Anaerobic gut fungi are an early branching family of fungi that are commonly found in the digestive tract of ruminants and monogastric herbivores. It is becoming increasingly clear that they are the primary colonizers of ingested plant biomass, and that they significantly contribute to the decomposition of plant biomass into fermentable sugars. As such, anaerobic fungi harbor a rich reservoir of undiscovered cellulolytic enzymes and enzyme complexes that can potentially transform the conversion of lignocellulose into bioenergy products. Despite their unique evolutionary history and cellulolytic activity, few species have been isolated and studied in great detail. As a result, their life cycle, cellular physiology, genetics, and cellulolytic metabolism remain poorly understood compared to aerobic fungi. To help address this limitation, this review briefly summarizes the current body of knowledge pertaining to anaerobic fungal biology, and describes progress made in the isolation, cultivation, molecular characterization, and long-term preservation of these microbes. We also discuss recent cellulase- and cellulosome-discovery efforts from gut fungi, and how these interesting, non-model microbes could be further adapted for biotechnology applications. © 2014 Wiley Periodicals, Inc.

  11. Adsorption of cellulase on cellulolytic enzyme lignin from lodgepole pine.

    Science.gov (United States)

    Tu, Maobing; Pan, Xuejun; Saddler, Jack N

    2009-09-09

    Enzymatic hydrolysis of lignocellulosic materials is significantly affected by cellulase adsorption onto the lignocellulosic substrates and lignin. The presence of lignin plays an important role in lignocellulosic hydrolysis and enzyme recycling. Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their adsorption onto cellulolytic enzyme lignin (CEL) from steam-exploded Lodgepole pine (SELP) and ethanol (organosolv)-pretreated Lodgepole pine (EPLP). The adsorption affinity of cellulase (Celluclast) onto isolated lignin (CEL-EPLP and CEL-SELP) was slightly higher than that from corresponding EPLP and SELP substrates on the basis of the Langmuir constants. Effects of temperature, ionic strength, and surfactant on cellulase adsorption onto isolated lignin were also explored in this study. Thermodynamic analysis of enzyme adsorption onto isolated lignin (Gibbs free energy change DeltaG(0) approximately -30 kJ/mol) indicated this adsorption was a spontaneous process. The addition of surfactant (0.2% w/v) could reduce the adsorption of cellulase onto CEL-SELP by 60%. Two types of adsorption isotherm were compared for cellulase adsorption onto isolated lignin. A Langmuir adsorption isotherm showed better fit for the experimental data than a Freundlich adsorption isotherm.

  12. Cellulolytic and xylanolytic enzymes from thermophilic Aspergillus terreus RWY.

    Science.gov (United States)

    Sharma, Reetika; Kocher, Gurvinder Singh; Bhogal, Ravinder Singh; Oberoi, Harinder Singh

    2014-12-01

    Thermophilic Aspergillus terreus RWY produced cellulases and xylanases in optimal concentrations at 45 °C in solid state fermentation process, though enzyme production was also observed at 50 and 55 °C. Filter paper cellulase (FP), endoglucanase (EG), β-glucosidase (BGL), cellobiohydrolase (CBH), xylanase, β-xylosidase, α-L-arabinofuranosidase and xylan esterase activities for A. terreus RWY at 45 °C in 72 h were 11.3 ± 0.65, 103 ± 6.4, 122.5 ± 8.7, 10.3 ± 0.66, 872 ± 22.5, 22.1 ± 0.75, 126.4 ± 8.4 and 907 ± 15.5 U (g-ds)(-1) , respectively. Enzyme was optimally active at temperatures and pH ranging between 50-60 °C and 4.0-6.0, respectively. The half life (T1/2 ) of 270 and 240 min at 70 and 75 °C, respectively for the enzyme indicates its stability at higher temperatures. The addition of MnCl2 , CoCl2 , and FeCl3 significantly enhanced cellulase activity. Enzyme demonstrated multiplicity by having seven, one and three isoform(s) for EG, CBH and BGL, respectively. Significant production of functionally active consortium of cellulolytic and xylanolytic enzymes from A. terreus RWY makes it a potential candidate in bioprocessing applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Reactivity improvement of cellulolytic enzyme lignin via mild hydrothermal modification.

    Science.gov (United States)

    Ma, Zhuoming; Tang, Jiafa; Li, Shujun; Suo, Enxiang

    2017-12-01

    Isolated by the cellulolytic enzyme lignin (CEL) process, water-alcohol (1:1, v/v) was introduced as co-solvent in the process of the hydrothermal treatment. The modification parameters such as reaction temperature and time, solid-to-liquid ratio, and catalysts (NaOH and NaOAlO 2 ) have been investigated in terms of the specific lignin properties, such as the phenolic hydroxyl content (OH phen ), DPPH free radical scavenging rate, and formaldehyde value. The CELs were also characterized by GPC, FT-IR and 1 H NMR spectroscopy, and Py-GC/MS. The key data are under optimal lignin modification conditions (solid-to-liquid ratio of 1:10 (w/v) and a temperature of 250°C for 60min) are: OH phen content: 2.50mmol/g; half maximal inhibitory concentration (IC 50 ) towards DPPH free radicals: 88.2mg/L; formaldehyde value: 446.9g/kg). Both base catalysts decrease the residue rate, but phenol reactivities of the products were also detracted. Py-GC/MS results revealed that modified lignin had a higher phenolic composition than the CEL did, especially the modified lignin without catalyst (ML), which represented 74.51% phenolic content. Copyright © 2017. Published by Elsevier Inc.

  14. Compositions for enhancing hydroysis of cellulosic material by cellulolytic enzyme compositions

    Science.gov (United States)

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew; Johansen, Katja Salomon

    2014-09-30

    The present invention relates to compositions comprising a GH61 polypeptide having cellulolytic enhancing activity and an organic compound comprising a carboxylic acid moiety, a lactone moiety, a phenolic moiety, a flavonoid moiety, or a combination thereof, wherein the combination of the GH61 polypeptide having cellulolytic enhancing activity and the organic compound enhances hydrolysis of a cellulosic material by a cellulolytic enzyme compared to the GH61 polypeptide alone or the organic compound alone. The present invention also relates to methods of using the compositions.

  15. Production of cellulolytic enzymes by fungal cultures. [Aspergillus, Trichoderma, Chaetomium, Stachybotrys, and Hypocrea

    Energy Technology Data Exchange (ETDEWEB)

    Pyc, R; Fiechter, A. Galas, E.

    1977-01-01

    Twelve fungal cultures belonging to the genera of Aspergillus, Trichoderma, Chaetomium, Stachybotrys, and Hypocrea were screened for the production of cellulolytic activity. All twelve were found to degrade xylan, avicel, and carboxymethylcellulose. More cellulolytic activity was obtained with shaken cultures than with still cultures and the addition of citrate-phosphate buffer to the media greatly depressed the levels of cellulolytic activity. Varying the composition of the mineral salts in the medium had no effect on the cellulolytic activity. The growth of Aspergillus wentii under controlled conditions in a bioreactor showed that the cellulolytic activity was not affected by the aeration rate or the type of stirrer. The rate of stirring, however, did effect the cellulolytic activity, as at lower stirring speeds considerable wall growth occurred which resulted in low levels of cellulolytic activity. Culture supernatant from Aspergillus wentii was found to hydrolyze from 30-32% of Solka-Floc and from 2-10% of corn cobs, wheat straw, and newsprint. The extensive hydrolysis of Solka-Floc indicates that with suitable treated cellulosic wastes and appropriate enzymes, appreciable amounts of sugars could be obtained.

  16. Enzymatic activity of the cellulolytic complex produced by trichoderma reesei. Enzymatic hydrolysis of cellulose

    International Nuclear Information System (INIS)

    Alfonsel Jaen, M.; Negro, M.J.; Saez, R.; Martin Moreno, C.

    1986-01-01

    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reese QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass from Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars productions, have been selected. Previous studies on enzymatic hydrolysis of O. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (author). 10 figs.; 10 refs

  17. Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose

    International Nuclear Information System (INIS)

    Alfonsel J, M.; Negro A, M. J.; Saez A, R.; Martin M, C.

    1986-01-01

    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs

  18. Multifunctional Cellulolytic Enzymes Outperform Processive Fungal Cellulases for Coproduction of Nanocellulose and Biofuels.

    Science.gov (United States)

    Yarbrough, John M; Zhang, Ruoran; Mittal, Ashutosh; Vander Wall, Todd; Bomble, Yannick J; Decker, Stephen R; Himmel, Michael E; Ciesielski, Peter N

    2017-03-28

    Producing fuels, chemicals, and materials from renewable resources to meet societal demands remains an important step in the transition to a sustainable, clean energy economy. The use of cellulolytic enzymes for the production of nanocellulose enables the coproduction of sugars for biofuels production in a format that is largely compatible with the process design employed by modern lignocellulosic (second generation) biorefineries. However, yields of enzymatically produced nanocellulose are typically much lower than those achieved by mineral acid production methods. In this study, we compare the capacity for coproduction of nanocellulose and fermentable sugars using two vastly different cellulase systems: the classical "free enzyme" system of the saprophytic fungus, Trichoderma reesei (T. reesei) and the complexed, multifunctional enzymes produced by the hot springs resident, Caldicellulosiruptor bescii (C. bescii). We demonstrate by comparative digestions that the C. bescii system outperforms the fungal enzyme system in terms of total cellulose conversion, sugar production, and nanocellulose production. In addition, we show by multimodal imaging and dynamic light scattering that the nanocellulose produced by the C. bescii cellulase system is substantially more uniform than that produced by the T. reesei system. These disparities in the yields and characteristics of the nanocellulose produced by these disparate systems can be attributed to the dramatic differences in the mechanisms of action of the dominant enzymes in each system.

  19. Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

    Directory of Open Access Journals (Sweden)

    R. Navanietha Krishnaraj

    2017-04-01

    Full Text Available Cellulolytic enzymes are promising candidates for the use of cellulose in any bioprocess operations and for the disposal of the cellulosic wastes in an environmentally benign manner. Cellulases from thermophiles have the advantage of hydrolyzing cellulose at wider range of operating conditions unlike the normal enzymes. Herein we report the modeled structures of cellulolytic enzymes (endoglucanase, cellobiohydrolase and ß-glucosidase from a thermophilic bacterium,Clostridium thermocellumand their validation using Root Mean Square Deviation (RMSD and Ramachandran plot analyses. Further, the molecular interactions of the modeled enzyme with cellulose were analyzed using molecular docking technique. The results of molecular docking showed that the endoglucanase, cellobiohydrolase and ß-glucosidase had the binding affinities of -10.7, -9.0 and -10.8 kcal/mol, respectively. A correlation between the binding affinity of the endoglucanase with cellulose and the enzyme activity was also demonstrated. The results showed that the binding affinities of cellulases with cellulose could be used as a tool to assess the hydrolytic activity of cellulases. The results obtained could be used in virtual screening of cellulolytic enzymes based on the molecular interactions with the substrate, and aid in developing systems biology models of thermophiles for industrial biotechnology applications.

  20. Evaluation of Myceliopthora thermophila as an Enzyme Factory for the Production of Thermophilic Cellulolytic Enzymes

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    Leonidas Matsakas

    2015-07-01

    Full Text Available Enzymatic hydrolysis is a key step in bioethanol production. Efficient hydrolysis requires a consortium of different enzymes that are able to hydrolyze cellulose and hemicellulose into fermentable sugars. Myceliopthora thermophila is a promising candidate for the production of thermophilic cellulolytic enzymes, the use of which could reduce the cost of ethanol production. The growth conditions of the fungus were optimized in order to achieve increased secretion of extracellular cellulases. Optimal conditions were found to be 7.0% w/v brewer’s spent grain as the carbon source and 0.4% w/v ammonium sulfate as the nitrogen source. The cellulases obtained were characterized for their optimum activity. The optimum temperature and pH for cellulase activity are 65 °C and pH 5.5, respectively. Studies on thermal inactivation of the crude extract showed that the cellulases of M. thermophila are stable for temperatures up to 60 °C. At this temperature the half-life was found to be as high as 27 h. Enzymatic hydrolysis of cellulose resulted in 31.4% hydrolysis yield at 60 °C after 24 h of incubation. Finally, the recalcitrance constant for cellulose and cellulose pretreated with ionic liquids was calculated to be 5.46 and 2.69, respectively.

  1. Temporal variations in microbial biomass C and cellulolytic enzyme activity in arable soils: effects of organic matter input

    DEFF Research Database (Denmark)

    Debosz, K.; Rasmussen, Peter Have; Pedersen, A. R.

    1999-01-01

    Temporal variations in soil microbial biomass C concentration and in activity of extracellular enzymes of the cellulolytic complex were investigated in a field experiment after eight years of cultivation with either low organic matter input (low-OM) or high organic matter input (high-OM). The cul......Temporal variations in soil microbial biomass C concentration and in activity of extracellular enzymes of the cellulolytic complex were investigated in a field experiment after eight years of cultivation with either low organic matter input (low-OM) or high organic matter input (high......-OM). The cultivation systems differed in whether their source of fertiliser was mainly mineral or organic, in whether a winter cover crop was grown, and whether straw was mulched or removed. Sampling occurred at approximately monthly intervals, over a period of two years. Distinct temporal variations in microbial......) and an endocellulase activity of 44.2 +/- 1.1 nmol g(-1) h(-1). (C) 1999 Elsevier Science B.V. All rights reserved....

  2. Hyperthermostable cellulolytic and hemicellulolytic enzymes and their biotechnological applications

    Directory of Open Access Journals (Sweden)

    Tipparat Hongpattarakere

    2002-07-01

    Full Text Available Hyperthermal cellulases and hemicellulases have been intensively studied due to their highly potential applications at extreme temperatures, which mimic industrial processes involving cellulose and hemicellulose degradation. More than 50 species of hyperthermophiles have been isolated, many of which possess hyperthermal enzymes required for hydrolyzing cellulose and hemicelluloses. Endoglucanases, exoglucanases, cellobiohydrolases, xylanases, β-glucosidase and β-galactosidase, which are produced by the hyperthermophiles, are resistant to boiling temperature. The characteristics of these enzymes and the ability to maintain their functional integrity at high temperature as well as their biotechnological application are discussed.

  3. Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

    Energy Technology Data Exchange (ETDEWEB)

    Vaheri, M.P.; Vaheri, M.E.O.; Kaupinen, V.S.

    1979-01-01

    Production and release of cellulolytic enzymes by T. reesei QM 9414 were studied under induced and non-induced conditions and glycerol, respectively, as the only C source. There was a base level of cell debris-bound hydrolytic activity against filter paper and p-nitrophenyl glycoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper- and CMC-hydrolyzing enzymes, which were actively released even in the early stages of cultivation. Beta-Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.

  4. Nutrient effects on production of cellulolytic enzymes by Aspergillus ...

    African Journals Online (AJOL)

    glucosidase) by Aspergillus niger on three media in liquid shake culture was compared. The culture filtrate of this organism exhibited relatively highest activity of all three enzymes and extracellular protein content at 7-day interval during the course of its ...

  5. Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

    2012-01-24

    The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  6. Thermostable amylolytic enzymes from a cellulolytic fungus Myceliophthora thermophila D14 (ATCC 48 104)

    Energy Technology Data Exchange (ETDEWEB)

    Sadhukhan, R K; Manna, S; Roy, S K; Chakrabarty, S L [Bose Research Inst., Calcutta (India). Dept. of Microbiology

    1990-09-01

    The production of amylolytic enzymes by a thermophilic cellulolytic fungus, Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45deg C. Among various nitrogenous compounds used, NaNO{sub 3} and KNO{sub 3} were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50deg C-60deg C) and displayed activity optima at 60deg C and pH 5.6. (orig.).

  7. Isolation of Cellulolytic Bacteria and Characterization of the Enzyme

    Directory of Open Access Journals (Sweden)

    Nisa Rachmania

    2009-04-01

    Full Text Available Four of cellulolitic bacteria isolates had beencharacterized. The determination of cellulase activity was conducted at the highest production time, using crudeenzymes with the modification of Miller methods (1959 on pure cellulose substrates such as CMC (Carboxymethylcellulose, Avicel and Filter paper Whatman No. 1 as well as agriculture waste such as rice straw, corn cob and bananapeel. Cellulase from C4-4, C5-1, C5-3 and C11-1 showed optimum activity at pH 5, 70°C, pH 3.5, 90°C, pH 5, 80°Cand pH 8, 70°C, respectively. Avicel is a appropriate substrate for C4-4 cellulase whereas CMC for the other three.C11-1 cellulase has the highest cellulase enzyme activity on rice straw substrate whereas C4-4 cellulase on banana peelsubstrates. C5-1 and C5-3 cellulase have relatively low cellulase activities in degrading substrates of agriculture waste.However, isolates of C5-1 and C5-3 have high cellulase activities on banana peel substrates.

  8. Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect β-N-acetyl-D-hexosaminidase.

    Directory of Open Access Journals (Sweden)

    Tian Liu

    Full Text Available The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp(490, Glu(328, Val(327 in OfHex1, and Trp(358, Tyr(131 and Ile(179 in BGlu1 were identified as being conserved in the +1 sugar binding site. OfHex1 Glu(328 together with Trp(490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K(m for (GlcNAc(2 and a 42-fold increment in K(i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu(368 and Asp(367 and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu(328 and Val(327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.

  9. Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall.

    Science.gov (United States)

    Dongowski, G; Sembries, S

    2001-09-01

    The action of three different commercial enzyme combinations on apple cell wall material has been examined in a model system under conditions of mash and pomace treatment by using an alcohol-insoluble substance prepared from apples. A part of the total dietary fiber, for example, galacturonan (pectin), appeared in the soluble fraction after enzymatic mash treatment. The soluble fraction increased intensely during pomace treatment. Furthermore, enzyme actions caused a change in the water-binding capacity of residues as well as changes in the monosaccharide composition and in the molecular weight distribution of saccharides in filtrates (soluble parts). The extent of decomposition of cell wall material and the increase of soluble oligomeric and/or polymeric dietary fiber components are caused by both the composition (pectinases, cellulases, and hemicellulases) and the activities of the enzyme preparations. The model experiments allow an insight into the reactions occurring during enzyme action on the plant cell wall, for example, during apple juice production using pectolytic and cellulolytic enzyme preparations.

  10. Biosynthesis of cellulolytic enzymes by Aspergillus niger A. n. 33 and its selectants

    Energy Technology Data Exchange (ETDEWEB)

    Czajkowska, D.; Hornecka, D.; Ilnicka-Olejniczak, O.

    1988-01-01

    The aim of the investigations was to obtain - from the parent strain Aspergillus niger A.n. 33 - selectants with an increased ability of cellulolytic enzymes biosynthesis. Own selection methods allowed to receive two selectants A.n. 33/2 and A.n. 33/20 characterized by enhanced activities of saccharifying cellulase (respectively 0.11 and 0.14 FPU/cm/sup 3/), endo-beta-1,4-glucanase (15.4 and 21.8 U/cm/sup 3/) and cellobiase (0.6 and 1.4 IU/cm/sup 3/) as compared with the parent strain (FPA - 0.09 IU, CMC - 8.2 U and CB - 0.1 IU/cm/sup 3/). Moreover, the selectants differed in shape and size of conidial heads, in shape and colour of conidia, as well as in structure and shape of hyphase. Enzyme preparations obtained after ultrafiltration of liquid cultures were characterized by following activities: FPA-4-16 IU, CMC-900-1800 U, CB-60-120 IU and xylanase-250-280 IU/cm/sup 3/.

  11. Temperature and pH optima of enzyme activities produced by cellulolytic thermophilic fungi in batch and solid-state cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grajek, W

    1986-01-01

    The temperature and pH optima of cellulolytic activities produced by thermophilic fungi in liquid and solid-state cultures were established. Some differences in optimal conditions for enzyme activities, which depended on culture methods, were confirmed. 10 references.

  12. Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

    Science.gov (United States)

    Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

    2015-03-01

    Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

  13. Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production.

    Science.gov (United States)

    Garcia-Kirchner, O; Muñoz-Aguilar, M; Pérez-Villalva, R; Huitrón-Vargas, C

    2002-01-01

    The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, beta-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly beta-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29 degrees C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.

  14. Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain.

    Science.gov (United States)

    Mattam, Anu Jose; Kuila, Arindam; Suralikerimath, Niranjan; Choudary, Nettem; Rao, Peddy V C; Velankar, Harshad Ravindra

    2016-01-01

    Lignocellulosic ethanol production involves major steps such as thermochemical pretreatment of biomass, enzymatic hydrolysis of pre-treated biomass and the fermentation of released sugars into ethanol. At least two different organisms are conventionally utilized for producing cellulolytic enzymes and for ethanol production through fermentation, whereas in the present study a single yeast isolate with the capacity to simultaneously produce cellulases and xylanases and ferment the released sugars into ethanol and xylitol has been described. A yeast strain isolated from soil samples and identified as Candida tropicalis MTCC 25057 expressed cellulases and xylanases over a wide range of temperatures (32 and 42 °C) and in the presence of different cellulosic substrates [carboxymethylcellulose and wheat straw (WS)]. The studies indicated that the cultivation of yeast at 42 °C in pre-treated hydrolysate containing 0.5 % WS resulted in proportional expression of cellulases (exoglucanases and endoglucanases) at concentrations of 114.1 and 97.8 U g(-1) ds, respectively. A high xylanase activity (689.3 U g(-1) ds) was also exhibited by the yeast under similar growth conditions. Maximum expression of cellulolytic enzymes by the yeast occurred within 24 h of incubation. Of the sugars released from biomass after pretreatment, 49 g L(-1) xylose was aerobically converted into 15.8 g L(-1) of xylitol. In addition, 25.4 g L(-1) glucose released after the enzymatic hydrolysis of biomass was fermented by the same yeast to obtain an ethanol titer of 7.3 g L(-1). During the present study, a new strain of C. tropicalis was isolated and found to have potential for consolidated bioprocessing (CBP) applications. The strain could grow in a wide range of process conditions (temperature, pH) and in the presence of lignocellulosic inhibitors such as furfural, HMF and acetic acid. The new yeast produced cellulolytic enzymes over a wide temperature range and in the presence of

  15. Linking Hydrolysis Performance to Trichoderma reesei Cellulolytic Enzyme Profile

    DEFF Research Database (Denmark)

    Lehmann, Linda Olkjær; Petersen, Nanna; I. Jørgensen, Christian

    2016-01-01

    Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, e.g. by spiking with single enzymes and monitoring hydrolysis performance. In this study a multivariate...... approach, partial least squares regression, was used to see if it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by Trichoderma reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed...

  16. Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose; Actividad enzimatica del complejo celulolitico producido por Trichoderma reesei. Hidrolisis enzimatica de la celulosa

    Energy Technology Data Exchange (ETDEWEB)

    Alfonsel, M; Negro, M J; Saez, R; Martin, C

    1986-07-01

    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs.

  17. Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1.

    Science.gov (United States)

    Lo, Yung-Chung; Huang, Chi-Yu; Cheng, Chieh-Lun; Lin, Chiu-Yue; Chang, Jo-Shu

    2011-09-01

    A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Interactions between Cellulolytic Enzymes with Native, Autohydrolysis, and Technical Lignins and the Effect of a Polysorbate Amphiphile in Reducing Nonproductive Binding.

    Science.gov (United States)

    Fritz, Consuelo; Ferrer, Ana; Salas, Carlos; Jameel, Hasan; Rojas, Orlando J

    2015-12-14

    Understanding enzyme-substrate interactions is critical in designing strategies for bioconversion of lignocellulosic biomass. In this study we monitored molecular events, in situ and in real time, including the adsorption and desorption of cellulolytic enzymes on lignins and cellulose, by using quartz crystal microgravimetry and surface plasmon resonance. The effect of a nonionic surface active molecule was also elucidated. Three lignin substrates relevant to the sugar platform in biorefinery efforts were considered, namely, hardwood autohydrolysis cellulolytic (HWAH), hardwood native cellulolytic (MPCEL), and nonwood native cellulolytic (WSCEL) lignin. In addition, Kraft lignins derived from softwoods (SWK) and hardwoods (HWK) were used as references. The results indicated a high affinity between the lignins with both, monocomponent and multicomponent enzymes. More importantly, the addition of nonionic surfactants at concentrations above their critical micelle concentration reduced remarkably (by over 90%) the nonproductive interactions between the cellulolytic enzymes and the lignins. This effect was hypothesized to be a consequence of the balance of hydrophobic and hydrogen bonding interactions. Moreover, the reduction of surface roughness and increased wettability of lignin surfaces upon surfactant treatment contributed to a lower affinity with the enzymes. Conformational changes of cellulases were observed upon their adsorption on lignin carrying preadsorbed surfactant. Weak electrostatic interactions were determined in aqueous media at pH between 4.8 and 5.5 for the native cellulolytic lignins (MPCEL and WSCEL), whereby a ∼20% reduction in the enzyme affinity was observed. This was mainly explained by electrostatic interactions (osmotic pressure effects) between charged lignins and cellulases. Noteworthy, adsorption of nonionic surfactants onto cellulose, in the form cellulose nanofibrils, did not affect its hydrolytic conversion. Overall, our results

  19. Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Yanase, Shuhei; Yamada, Ryosuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Hasunuma, Tomohisa; Tanaka, Tsutomu; Fukuda, Hideki [Kobe Univ. (Japan). Organization of Advanced Science and Technology

    2010-09-15

    To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 C and 37 C, while the activity of cellulolytic enzymes is highest at around 50 C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus {beta}-glucosidase on the cell surface, which successfully converts a cellulosic {beta}-glucan to ethanol directly at 48 C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of {beta}-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface. (orig.)

  20. Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

    DEFF Research Database (Denmark)

    Andric, Pavle; Meyer, Anne S.; Jensen, Peter Arendt

    2010-01-01

    Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and glucose...... on enzyme-catalyzed cellulose hydrolysis reactions impose significant limitations on the efficiency of lignocellulose conversion especially at high-biomass dry matter conditions. To provide the base for selecting the optimal reactor conditions, this paper reviews the reaction kinetics, mechanisms......, and significance of this product inhibition, notably the cellobiose and glucose inhibition, on enzymatic cellulose hydrolysis. Particular emphasis is put on the distinct complexity of cellulose as a substrate, the multi-enzymatic nature of the cellulolytic degradation, and the particular features of cellulase...

  1. Cellulolytic Enzymes Production via Solid-State Fermentation: Effect of Pretreatment Methods on Physicochemical Characteristics of Substrate

    Directory of Open Access Journals (Sweden)

    Khushal Brijwani

    2011-01-01

    Full Text Available We investigated the effect of pretreatment on the physicochemical characteristics—crystallinity, bed porosity, and volumetric specific surface of soybean hulls and production of cellulolytic enzymes in solid-state fermentation of Trichoderma reesei and Aspergillus oryzae cultures. Mild acid and alkali and steam pretreatments significantly increased crystallinity and bed porosity without significant change inholocellulosic composition of substrate. Crystalline and porous steam-pretreated soybean hulls inoculated with T. reesei culture had 4 filter paper units (FPU/g-ds, 0.6 IU/g-ds β-glucosidase, and 45 IU/g-ds endocellulase, whereas untreated hulls had 0.75 FPU/g-ds, 0.06 IU/g-ds β-glucosidase, and 7.29 IU/g-ds endocellulase enzyme activities. In A. oryzae steam-pretreated soybean hulls had 47.10 IU/g-ds endocellulase compared to 30.82 IU/g-ds in untreated soybean hulls. Generalized linear statistical model fitted to enzyme activity data showed that effects of physicochemical characteristics on enzymes production were both culture and enzyme specific. The paper shows a correlation between substrate physicochemical properties and enzyme production.

  2. Cellulolytic Enzymes Production via Solid-State Fermentation: Effect of Pretreatment Methods on Physicochemical Characteristics of Substrate.

    Science.gov (United States)

    Brijwani, Khushal; Vadlani, Praveen V

    2011-01-01

    We investigated the effect of pretreatment on the physicochemical characteristics-crystallinity, bed porosity, and volumetric specific surface of soybean hulls and production of cellulolytic enzymes in solid-state fermentation of Trichoderma reesei and Aspergillus oryzae cultures. Mild acid and alkali and steam pretreatments significantly increased crystallinity and bed porosity without significant change inholocellulosic composition of substrate. Crystalline and porous steam-pretreated soybean hulls inoculated with T. reesei culture had 4 filter paper units (FPU)/g-ds, 0.6 IU/g-ds β-glucosidase, and 45 IU/g-ds endocellulase, whereas untreated hulls had 0.75 FPU/g-ds, 0.06 IU/g-ds β-glucosidase, and 7.29 IU/g-ds endocellulase enzyme activities. In A. oryzae steam-pretreated soybean hulls had 47.10 IU/g-ds endocellulase compared to 30.82 IU/g-ds in untreated soybean hulls. Generalized linear statistical model fitted to enzyme activity data showed that effects of physicochemical characteristics on enzymes production were both culture and enzyme specific. The paper shows a correlation between substrate physicochemical properties and enzyme production.

  3. PRODUCTION AND CHARACTERIZATION OF CELLULOLYTIC ENZYMES BY ASPERGILLUS NIGER AND RHIZOPUS SP . BY SOLID STATE FERMENTATION OF PRICKLY PEAR

    Directory of Open Access Journals (Sweden)

    TAMIRES CARVALHO DOS SANTOS

    2016-01-01

    Full Text Available Prickly palm cactus husk was used as a solid - state fermentation support substrate for the production of cellulolytic enzymes using Aspergillus niger and Rhizopus sp. A Box - Behnken design was used to evaluate the effects of water activity, fermentation time and temperature on endoglucanase and total cellulase production. Response Surface Methodology showed that optimum conditions for endoglucanase production were achieved at after 70.35 h of fermentation at 29.56°C and a water activity of 0.875 for Aspergillus niger and after 68.12 h at 30.41°C for Rhizopus sp. Optimum conditions for total cellulase production were achieved after 74.27 h of fermentation at 31.22°C for Aspergillus niger and after 72.48 h and 27.86°C for Rhizopus sp . Water activity had a significant effect on Aspergillus niger endoglucanase production only. In industrial applications, enzymatic characterization is important for optimizing variables such as temperature and pH. In this study we showed that endoglucanase and total cellulase had a high level of thermostability and pH stability in all the enzymatic extracts. Enzymatic deactivation kinetic experiments indicated that the enzymes remained active after the freezing of the crude extract. Based on the results, bioconversion of cactus is an excellent alternative for the production of thermostable enzymes.

  4. Characterising Complex Enzyme Reaction Data.

    Directory of Open Access Journals (Sweden)

    Handan Melike Dönertaş

    Full Text Available The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG. Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution.

  5. Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia

    Directory of Open Access Journals (Sweden)

    W. P. Lokapirnasari

    2015-03-01

    Full Text Available Aim: This study aims to produce and assay cellulolytic enzyme activity (endo-(1,4-β-D-glucanase, exo-(1,4-β-Dglucanase, and β-glucosidase, at optimum temperature and optimum pH of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. Materials and Methods: To produce enzyme from a single colony of E. cloacae WPL 214, 98 × 1010 CFU/ml of isolates was put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C in accordance with growth time and optimum temperature of E. cloacae WPL 214. Further on, culture was centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulose enzyme activity was withdrawn to assay endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase, and β-glucosidase. Results: Cellulase enzyme of E. cloacae WPL 214 isolates had endoglucanase activity of 0.09 U/ml, exoglucanase of 0.13 U/ml, and cellobiase of 0.10 U/ml at optimum temperature 35°C and optimum pH 5. Conclusion: E. cloacae WPL 214 isolated from bovine rumen fluid waste produced cellulose enzyme with activity as cellulolytic enzyme of endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase and β-glucosidase.

  6. Production of Cellulolytic and Hemicellulolytic Enzymes From Aureobasidium pulluans on Solid State Fermentation

    Science.gov (United States)

    Leite, Rodrigo Simões Ribeiro; Bocchini, Daniela Alonso; da Silva Martins, Eduardo; Silva, Dênis; Gomes, Eleni; da Silva, Roberto

    This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme remaining 100% active when incubated at 75°C for 1 h.

  7. Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production?

    DEFF Research Database (Denmark)

    Cannella, David; Jørgensen, Henning

    2014-01-01

    proven essential for economic feasibility at industrial scale. Historically, simultaneous saccharification and fermentation (SSF) was found to give better ethanol yields compared to separate hydrolysis and fermentation (SHF), but data in literature are typically based on operating the process at low dry...... matter conditions. In this work the impact of selected enzyme preparation and processing strategy (SHF, presaccharification and simultaneous saccharification and fermentation—PSSF, and SSF) on final ethanol yield and overall performance was investigated with pretreated wheat straw up to 30% DM...... cellulose to around 94%, revealing that the most relevant products could be accounted for. One observation was the presence of oxidized sugar (gluconic acid) upon enzymatic hydrolysis with the latest enzyme preparation. Experiments showed gluconic acid formation by recently discovered enzymatic class...

  8. Production and characterization of cellulolytic enzymes from Trichoderma reesei grown on various carbon sources

    Energy Technology Data Exchange (ETDEWEB)

    Warzywoda, Michel; Labre, Elisabeth; Pourquie, Jacques [Institut Francais du Petrole (IFP), 92 - Rueil-Malmaison (France)

    1992-01-01

    Ethanol production from lignocellulosics is considered, using a process in which biomass is first pretreated by steam explosion, yielding freely water-extractible pentoses and a cellulose-rich residue which can be further hydrolyzed by cellulases into glucose to be fermented into ethanol. Results that are reported show that both the pentose extracts and the glucose-rich hydrolyzates can be used as carbon sources for cellulase production by Trichoderma reesei. When compared with lactose as the main carbon source, pentose extracts support lower but satisfactory protein productions which are characterized by an increase in hemicellulolytic activities, which significantly improves the saccharifying potential of these enzyme preparations. (author).

  9. Biochemical characterization of thermostable cellulase enzyme from ...

    African Journals Online (AJOL)

    user

    2012-05-29

    May 29, 2012 ... tested for their ability to produce cellulase complex enzyme by growing on a defined substrates as well ... In the current industrial processes, cellulolytic enzymes ... energy sources such as glucose, ethanol, hydrogen and.

  10. Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

    Energy Technology Data Exchange (ETDEWEB)

    Saddler, J N

    1982-11-01

    Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase (1,4-(1,3;1,4)-beta-D-glucan 4- glucanohydrolase, EC 3.2.1.4) activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) activity was approximately six to nine times greater. (Refs. 26).

  11. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

    2013-12-01

    Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

  12. Composition of cellulase complex of Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Golovchenko, N P; Chuvil' skaya, N A; Akimenko, V K

    1985-01-01

    It is thought that the anaerobic thermophilic cellulolytic bacterium C. thermocellum has the potential for direct industrial bioconversion of cellulose into ethanol. Therefore, much attention has been given to the study of the cellulolytic properties of the culture and to the characteristics of the cellulose complex, which is still not completely understood. Hence, the activity and location of various cellulolytic enzymes of C. thermocellum were determined. C. thermocellum has 6 known cellulolytic enzymes. Endoglucanase, cellobiohydrolase and exoglucosidase are extracellular enzymes (99-100 percent of the activity is located outside the cells) while cellulobiases, cellobiose phosphorylase and cellodextrine phosphorylase are inside the cells (80-90% of the activity). 25 references.

  13. Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

    Science.gov (United States)

    Teeravivattanakit, Thitiporn; Baramee, Sirilak; Phitsuwan, Paripok; Sornyotha, Somphit; Waeonukul, Rattiya; Pason, Patthra; Tachaapaikoon, Chakrit; Poomputsa, Kanokwan; Kosugi, Akihiko; Sakka, Kazuo; Ratanakhanokchai, Khanok

    2017-11-15

    Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii β-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment. IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It

  14. 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.

  15. Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

    Science.gov (United States)

    Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

    2010-12-10

    Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Composition and microstructure alteration of triticale grain surface after processing by enzymes of cellulase complex

    Directory of Open Access Journals (Sweden)

    Elena Kuznetsova

    2016-01-01

    Full Text Available It is found that the pericarp tissue of grain have considerable strength and stiffness, that has an adverse effect on quality of whole-grain bread. Thereby, there exists the need for preliminary chemical and biochemical processing of durable cell walls before industrial use. Increasingly used in the production of bread finds an artificial hybrid of the traditional grain crops of wheat and rye - triticale, grain which has high nutritional value. The purpose of this research was to evaluate the influence of cellulose complex (Penicillium canescens enzymes on composition and microstructure alteration of triticale grain surface, for grain used in baking. Triticale grain was processed by cellulolytic enzyme preparations with different composition (producer is Penicillium canescens. During experiment it is found that triticale grain processing by enzymes of cellulase complex leads to an increase in the content of water-soluble pentosans by 36.3 - 39.2%. The total amount of low molecular sugars increased by 3.8 - 10.5 %. Studies show that under the influence of enzymes the microstructure of the triticale grain surface is changing. Microphotographs characterizing grain surface structure alteration in dynamic (every 2 hours during 10 hours of substrate hydrolysis are shown. It is found that the depth and direction of destruction process for non-starch polysaccharides of grain integument are determined by the composition of the enzyme complex preparation and duration of exposure. It is found, that xylanase involved in the modification of hemicelluloses fiber having both longitudinal and radial orientation. Hydrolysis of non-starch polysaccharides from grain shells led to increase of antioxidant activity. Ferulic acid was identified in alcoholic extract of triticale grain after enzymatic hydrolysis under the influence of complex preparation containing cellulase, xylanase and β-glucanase. Grain processing by independent enzymes containing in complex

  17. Bioconversion process of rice straw by thermotolerant cellulolytic ...

    African Journals Online (AJOL)

    Administrator

    2011-09-26

    state fermentation for bioethanol production is a focus of current attention. ... Optimization of fermentation conditions showed highest cellulolytic enzymes ... using dilute acid pretreated rice straw hydrolysate with initial soluble ...

  18. An innovative approach for hyperproduction of cellulolytic and ...

    African Journals Online (AJOL)

    The present work describes the production of cellulolytic enzymes and hemicellulolytic enzyme (xylanase) along with total extracellular protein by Aspergillus niger and Trichoderma viride using submerged fermentation. Among seven different kinds of experiments, secretion rate of protein and enzymes was investigated by ...

  19. Characterization and Identification of Cellulolytic Bacteria from gut of Worker Macrotermes gilvus

    Directory of Open Access Journals (Sweden)

    Andri Ferbiyanto

    2015-10-01

    Full Text Available As a social insect, termite colony consists of three castes, i.e. reproductive, soldier, and worker castes. In their role of cellulose digestion, the worker termites use two sources of cellulolytic enzyme that include cellulases produced by the termite and the gut symbions. Macrotermes gilvus classified in mound builder termite, mostly depend on cellulolytic bacteria for cellulose digestion. This study aims to characterize cellulolytic bacteria of termite gut symbionts of worker M. gilvus and to identify the cellulolytic bacteria based on sequences of 16S ribosomal RNA (rRNA gene. Cellulolytic bacteria of termite gut were isolated and cultured in CMC (Carboxymethyl cellulose media. The biochemical characters of bacterial isolates were assayed using Microbact 12A and 12B. Cellulolytic activity was determined based on formation of clear zone and cellulolytic index on CMC plate media. The bacterial isolate that has the highest cellulolytic index was analyzed for its 16S rRNA gene sequences. Four isolates of cellulolytic bacteria were successfully isolated from gut of M. gilvus with aerobic and anaerobic conditions. The highest formation of cellulolytic index (2.5 was revealed by RA2. BLAST-N (Basic Local Alignment Search Tool for Nucleotides result of 16S rRNA gene sequences of RU4 and RA2 isolates showed that the isolate has similarity with Bacillus megaterium and Paracoccus yeei, respectively. This result indicated that RA2 isolate was P. yeei, a cellulolytic bacterium of a termite gut of M. gilvus.

  20. 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 ...

  1. Redistribution of mineral elements in wheat grain when applying the complex enzyme preparations based on phytase

    Directory of Open Access Journals (Sweden)

    Elena Kuznetsova

    2016-01-01

    Full Text Available Biogenic minerals play an important role in the whole human nutrition, but they are included in the grain of the phytates that reduces their bioavailability. Whole wheat bread is generally considered a healthy food, but the presence of mineral elements in it is insignificant, because of weak phytate degradation. From all sources of exogenous phytase the most productive are microscopic fungi. To accelerate the process of transition hard mineral elements are mobilized to implement integrated cellulolytic enzyme preparation based on the actions of phytase (producer is Penicillium canescens. Phytase activity was assessed indirectly by the rate of release of phosphate from the substrate. It has been established that the release rate of the phosphoric acid substrate is dependent on the composition of the drug and the enzyme complex is determined by the presence of xylanase. The presented experimental data shows that a cellulase treatment of the grain in conjunction with the β-glucanase or xylanase leading to an increase in phytase activity could be 1.4 - 2.3 times as compared with the individual enzymes. As a result of concerted action of enzymes complex preparation varies topography grain, increase the pore sizes in seed and fruit shells that facilitate the penetration of the enzyme phytase in the aleurone layer to the site of phytin hydrolysis and leads to an increase in phytase activity. In terms of rational parameters of enzymatic hydrolysis, the distribution of mineral elements in the anatomical parts of the grain after processing complex enzyme preparation with the help of X-ray detector EMF miniCup system in a scanning electron microscope JEOL JSM 6390 were investigated. When processing enzyme preparation wheat trend in the distribution of mineral elements, characteristic of grain - the proportion of these elements in the aleurone layer decreases, and in the endosperm increases. Because dietary fiber and phytate found together in the

  2. Ligninolytic enzyme complex of Armillaria spp

    Czech Academy of Sciences Publication Activity Database

    Stoychev, I.; Nerud, František

    2000-01-01

    Roč. 45, č. 3 (2000), s. 248-250 ISSN 0015-5632 Institutional research plan: CEZ:AV0Z5020903 Keywords : lignin olytic enzyme * lignin peroxidase Subject RIV: EE - Microbiology, Virology Impact factor: 0.752, year: 2000

  3. The effect of lignin removal by alkaline peroxide pretreatment on the susceptibility of corn stover to purified cellulolytic and xylanolytic enzymes.

    Science.gov (United States)

    Selig, Michael J; Vinzant, Todd B; Himmel, Michael E; Decker, Stephen R

    2009-05-01

    Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.

  4. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

    Science.gov (United States)

    McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-01-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

  5. Microbiota Dynamics Associated with Environmental Conditions and Potential Roles of Cellulolytic Communities in Traditional Chinese Cereal Starter Solid-State Fermentation.

    Science.gov (United States)

    Li, Pan; Liang, Hebin; Lin, Wei-Tie; Feng, Feng; Luo, Lixin

    2015-08-01

    Traditional Chinese solid-state fermented cereal starters contain highly complex microbial communities and enzymes. Very little is known, however, about the microbial dynamics related to environmental conditions, and cellulolytic communities have never been proposed to exist during cereal starter fermentation. In this study, we performed Illumina MiSeq sequencing combined with PCR-denaturing gradient gel electrophoresis to investigate microbiota, coupled with clone library construction to trace cellulolytic communities in both fermentation stages. A succession of microbial assemblages was observed during the fermentation of starters. Lactobacillales and Saccharomycetales dominated the initial stages, with a continuous decline in relative abundance. However, thermotolerant and drought-resistant Bacillales, Eurotiales, and Mucorales were considerably accelerated during the heating stages, and these organisms dominated until the end of fermentation. Enterobacteriales were consistently ubiquitous throughout the process. For the cellulolytic communities, only the genera Sanguibacter, Beutenbergia, Agrobacterium, and Erwinia dominated the initial fermentation stages. In contrast, stages at high incubation temperature induced the appearance and dominance of Bacillus, Aspergillus, and Mucor. The enzymatic dynamics of amylase and glucoamylase also showed a similar trend, with the activities clearly increased in the first 7 days and subsequently decreased until the end of fermentation. Furthermore, β-glucosidase activity continuously and significantly increased during the fermentation process. Evidently, cellulolytic potential can adapt to environmental conditions by changes in the community structure during the fermentation of starters. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Choosing the Best Enzyme Complex Structure Made Easy.

    Science.gov (United States)

    Das, Sayoni; Orengo, Christine

    2018-04-03

    In this issue of Structure, Tyzack et al. (2018) present a study of enzyme-ligand complexes in the PDB and show that the molecular similarity of bound and cognate ligands can be used to choose the most biologically appropriate complex structure for analysis when multiple structures are available. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Enzyme-lipid complex. Koso-shishitsu fukugotai

    Energy Technology Data Exchange (ETDEWEB)

    Okahata, Y; Ijiro, K [Tokyo Inst. of Technology., Tokyo (Japan)

    1990-08-01

    Enzyme, as unstable against organic solvent, being to be designed not to be quenched, organic solvent was tried to be made soluble by making enzyme-lipid complex. By mixing aqueous solution of enzyme with aqueous dispersion liquid of lipid, white powder was obtaind. Enzyme has monomolecular film through which reaction substance passes. Lipase-lipid complex, of which monomolecular film is qualified by hydrogen and other soft linkages, homogeneously dissolves in organic solvent and has a high activity, not given by the conventional qualification method. That activity being applied, asymmetrical esterificating reaction of alcohol could be done in organic solvent, containing high concentration reactive substance. While substance selectivity, not known in water, was obtained. Through reaction of amine with amino acid dielectrics in isooctane solvent by {alpha}-chymotrypsin-lipid complex, was indicated an exact substance selectivity. Enzyme-lipid complex dissolving in organic solvent, monomolecular film can be formed without being quenched on aqueous surface, which film can be utilized as sensor film. 10 refs., 5 figs. 1 tab.

  8. Cellulolytic potential of thermophilic species from four fungal orders

    DEFF Research Database (Denmark)

    Busk, Peter Kamp; Lange, Lene

    2013-01-01

    and in characterization of their industrially useful enzymes. In the present study we investigated the cellulolytic potential of 16 thermophilic fungi from the three ascomycete orders Sordariales, Eurotiales and Onygenales and from the zygomycete order Mucorales thus covering all fungal orders that include thermophiles....... Thermophilic fungi are the only described eukaryotes that can grow at temperatures above 45 ºC. All 16 fungi were able to grow on crystalline cellulose but their secreted enzymes showed widely different cellulolytic activities, pH optima and thermostabilities. Interestingly, in contrast to previous reports, we......Elucidation of fungal biomass degradation is important for understanding the turnover of biological materials in nature and has important implications for industrial biomass conversion. In recent years there has been an increasing interest in elucidating the biological role of thermophilic fungi...

  9. Characterization of Cellulolytic Bacterial Cultures Grown in Different Substrates

    Directory of Open Access Journals (Sweden)

    Mohamed Idris Alshelmani

    2013-01-01

    Full Text Available Nine aerobic cellulolytic bacterial cultures were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ and the American Type Culture Collection (ATCC. The objectives of this study were to characterize the cellulolytic bacteria and to determine the optimum moisture ratio required for solid state fermentation (SSF of palm kernel cake (PKC. The bacteria cultures were grown on reconstituted nutrient broth, incubated at 30∘C and agitated at 200 rpm. Carboxymethyl cellulase, xylanase, and mannanase activities were determined using different substrates and after SSF of PKC. The SSF was conducted for 4 and 7 days with inoculum size of 10% (v/w on different PKC concentration-to-moisture ratios: 1 : 0.2, 1 : 0.3, 1 : 0.4, and 1 : 0.5. Results showed that Bacillus amyloliquefaciens 1067 DSMZ, Bacillus megaterium 9885 ATCC, Paenibacillus curdlanolyticus 10248 DSMZ, and Paenibacillus polymyxa 842 ATCC produced higher enzyme activities as compared to other bacterial cultures grown on different substrates. The cultures mentioned above also produced higher enzyme activities when they were incubated under SSF using PKC as a substrate in different PKC-to-moisture ratios after 4 days of incubation, indicating that these cellulolytic bacteria can be used to degrade and improve the nutrient quality of PKC.

  10. Cellulolytic and xylanolytic activities of common indoor fungi

    DEFF Research Database (Denmark)

    Andersen, Birgitte; Poulsen, Rehab; Hansen, Gustav Hammerich

    2016-01-01

    Moldy building materials, such as chip wood and gypsum, should be a good source for fungal strains with high production of lignocellulolytic enzymes. Screening of 21 common indoor fungal strains showed, contrary to the expected, that the Chaetomium and Stachybotrys strains had little...... or no cellulolytic and xylanolytic activities using AZCL-assays. On the other hand, both Cladosporium sphaerospermum and Penicillium chrysogenum showed the highest cellulase, β-glucosidase, mannase, β-galactanase and arabinanase activities and would be good candidates for over-producers of enzymes needed...

  11. Polyelectrolyte Complex Optimization for Macrophage Delivery of Redox Enzyme Nanoparticles

    Science.gov (United States)

    Zhao, Yuling; Haney, Matthew J.; Klyachko, Natalia L.; Li, Shu; Booth, Stephanie L.; Higginbotham, Sheila M.; Jones, Jocelyn; Zimmerman, Matthew C.; Mosley, R. Lee; Kabanov, Alexander V.; Gendelman, Howard E.; Batrakova, Elena V.

    2011-01-01

    Background We posit that cell-mediated drug delivery can improve transport of therapeutic enzymes to the brain and decrease inflammation and neurodegeneration induced during Parkinson’s disease. Our prior work demonstrated that macrophages loaded with nanoformulated catalase (“nanozyme”) protect the nigrostriatum in a murine model of Parkinson’s disease. Packaging of catalase into block ionomer complex with a synthetic polyelectrolyte block copolymers protects the enzyme degradation in macrophages. Methods We examined relationships between the composition and structure of block ionomer complexes, their physicochemical characteristics, and loadings, release rates, and catalase activity in bone marrow-derived macrophages. Results Formation of block-ionomer complexes resulted in improved aggregation stability. Block ionomer complexes with ε-polylisine, and poly-L-glutamic acid -poly(ethylene glycol) demonstrated the least cytotoxicity and high loading and release rates, however, did not efficiently protect catalase inside macrophages. Conclusion nanozymes with polyethyleneimine- and poly(L-lysine)10-poly(ethylene glycol) provided the best protection of enzymatic activity for cell-mediated drug delivery. PMID:21182416

  12. Multifaceted roles of metabolic enzymes of the Paracoccidioides species complex

    Directory of Open Access Journals (Sweden)

    Caroline Maria Marcos

    2014-12-01

    Full Text Available Paracoccidioides species are dimorphic fungi, and are the etiologic agents of paracoccidioidomycosis (PCM, a serious disease of multiple organs. The large number of tissues colonized by this fungus suggests the presence of a variety of surface molecules involved in adhesion. A surprising finding is that the majority of enzymes in the glycolytic pathway, tricarboxylic acid (TCA cycle and glyoxylate cycle in Paracoccidioides spp. has adhesive properties that aid in the interaction with the host extracellular matrix, and so act as ‘moonlighting’ proteins. Moonlighting proteins have multiple functions and add another dimension to cellular complexity, while benefiting cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play roles in bacterial pathogens, either by acting as proteins secreted in a conventional pathway or not and/or as cell surface component that facilitate adhesion or adherence . This review outlines the multifuncionality exposed by a variety of Paracoccidioides spp. enzymes including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase and enolase. The roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host are discussed.

  13. DNA-directed control of enzyme-inhibitor complex formation: a modular approach to reversibly switch enzyme activity

    NARCIS (Netherlands)

    Janssen, B.M.G.; Engelen, W.; Merkx, M.

    2015-01-01

    DNA-templated reversible assembly of an enzyme–inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-ß-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template

  14. NREL Explains the Higher Cellulolytic Activity of a Vital Microorganism

    Energy Technology Data Exchange (ETDEWEB)

    2016-06-01

    The discovery of a new mode of action by C. thermocellum to convert biomass to biofuels is significant because the bacterium is already recognized as one of the most effective in the biosphere. Researchers found that, in addition to using common cellulase degradation mechanisms attached to cells, C. thermocellum also uses a new category of cell-free scaffolded enzymes. The new discovery will influence the strategies used to improve the cellulolytic activity of biomass degrading microbes going forward. Better understanding of this bacterium could lead to cheaper production of ethanol and drop-in fuels. Also, this discovery demonstrates that nature's biomass conversion behaviors are not fully understood and remain as opportunities for future microbial/enzyme engineering efforts.

  15. Comparative study of cellulolytic activity of three rumen fungi on different substrates

    Directory of Open Access Journals (Sweden)

    Atanasova-Pančevska Natalija

    2011-01-01

    Full Text Available Anaerobic chytridiomycete fungi are found in the gastrointestinal tracts of many domesticated ruminant and nonruminant herbivores and of a wide variety of wild herbivorous mammals. They produce high levels of cellulases and hemicellulases; these enzymes are regulated by substrate (especially soluble sugars available to the organisms. The aim of this paper was to do a comparative study of cellulolytic activity of three rumen fungi on carboxymethyl cellulose and Avicel. The capacity of enzymes was determined by monitoring the growth on carboxymethyl cellulose (CMC and Avicel. Enzyme activity was detected extracellularly in culture supernatants after vegetative growth. All of the isolates degraded CMC and avicel, and exhibited cellulolytic activities (carboxymethyl cellulose-(CMC-ase and avicelase.

  16. Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

    Directory of Open Access Journals (Sweden)

    Yoko Kimata-Ariga

    Full Text Available Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT, nitrite reductase (NiR and glutamine synthetase (GS, separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE. GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa and multiple sizes (>120 kDa, respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

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

    Science.gov (United States)

    Saadeddin, Anas

    2014-06-01

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

  18. Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

    Science.gov (United States)

    2012-01-01

    Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis

  19. Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

    Directory of Open Access Journals (Sweden)

    Amore Antonella

    2012-12-01

    Full Text Available Abstract Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose

  20. SACCHARIFICATION OF CORNCOB USING CELLULOLYTIC BACTERIA FOR BIOETHANOL PRODUCTION

    Directory of Open Access Journals (Sweden)

    TITI CANDRA SUNARTI

    2010-08-01

    Full Text Available The use of cellulose degrading enzyme (cellulases for hydrolysis of lignocellulosic material is a part of bioethanol production process. In this experiment, delignified corncob, its cellulose fraction and alpha cellulose were used as substrates to produce fermentable sugar by using three local isolates of celluloytic bacteria (C5-1, C4-4, C11-1 and Cmix ; mixed cultures of three isolates, and Saccharomyces cereviseae to produce ethanol. The results showed that all isolates of cellulolytic bacteria can grow on cellulose fraction better than on delignified corncob, and alpha cellulose. The highest hydrolytic activity produced from cellulose fraction was by isolate C4-4, which liberated 3.50 g/l of total sugar. Ethanol can be produced by mixed culture of bacteria and yeast, but because of competitive growth, the fermentation only produced 0.39-0.47 g/l of ethanol.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  3. Isolation and characterization of aerobic microorganisms with cellulolytic activity in the gut of endogeic earthworms.

    Science.gov (United States)

    Fujii, Katsuhiko; Ikeda, Kana; Yoshida, Seo

    2012-09-01

    The ability of earthworms to decompose lignocellulose involves the assistance of microorganisms in their digestive system. While many studies have revealed a diverse microbiota in the earthworm gut, including aerobic and anaerobic microorganisms, it remains unclear which of these species contribute to lignocellulose digestion. In this study, aerobic microorganisms with cellulolytic activity isolated from the gut of two endogeic earthworms, Amynthas heteropoda (Megascolecidae) and Eisenia fetida (Lumbricidae) were isolated by solid culture of gut homogenates using filter paper as a carbon source. A total of 48 strains, including four bacterial and four fungal genera, were isolated from two earthworm species. Characterization of these strains using enzyme assays showed that the most representative ones had exocellulase and xylanase activities, while some had weak laccase activity. These findings suggest that earthworms digest lignocellulose by exploiting microbial exocellulase and xylanase besides their own endocellulase. Phylogenetic analysis showed that among the cellulolytic isolates in both earthworm species Burkholderia and Chaetomium were the dominant bacterial and fungal members.

  4. Diameter dependent electron transfer kinetics in semiconductor-enzyme complexes.

    Science.gov (United States)

    Brown, Katherine A; Song, Qing; Mulder, David W; King, Paul W

    2014-10-28

    Excited state electron transfer (ET) is a fundamental step for the catalytic conversion of solar energy into chemical energy. To understand the properties controlling ET between photoexcited nanoparticles and catalysts, the ET kinetics were measured for solution-phase complexes of CdTe quantum dots and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) using time-resolved photoluminescence spectroscopy. Over a 2.0-3.5 nm diameter range of CdTe nanoparticles, the observed ET rate (kET) was sensitive to CaI concentration. To account for diameter effects on CaI binding, a Langmuir isotherm and two geometric binding models were created to estimate maximal CaI affinities and coverages at saturating concentrations. Normalizing the ET kinetics to CaI surface coverage for each CdTe diameter led to k(ET) values that were insensitive to diameter, despite a decrease in the free energy for photoexcited ET (ΔGET) with increasing diameter. The turnover frequency (TOF) of CaI in CdTe-CaI complexes was measured at several molar ratios. Normalization for diameter-dependent changes in CaI coverage showed an increase in TOF with diameter. These results suggest that k(ET) and H2 production for CdTe-CaI complexes are not strictly controlled by ΔG(ET) and that other factors must be considered.

  5. Escherichia coli pyruvate dehydrogenase complex: particle masses of the complex and component enzymes measured by scanning transmission electron microscopy

    International Nuclear Information System (INIS)

    CaJacob, C.A.; Frey, P.A.; Hainfeld, J.F.; Wall, J.S.; Yang, H.

    1985-01-01

    Particle masses of the Escherichia coli pyruvate dehydrogenase (PDH) complex and its component enzymes have been measured by scanning transmission electron microscopy (STEM). The particle mass of PDH complex measured by STEM is 5.28 X 10(6) with a standard deviation of 0.40 X 10(6). The masses of the component enzymes are 2.06 X 10(5) for the dimeric pyruvate dehydrogenase (E1), 1.15 X 10(5) for dimeric dihydrolipoyl dehydrogenase (E3), and 2.20 X 10(6) for dihydrolipoyl transacetylase (E2), the 24-subunit core enzyme. STEM measurements on PDH complex incubated with excess E3 or E1 failed to detect any additional binding of E3 but showed that the complex would bind additional E1 under forcing conditions. The additional E1 subunits were bound too weakly to represent binding sites in an isolated or isolable complex. The mass measurements by STEM are consistent with the subunit composition 24:24:12 when interpreted in the light of the flavin content of the complex and assuming 24 subunits in the core enzyme (E2)

  6. Selection and molecular characterization of cellulolytic-xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites.

    Science.gov (United States)

    Okeke, Benedict C; Hall, Rosine W; Nanjundaswamy, Ananda; Thomson, M Sue; Deravi, Yasaman; Sawyer, Leah; Prescott, Andrew

    2015-06-01

    Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass are a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences from the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120 h incubation was 121.08 units/L/h and 348 units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83 units/L/h and 359 units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  7. Process relevant screening of cellulolytic organisms for consolidated bioprocessing.

    Science.gov (United States)

    Antonov, Elena; Schlembach, Ivan; Regestein, Lars; Rosenbaum, Miriam A; Büchs, Jochen

    2017-01-01

    Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics. The screening of five different cellulase producers with the freeze assay identified Trichoderma   reesei and Penicillium   verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P.   verruculosum but a similar cellulase production rate afterwards, compared to T.   reesei . The freeze assay measurement depicted that P.   verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the

  8. Biochanin A improves fiber fermentation by cellulolytic bacteria

    Science.gov (United States)

    The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity. When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39 °C) with ground hay, cellulolytic bacteria proliferated, short chain fatty...

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1995-01-01

    A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent-insoluble c...... intracellularly. I therefore propose that, in the enterocyte, the brush border enzymes are targeted directly from the trans-Golgi network toward the apical cell surface......., and their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the enrichment...

  11. Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae.

    Science.gov (United States)

    Liu, Zhuo; Inokuma, Kentaro; Ho, Shih-Hsin; den Haan, Riaan; van Zyl, Willem H; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-06-01

    Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Isolation of Cellulolytic Bacteria and Characterization of the Enzyme

    OpenAIRE

    Nisa Rachmania; Titi Candra Sunarti; Besty Maranatha; Wahyu Widosari; Anja Meryandini; Hasrul Satria

    2009-01-01

    Four of cellulolitic bacteria isolates had beencharacterized. The determination of cellulase activity was conducted at the highest production time, using crudeenzymes with the modification of Miller methods (1959) on pure cellulose substrates such as CMC (Carboxymethylcellulose), Avicel and Filter paper Whatman No. 1 as well as agriculture waste such as rice straw, corn cob and bananapeel. Cellulase from C4-4, C5-1, C5-3 and C11-1 showed optimum activity at pH 5, 70°C, pH 3.5, 90°C, pH 5, 80°...

  13. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    Science.gov (United States)

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

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

    Directory of Open Access Journals (Sweden)

    J. Q. Yi

    2013-08-01

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

  15. Rock phosphate solubilizing and cellulolytic actinomycete isolates of earthworm casts

    Science.gov (United States)

    Mba, Caroline C.

    1994-03-01

    Four microbial isolates, OP2, OP3, OP6, and OP7, of earthworm casts of Pontoscolex corethrurus were found to be acid tolerant actinomycetes and efficient rock phosphate (RP) solubilizers that could grow fast on NH4Cl-enriched or N-free carboxymethyl cellulose or glucose as sole carbon source. CMC (carboxymethyl cellulose) induced production of extracellular cellulase enzyme and the production of reducing sugar in all the isolates. RP solubilizing power was observed to be inversely related to glucose consumption. The most efficient RP solubilizer was found to consume the least glucose. Growth was faster on cellulose than on glucose media. N-free CMC induced greater glucose production than NH4Cl-enriched CMC medium. Both CMC and glucose media were acidified by all the isolates, however, RP solubilizing power decreased with acidification. Solubilization power was greatest with isolate OP7, which also produced the greatest amount of reducing sugar per gram CMC. Both RP solubilizing power and the cellulolytic efficiency varied among isolates. A minimum of 631 µg P/0.1 g RP and a maximum of 951.4 µg P/0.1 g RP was recorded.

  16. Isolation and characterization of efficient cellulolytic fungi from ...

    African Journals Online (AJOL)

    Isolation and characterization of efficient cellulolytic fungi from degraded wood and industrial samples. Abreham Bekele, Tariku Abena, Adane Habteyohannes, Addisalem Nugissie, Fitala Gudeta, Tigist Getie, Musin Kelel, Admas Berhanu ...

  17. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

    Directory of Open Access Journals (Sweden)

    Chang Jui-Jen

    2012-07-01

    Full Text Available Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO, that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei, a beta-glucosidase (from a cow rumen fungus, a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

  18. Screening Cellulolytic Bacteria from the Digestive Tract Snail (Achatina fulica and Test the Ability of Cellulase Activity

    Directory of Open Access Journals (Sweden)

    Wijanarka Wijanarka

    2016-11-01

    Full Text Available On the research of enzyme production levels observed cellulase produced by bacteria in the digestive tract of the isolation of the Snail (Achatina fulica. Isolation of bacteria based on the ability of bacteria to grow on CMC media. The purpose of this study was to determine cellulase activity by cellulolytic bacteria. Some bacterial isolates were identified as cellulolytic bacteria, they were KE-B1, KE-B2, KE-B3, KE-B4, KE-B5, and KE-B6. Isolates KE-B6 was the best isolates. Furthermore KE-B6 isolates were grown on media production to determine the pattern of growth and enzyme activity. Measurement of cell growth was conducted by inoculating starter aged 22 hours at CMC production of liquid medium. Cellulase enzyme activity measurements was performed by the DNS method. The results showed that the highest activity by new isolate bacteria KE-B6 and its value of the activity of 0.4539 U/mL, growth rate (µ 0.377/hour and generation time (g 1.84 hour. This research expected cellulase of producing bacteria were easy, inexpensive and efficient. This enzyme can be used as an enzyme biolytic once expected to replace expensive commercial enzyme. The biotylic enzyme can be applied to strains improvement (protoplast fusion.How to CiteWijanarka, W., Kusdiyantini, E. & Parman, S. (2016. Screening Cellulolytic Bacteria from the Digestive Tract Snail (Achatina fulica and Test the Ability of Cellulase Activity. Biosaintifika: Journal of Biology & Biology Education, 8(3, 386-392. 

  19. Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.

    Directory of Open Access Journals (Sweden)

    Shilpa Aggarwal

    2010-04-01

    Full Text Available It is widely accepted that the highly error prone replication process of influenza A virus (IAV, together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol, is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT of human immunodeficiency virus (HIV-1 and murine leukemia virus (MuLV, which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg(++ with Mn(++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn(++. Finally, when the IAV nucleoprotein (NP was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity.Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of

  20. Biodegradation of Palm Kernel Cake by Cellulolytic and Hemicellulolytic Bacterial Cultures through Solid State Fermentation

    Directory of Open Access Journals (Sweden)

    Mohamed Idris Alshelmani

    2014-01-01

    Full Text Available Four cellulolytic and hemicellulolytic bacterial cultures were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ and the American Type Culture Collection (ATCC. Two experiments were conducted; the objective of the first experiment was to determine the optimum time period required for solid state fermentation (SSF of palm kernel cake (PKC, whereas the objective of the second experiment was to investigate the effect of combinations of these cellulolytic and hemicellulolytic bacteria on the nutritive quality of the PKC. In the first experiment, the SSF was lasted for 12 days with inoculum size of 10% (v/w on different PKC to moisture ratios. In the second experiment, fifteen combinations were created among the four microbes with one untreated PKC as a control. The SSF lasted for 9 days, and the samples were autoclaved, dried, and analyzed for proximate analysis. Results showed that bacterial cultures produced high enzymes activities at the 4th day of SSF, whereas their abilities to produce enzymes tended to be decreased to reach zero at the 8th day of SSF. Findings in the second experiment showed that hemicellulose and cellulose was significantly P<0.05 decreased, whereas the amount of reducing sugars were significantly P<0.05 increased in the fermented PKC (FPKC compared with untreated PKC.

  1. Effects of Maize Source and Complex Enzymes on Performance and Nutrient Utilization of Broilers

    Directory of Open Access Journals (Sweden)

    Defu Tang

    2014-12-01

    Full Text Available The objective of this study was to investigate the effect of maize source and complex enzymes containing amylase, xylanase and protease on performance and nutrient utilization of broilers. The experiment was a 4×3 factorial design with diets containing four source maize samples (M1, M2, M3, and M4 and without or with two kinds of complex enzyme A (Axtra XAP and B (Avizyme 1502. Nine hundred and sixty day old Arbor Acres broiler chicks were used in the trial (12 treatments with 8 replicate pens of 10 chicks. Birds fed M1 diet had better body weight gain (BWG and lower feed/gain ratio compared with those fed M3 diet and M4 diet (p0.05, respectively. The fresh feces output was significantly decreased by the addition of enzyme B (p<0.05. Maize source affects the nutrients digestibility and performance of broilers, and a combination of amylase, xylanase and protease is effective in improving the growth profiles of broilers fed maize-soybean-rapeseed-cotton mixed diets.

  2. DICER-ARGONAUTE2 complex in continuous fluorogenic assays of RNA interference enzymes.

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    Mark A Bernard

    Full Text Available Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC have been hindered by lack of methods for continuous monitoring of enzymatic activity. "Quencherless" fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS and hypoxia-inducible factor 1-α subunit (HIF1A. Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM with substrate inhibition kinetics (Ki=105 nM, demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER bound in the active site of DICER may undergo direct transfer (as AGO2 substrate to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29, suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a

  3. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.

    Directory of Open Access Journals (Sweden)

    Kathrin Roderer

    Full Text Available The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS. The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

  4. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.

    Science.gov (United States)

    Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

    2014-01-01

    The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

  5. Utilization of mixed cellulolytic microbes from termite extract, elephant faecal solution and buffalo ruminal fluid to increase in vitro digestibility of King Grass

    Directory of Open Access Journals (Sweden)

    Agung Prabowo

    2007-06-01

    Full Text Available Cellulose is a compound of plant cell walls which is difficult to be degraded because it composed of glucose monomers linked by β-(1.4-bound. It will be hydrolysed by cellulase enzyme secreted by cellulolytic microbes. The effective digestion of cellulose needs high activity of cellulase enzyme. This research aims to increase in vitro king grass digestibility utilizing mixed cellulolytic microbes of termite extract, elephant faecal solution, and buffalo ruminal fluid. Twelve syringes contained gas test media were randomly divided into four treatments based on sources of microbe (SM, namely: S (SM: cattle ruminal fluid [S], RGK (SM: mixed cellulolytic microbes of termite extract, elephant faecal solution, and buffalo ruminal fluid [RGK], with composition 1 : 1 : 1, S-RGK (SM: S + RGK, with composition 1:1, and TM (without given treatment microbe. Digestibility was measured using gas test method. Average of gas production treatment of S-RGK (70.2 + 0.6 ml was higher and significantly different (P<0.01 compared to treatment of S (60.3 + 0.8 ml, RGK (40.8 + 2.3 ml, and TM (13.3 + 2.0 ml. Utilization of mixed cellulolytic microbes of termite extract, elephant faecal solution, and buffalo ruminal fluid (RGK that combined with microbes of cattle ruminal fluid (S could increase in vitro digestibility of king grass.

  6. Influence of a soil enzyme on iron-cyanide complex speciation and mineral adsorption.

    Science.gov (United States)

    Zimmerman, Andrew R; Kang, Dong-Hee; Ahn, Mi-Youn; Hyun, Seunghun; Banks, M Katherine

    2008-01-01

    Cyanide is commonly found as ferrocyanide [Fe(II)(CN)(6)](-4) and in the more mobile form, ferricyanide [Fe(III)(CN)(6)](-3) in contaminated soils and sediments. Although soil minerals may influence ferrocyanide speciation, and thus mobility, the possible influence of soil enzymes has not been examined. In a series of experiments conducted under a range of soil-like conditions, laccase, a phenoloxidase enzyme derived from the fungi Trametes versicolor, was found to exert a large influence on iron-cyanide speciation and mobility. In the presence of laccase, up to 93% of ferrocyanide (36-362ppm) was oxidized to ferricyanide within 4h. No significant effect of pH (3.6 and 6.2) or initial ferrocyanide concentration on the extent or rate of oxidation was found and ferrocyanide oxidation did not occur in the absence of laccase. Relative to iron-cyanide-mineral systems without laccase, ferrocyanide adsorption to aluminum hydroxide and montmorillonite decreased in the presence of laccase and was similar to or somewhat greater than that of ferricyanide without laccase. Laccase-catalyzed conversion of ferrocyanide to ferricyanide was extensive though up to 33% of the enzyme was mineral-bound. These results demonstrate that soil enzymes can play a major role in ferrocyanide speciation and mobility. Biotic soil components must be considered as highly effective oxidation catalysts that may alter the mobility of metals and metal complexes in soil. Immobilized enzymes should also be considered for use in soil metal remediation efforts.

  7. Compound complex enzymes and proteins of Stipa capillata from Semipalatinsk polygon

    International Nuclear Information System (INIS)

    Sarsenbaev, K.N.; Esnazarov, U.; Sarsenbaeva, M.V.; Seisebaev, A.

    2002-01-01

    The effects of low and high doses of irradiation near Semipalatinsk Atomic lake on the compound complex of different enzymes and proteins of leaves from different population of Stipa capillata are considered. 36 samples of Stipa capillata were analyzed by the iso-electrofocusing methods, native and SDS-electrophoresis. Levels of radioactivity effect on compound complex of peroxidase, esterase, acid phosphates and soluble proteins were found. SDS-PAGE and IEF methods did not show difference in peptides spectra between 36 populations of examined species. It means, that difference between contaminated and non-contaminated populations not so big as was expected. Compound complex soluble protein of Stipa capillata leaves changes under chronic doses of radioactivity. The difference in spectra between control and contaminated leaves make up 3-6 bands. Control leaves have more high molecular weight proteins than contaminated ones. Appearance of new bands is one of ways of plant adaptation. New components of enzymes spectra and soluble proteins were found. It was suggested, that gene mutation or post-translation modification of these proteins are result of chronic irradiation. To prove exactly genetic nature of this alteration aminoacids sequence for these proteins the DNA sequence of different Stipa capillata populations genomes were compared

  8. Cellulolytic and xylanolytic potential of high β-glucosidase-producing Trichoderma from decaying biomass.

    Science.gov (United States)

    Okeke, Benedict C

    2014-10-01

    Availability, cost, and efficiency of microbial enzymes for lignocellulose bioconversion are central to sustainable biomass ethanol technology. Fungi enriched from decaying biomass and surface soil mixture displayed an array of strong cellulolytic and xylanolytic activities. Strains SG2 and SG4 produced a promising array of cellulolytic and xylanolytic enzymes including β-glucosidase, usually low in cultures of Trichoderma species. Nucleotide sequence analysis of internal transcribed spacer 2 (ITS2) region of rRNA gene revealed that strains SG2 and SG4 are closely related to Trichoderma inhamatum, Trichoderma piluliferum, and Trichoderma aureoviride. Trichoderma sp. SG2 crude culture supernatant correspondingly displayed as much as 9.84 ± 1.12, 48.02 ± 2.53, and 30.10 ± 1.11 units mL(-1) of cellulase, xylanase, and β-glucosidase in 30 min assay. Ten times dilution of culture supernatant of strain SG2 revealed that total activities were about 5.34, 8.45, and 2.05 orders of magnitude higher than observed in crude culture filtrate for cellulase, xylanase, and β-glucosidase, respectively, indicating that more enzymes are present to contact with substrates in biomass saccharification. In parallel experiments, Trichoderma species SG2 and SG4 produced more β-glucosidase than the industrial strain Trichoderma reesei RUT-C30. Results indicate that strains SG2 and SG4 have potential for low cost in-house production of primary lignocellulose-hydrolyzing enzymes for production of biomass saccharides and biofuel in the field.

  9. Complex Enzyme-Assisted Extraction Releases Antioxidative Phenolic Compositions from Guava Leaves.

    Science.gov (United States)

    Wang, Lu; Wu, Yanan; Liu, Yan; Wu, Zhenqiang

    2017-09-30

    Phenolics in food and fruit tree leaves exist in free, soluble-conjugate, and insoluble-bound forms. In this study, in order to enhance the bioavailability of insoluble-bound phenolics from guava leaves (GL), the ability of enzyme-assisted extraction in improving the release of insoluble-bound phenolics was investigated. Compared to untreated GL, single xylanase-assisted extraction did not change the composition and yield of soluble phenolics, whereas single cellulase or β -glucosidase-assisted extraction significantly enhanced the soluble phenolics content of PGL. However, complex enzyme-assisted extraction (CEAE) greatly improved the soluble phenolics content, flavonoids content, ABTS, DPPH, and FRAP by 103.2%, 81.6%, 104.4%, 126.5%, and 90.3%, respectively. Interestingly, after CEAE, a major proportion of phenolics existed in the soluble form, and rarely in the insoluble-bound form. Especially, the contents of quercetin and kaempferol with higher bio-activity were enhanced by 3.5- and 2.2-fold, respectively. More importantly, total soluble phenolics extracts of GL following CEAE exhibited the highest antioxidant activity and protective effect against supercoiled DNA damage. This enzyme-assisted extraction technology can be useful for extracting biochemical components from plant matrix, and has good potential for use in the food and pharmaceutical industries.

  10. Complex Enzyme-Assisted Extraction Releases Antioxidative Phenolic Compositions from Guava Leaves

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2017-09-01

    Full Text Available Phenolics in food and fruit tree leaves exist in free, soluble-conjugate, and insoluble-bound forms. In this study, in order to enhance the bioavailability of insoluble-bound phenolics from guava leaves (GL, the ability of enzyme-assisted extraction in improving the release of insoluble-bound phenolics was investigated. Compared to untreated GL, single xylanase-assisted extraction did not change the composition and yield of soluble phenolics, whereas single cellulase or β-glucosidase-assisted extraction significantly enhanced the soluble phenolics content of PGL. However, complex enzyme-assisted extraction (CEAE greatly improved the soluble phenolics content, flavonoids content, ABTS, DPPH, and FRAP by 103.2%, 81.6%, 104.4%, 126.5%, and 90.3%, respectively. Interestingly, after CEAE, a major proportion of phenolics existed in the soluble form, and rarely in the insoluble-bound form. Especially, the contents of quercetin and kaempferol with higher bio-activity were enhanced by 3.5- and 2.2-fold, respectively. More importantly, total soluble phenolics extracts of GL following CEAE exhibited the highest antioxidant activity and protective effect against supercoiled DNA damage. This enzyme-assisted extraction technology can be useful for extracting biochemical components from plant matrix, and has good potential for use in the food and pharmaceutical industries.

  11. Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1999-12-01

    To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD ({approx} +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT ({approx} -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

  12. Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme - onset of the enzymatic repair process

    International Nuclear Information System (INIS)

    Pinak, Miroslav

    1999-12-01

    To describe the first step of the enzymatic repair process (formation of complex enzyme-DNA), in which the thymine dimer (TD) part is removed from DNA, the 500 picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center - Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation the research was focused on the role of electrostatic energy in formation of complex enzyme-DNA. It is found, that during the first 100 ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Walls interactions with TD part of DNA and forms complex that lasts stabile for 500 ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD (∼ +10 kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stabile. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT (∼ -11 kcal/mol), in contrary to the positive one in the system with TD, doesn't drive enzyme towards DNA and complex is not formed. (author)

  13. A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

    Energy Technology Data Exchange (ETDEWEB)

    Budayova-Spano, Monika, E-mail: spano@embl-grenoble.fr [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Bonneté, Françoise; Ferté, Natalie [Centre de Recherche en Matière Condensée et Nanosciences, Campus de Luminy, Case 913, 13288 Marseille (France); El Hajji, Mohamed [Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier (France); Meilleur, Flora [Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Blakeley, Matthew Paul [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Castro, Bertrand [Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier (France); European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France)

    2006-03-01

    Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D{sub 2}O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map. Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grown in D{sub 2}O) with volume 1.8 mm{sup 3}. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.

  14. Effects of enzyme complex SSF (solid state fermentation in pellet diets for Nile tilapia

    Directory of Open Access Journals (Sweden)

    Guilherme de Souza Moura

    2012-10-01

    Full Text Available The effects of enzyme complex SSF (solid state fermentation on growth performance and the availability of sucrose and monosaccharides in the chyme of Nile were involved. The study included 360 fish (70g±4.43 in a completely randomized design with six dietary treatments (0, 50, 100, 150, 200 and 250 ppm of SSF arranged in six replicates, with 10 fish per replicate. Every 15 days, one tilapia of each experimental unit was sacrificed for analyses of carbohydrate in the chyme. On day 60 of the experiment, the performance parameters were measured. There was a linear effect according to treatment for final weight and weight gain. For the other performance parameters, there were no differences. There was quadratic effect for sucrose and glucose in function of the treatment, whereas the fructose levels increased linearly. The addition of 150 ppm of the enzyme complex SSF in the feed improves the performance of Nile tilapia and increases the availability of sucrose and monosaccharides in the chyme.

  15. Isolation of microbial native Stumps with cellulolytic activity of a compost process

    International Nuclear Information System (INIS)

    Jaramillo G, Marisol; Ruiz V Orlando Simon; Yepes P, Maria del Socorro; Montoya C, Olga Ines

    2003-01-01

    The isolation, selection adaptation and handling of native microorganisms coming from organic waste are an alternative to avoid the accumulation and the lack of the proper use of these undesirable materials. This organic waste is a source for obtaining microbial strains, which are potentially producers of Industrial enzymes and, at the same time, it works as substrate so that these organisms can transform it into compost or organic manure. In this work, 39 native strains of microorganisms with potential cellulolytic activity coming from the organic waste of the urban and rural sector, from the Compost Plant of Marinilla Antioquia) municipality, were isolated, evaluated and purified. The waste was previously selected and then submitted to an aerobic degradation or compost. The microbial strains were isolated in a selective medium with carboxymethyl cellulose (CMC), of the phases mesophile, termophile, cooling and maturation of the compost process. Eighty-two percent (82%)of the obtained colonies were identified, in principle as Bacillus, because of their morphology and their reaction to the Gram coloration. The fungi population was seen only during the cooling phase. Then, the potential cellulolytic activity was evaluated qualitatively in a solid medium with the Congo Red coloration, with which the Beta-endoglucanase activity was evaluated through the formation of clarified zones. Such staining was applied in two mediums with CMC with and without glucose It was observed that 33.3% of the isolated organisms produced the enzyme In both mediums; however, 25.6% of microorganisms did not show the production of this enzyme, and only 15.8% did not require the inducers to produce it

  16. Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes.

    Science.gov (United States)

    Petasis, Doros T; Hendrich, Michael P

    2015-01-01

    Electron paramagnetic resonance (EPR) spectroscopy has long been a primary method for characterization of paramagnetic centers in materials and biological complexes. Transition metals in biological complexes have valence d-orbitals that largely define the chemistry of the metal centers. EPR spectra are distinctive for metal type, oxidation state, protein environment, substrates, and inhibitors. The study of many metal centers in proteins, enzymes, and biomimetic complexes has led to the development of a systematic methodology for quantitative interpretation of EPR spectra from a wide array of metal containing complexes. The methodology is now contained in the computer program SpinCount. SpinCount allows simulation of EPR spectra from any sample containing multiple species composed of one or two metals in any spin state. The simulations are quantitative, thus allowing determination of all species concentrations in a sample directly from spectra. This chapter will focus on applications to transition metals in biological systems using EPR spectra from multiple microwave frequencies and modes. © 2015 Elsevier Inc. All rights reserved.

  17. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Science.gov (United States)

    Wogulis, Mark; Sweeney, Matthew; Heu, Tia

    2017-06-14

    The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

  18. Cellulolytic activities of wild type fungi isolated from decayed wood ...

    African Journals Online (AJOL)

    Prof. Ogunji

    amongst the fungal isolates while M. mucedo had the least cellulolytic ... across plant taxa, high cellulose content; typically in the range of ... antibiotic mixture made up of distilled water, 50 ml and Erythromycin: 500mg was .... cellulases including β-glucosidase were produced from Penicillium, Aspergillus and Trichoderma ...

  19. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Science.gov (United States)

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2013-10-29

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused CBHI.1 protein. Plate enzyme ...

  1. Influence of the complexity of radiation-induced DNA damage on enzyme recognition

    International Nuclear Information System (INIS)

    Palmer, Philip

    2002-01-01

    Ionising radiation is unique in inducing DNA clustered damage together with the simple isolated lesions. Understanding how these complex lesions are recognised and repaired by the cell is key to understanding the health risks associated with radiation exposure. This study focuses on whether ionising radiation-induced complex single-strand breaks (SSB) are recognised by DNA-PK and PARP, and whether the complexity of DSB influence their ligation by either DNA ligase lV/XRCC4 (LX) complex or T4 DNA ligase. Plasmid DNA, irradiated in aqueous solution using sparsely ionising γ-rays and densely ionising α-particles produce different yields of complex DNA damages, used as substrates for in vitro DNA-PK and PARP activity assays. The activity of DNA-PK to phosphorylate a peptide was determined using HF19 cell nuclear extracts as a source of DNA-PK. PARP ADP-ribosylation activity was determined using purified PARP enzyme. The activation of DNA-PK and PARP by irradiated DNA is due to SSB and not the low yield of DSB (linear plasmid DNA <10%). A ∼2 fold increase in DNA-PK activation and a ∼3-fold reduction in PARP activity seen on increasing the ionising density of the radiation (proportion of complex damage) are proposed to reflect changes in the complexity of SSB and may relate to damage signalling. Complex DSB synthesised as double-stranded oligonucleotides, with a 2 bp 5'-overhang, and containing modified lesions, 8-oxoguanine and abasic sites, at known positions relative to the termini were used as substrates for in vitro ligation by DNA ligase IV/XRCC4 or T4 ligase. The presence of a modified lesion 2 or 3 bp but not 4 bp from the 3'-termini and 2 or 6 bp from the 5'-termini caused a drastic reduction in the extent of ligation. Therefore, the presence of modified lesions near to the termini of a DSB may compromise their rejoining by non-homologous end-joining (NHEJ) involving the LX complex. (author)

  2. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    Science.gov (United States)

    Barabote, Ravi D.; Xie, Gary; Leu, David H.; Normand, Philippe; Necsulea, Anamaria; Daubin, Vincent; Médigue, Claudine; Adney, William S.; Xu, Xin Clare; Lapidus, Alla; Parales, Rebecca E.; Detter, Chris; Pujic, Petar; Bruce, David; Lavire, Celine; Challacombe, Jean F.; Brettin, Thomas S.; Berry, Alison M.

    2009-01-01

    We present here the complete 2.4-Mb genome of the cellulolytic actinobacterial thermophile Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized and elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls, and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. Several of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymes are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and noncoding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot-springs-dwelling prokaryote include a low occurrence of pseudogenes or mobile genetic elements, an unexpected complement of flagellar genes, and the presence of three laterally acquired genomic islands of likely ecophysiological value. PMID:19270083

  3. Dynamic Modelling Reveals 'Hotspots' on the Pathway to Enzyme-Substrate Complex Formation.

    Directory of Open Access Journals (Sweden)

    Shane E Gordon

    2016-03-01

    Full Text Available Dihydrodipicolinate synthase (DHDPS catalyzes the first committed step in the diaminopimelate pathway of bacteria, yielding amino acids required for cell wall and protein biosyntheses. The essentiality of the enzyme to bacteria, coupled with its absence in humans, validates DHDPS as an antibacterial drug target. Conventional drug design efforts have thus far been unsuccessful in identifying potent DHDPS inhibitors. Here, we make use of contemporary molecular dynamics simulation and Markov state models to explore the interactions between DHDPS from the human pathogen Staphylococcus aureus and its cognate substrate, pyruvate. Our simulations recover the crystallographic DHDPS-pyruvate complex without a priori knowledge of the final bound structure. The highly conserved residue Arg140 was found to have a pivotal role in coordinating the entry of pyruvate into the active site from bulk solvent, consistent with previous kinetic reports, indicating an indirect role for the residue in DHDPS catalysis. A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways. By means of umbrella sampling we show that these binding intermediates are thermodynamically metastable, consistent with both the available experimental data and the substrate binding model presented in this study. Our results provide insight into an important enzyme-substrate interaction in atomistic detail that offers the potential to be exploited for the discovery of more effective DHDPS inhibitors and, in a broader sense, dynamic protein-drug interactions.

  4. Sunflower Meal and Supplementation of an Enzyme Complex in Layer Diets

    Directory of Open Access Journals (Sweden)

    WAG Araújo

    2015-09-01

    Full Text Available ABSTRACTThe objective of this experiment was to evaluate the performance of 64- to 79-wk-old laying hens fed diets supplemented with an enzyme complex (EC and containing increasing sunflower meal (SFM levels. A total of 384 Hy-Line Brown layers were distributed according to a randomized block design in a 4 × 2 factorial arrangement (four levels of SFM, and inclusion or not of EC, with eight replicates of six birds each unit. The levels of SFM inclusion were 0, 8, 16 and 24%, utilized in two distinct diets. Diets were calculated to meet all the nutritional requirements of birds, except for the nutrients that would be made available by the nutritional matrix of the enzyme complex, with or without utilization of EC. The parameters analyzed were feed intake (g/bird/day, egg production (%/bird/day, egg weight, egg mass (g/bird/day, feed conversion ratio per egg mass, feed conversion ratio per dozen eggs, body weight gain, egg components (yolk, albumen and eggshell and the economic efficiency index (EEI. There was no interaction between EC addition and the SFM levels in the diet. The addition of EC in the diets of laying hens did not affect egg productive or components parameters. The increase in the SFM levels in the diet presented quadratic effect on egg production and feed conversion ratio per dozen eggs, with calculated optimal sunflower meal inclusion levels of 6.72% and 5.83%, respectively, for each parameter. The best economic efficiency per dozen eggs was obtained with the diet with 16.0% SFM and EC inclusion, whereas per egg mass with the diet with of 24.0% SFM and no EC addition.

  5. Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees.

    Science.gov (United States)

    Yang, Jiang-Ke; Zhang, Jing-Jing; Yu, Heng-Yu; Cheng, Jian-Wen; Miao, Li-Hong

    2014-02-01

    Cellulolytic bacteria in forest soil provide carbon sources to improve the soil fertility and sustain the nutrient balance of the forest ecological system through the decomposition of cellulosic remains. These bacteria can also be utilized for the biological conversion of biomass into renewable biofuels. In this study, the community compositions and activities of cellulolytic bacteria in the soils of forests planted with broad-leaved deciduous (Chang Qing Garden, CQG) and broad-leaved evergreen (Forest Park, FP) trees in Wuhan, China were resolved through restriction fragment length polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. All of the isolates exhibited 35 RFLP fingerprint patterns and were clustered into six groups at a similarity level of 50 %. The phylogeny analysis based on the 16S rRNA gene sequence revealed that these RFLP groups could be clustered into three phylogenetic groups and further divided into six subgroups at a higher resolution. Group I consists of isolates from Bacillus cereus, Bacillus subtilis complex (I-A) and from Paenibacillus amylolyticus-related complex (I-B) and exhibited the highest cellulase activity among all of the cellulolytic bacteria isolates. Cluster II consists of isolates belonging to Microbacterium testaceum (II-A), Chryseobacterium indoltheticum (II-B), and Flavobacterium pectinovorum and the related complex (II-C). Cluster III consists of isolates belonging to Pseudomonas putida-related species. The community shift with respect to the plant species and the soil properties was evidenced by the phylogenetic composition of the communities. Groups I-A and I-B, which account for 36.0 % of the cellulolytic communities in the CQG site, are the dominant groups (88.4 %) in the FP site. Alternatively, the ratio of the bacteria belonging to group III (P. putida-related isolates) shifted from 28.0 % in CQG to 4.0 % in FP. The soil nutrient analysis revealed that the CQG site planted with deciduous broad

  6. Enzyme-coupled nanoparticles-assisted laser desorption ionization mass spectrometry for searching for low-mass inhibitors of enzymes in complex mixtures.

    Science.gov (United States)

    Salwiński, Aleksander; Da Silva, David; Delépée, Raphaël; Maunit, Benoît

    2014-04-01

    In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: 'ion fading' (IF-ENALDI), based on superficial adsorption of inhibitors and 'ion hunting' (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme-inhibitor pairs: tyrosinase-glabridin and trypsin-leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin-leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

  7. Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

    Science.gov (United States)

    Suwannarangsee, Surisa; Bunterngsook, Benjarat; Arnthong, Jantima; Paemanee, Atchara; Thamchaipenet, Arinthip; Eurwilaichitr, Lily; Laosiripojana, Navadol; Champreda, Verawat

    2012-09-01

    Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution

    International Nuclear Information System (INIS)

    Elegheert, Jonathan; Hemel, Debbie van den; Dix, Ina; Stout, Jan; Van Beeumen, Jozef; Brigé, Ann; Savvides, Savvas N.

    2009-01-01

    Of the four old yellow enzyme homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Shewanella oneidensis is an environmentally versatile Gram-negative γ-proteobacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 Å resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30–1.60 Å resolution, thus providing a rare opportunity to dissect the structure–function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride–Meisenheimer complex

  9. Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia

    OpenAIRE

    Avellaneda-Torres,Lizeth Manuela; Pulido,Claudia Patricia Guevara; Rojas,Esperanza Torres

    2014-01-01

    A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as ...

  10. Catalytic oxidation of concentrated orange oil phase by synthetic metallic complexes biomimetic to MMO enzyme.

    Science.gov (United States)

    Fernandes, Ilizandra A; Esmelindro, Maria Carolina; Corazza, Marcos L; Franceschi, Elton; Treichel, Helen; de Oliveira, Debora; Frizzo, Caren D; Oliveira, J Vladimir

    2010-07-01

    This paper reports the catalytic oxidation of the concentrated orange oil phase using the complexes [Fe(III)(BMPP)Cl(micro-O)Fe(III)Cl(3)], [Cu(II)(BTMEA)(2)Cl]Cl and [Co(II)(BMPP)]Cl(2) biomimetic to methane monooxygenase enzyme as catalysts and hydrogen peroxide as oxidant. The reaction products of oil oxidation, mainly nootkatone, were identified by gas chromatography/mass spectrometry. A screening of catalysts was performed through a full 2(3) experimental design, varying the temperature from 30 to 70 degrees C, the catalyst concentration from 7.0 x 10(-4) to 1.5 x 10(-3) mol L(-1) and the oxidant/substrate molar ratio from 1:1 to 3:1. The results of reaction kinetics employing the most promising catalysts showed that conversions to nootkatone of up to 8% were achieved after 16 h at 70 degrees C. The results obtained in this study in terms of nootkatone production should be considered encouraging, since a real, industrially collected, raw material, instead of pure valencene, was employed in the reaction experiments, with a final content about ten times that present in the original concentrated oil.

  11. Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia.

    Science.gov (United States)

    Avellaneda-Torres, Lizeth Manuela; Pulido, Claudia Patricia Guevara; Rojas, Esperanza Torres

    2014-01-01

    A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

  12. Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia

    Directory of Open Access Journals (Sweden)

    Lizeth Manuela Avellaneda-Torres

    2014-12-01

    Full Text Available A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP, Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS of ribosomal DNA for fungi. Multivariate statistical analysis (MVA was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

  13. Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions.

    Science.gov (United States)

    Mehta, Angad P; Abdelwahed, Sameh H; Mahanta, Nilkamal; Fedoseyenko, Dmytro; Philmus, Benjamin; Cooper, Lisa E; Liu, Yiquan; Jhulki, Isita; Ealick, Steven E; Begley, Tadhg P

    2015-02-13

    In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Wieczorek Andrew S

    2010-09-01

    Full Text Available Abstract Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA. Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase, and were

  15. Coordination of manganous ion at the active site of pyruvate, phosphate dikinase: the complex of oxalate with the phosphorylated enzyme

    International Nuclear Information System (INIS)

    Kofron, J.L.; Ash, D.E.; Reed, G.H.

    1988-01-01

    Electron paramagnetic resonance spectroscopy has been used to investigate the structure of the complex of manganous ion with the phosphorylated form of pyruvate, phosphate dikinase (E/sub p/) and the inhibitor oxalate. Oxalate, an analogue of the enolate of pyruvate, is competitive with respect to pyruvate in binding to the phosphorylated form of the enzyme. Superhyperfine coupling between the unpaired electrons of Mn(I) and ligands specifically labeled with 17 O has been used to identify oxygen ligands to Mn(II) in the complex with oxalate and the phosphorylated form of the enzyme. Oxalate binds at the active site as a bidentate chelate with Mn(II). An oxygen from the 3'-N-phosphohistidyl residue of the protein is in the coordination sphere of Mn(II), and at least two water molecules are also bound to Mn(II) in the complex. Oxalate also binds directly to Mn(II) in a complex with nonphosphorylated enzyme. The structure for the E/sub p/-Mn(II)-oxalate complex implies that simultaneous coordination of a phospho group and of the attacking nucleophile to the divalent cation is likely an important factor in catalysis of this phospho-transfer reaction

  16. Cellulolytic (cel) genes of Clostridium thermocellum F7 and the proteins encoded by them

    International Nuclear Information System (INIS)

    Piruzyan, E.S.; Mogutov, M.A.; Velikodvorskaya, G.A.; Pushkarskaya, T.A.

    1988-01-01

    This study is concerned with genes cell, ce12, and ce13 encoding the endoglucanase of the cellulolytic complex of the anaerobic thermophilic Clostridium thermocellum F7 bacteria, these genes having been closed by us earlier. The authors present the characteristics of proteins synthesized by the cel genes in the minicell system of the strain Escherichia coli K-12 X925. The molecular weights of the proteins encoded by genes cell, ce12, and ce13 are 30,000, 45,000, and 50,000 dalton, respectively. The study of the homology of the cloned section of the C. thermocellum DNA containing the endoglucanase genes, using Southern's blot-hybridization method, did not reveal their physical linkage in the genome. The authors detected a plasmid with a size of about 30 kb in the cells of the C. thermocellum F7 strain investigated

  17. Functional and composition differences between mitochondrial complex II in Arabidopsis and rice are correlated with the complex genetic history of the enzyme.

    Science.gov (United States)

    Huang, Shaobai; Taylor, Nicolas L; Narsai, Reena; Eubel, Holger; Whelan, James; Millar, A Harvey

    2010-02-01

    Complex II plays a central role in mitochondrial metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. However, the composition and function of the plant enzyme has been elusive and differs from the well-characterised enzymes in mammals and bacteria. Herewith, we demonstrate that mitochondrial Complex II from Arabidopsis and rice differ significantly in several aspects: (1) Stability-Rice complex II in contrast to Arabidopsis is not stable when resolved by native electrophoresis and activity staining. (2) Composition-Arabidopsis complex II contains 8 subunits, only 7 of which have homologs in the rice genome. SDH 1 and 2 subunits display high levels of amino acid identity between two species, while the remainder of the subunits are not well conserved at a sequence level, indicating significant divergence. (3) Gene expression-the pairs of orthologous SDH1 and SDH2 subunits were universally expressed in both Arabidopsis and rice. The very divergent genes for SDH3 and SDH4 were co-expressed in both species, consistent with their functional co-ordination to form the membrane anchor. The plant-specific SDH5, 6 and 7 subunits with unknown functions appeared to be differentially expressed in both species. (4) Biochemical regulation -succinate-dependent O(2) consumption and SDH activity of isolated Arabidopsis mitochondria were substantially stimulated by ATP, but a much more minor effect of ATP was observed for the rice enzyme. The ATP activation of succinate-dependent reduction of DCPIP in frozen-thawed and digitonin-solubilised mitochondrial samples, and with or without the uncoupler CCCP, indicate that the differential ATP effect on SDH is not via the protonmotive force but likely due to an allosteric effect on the plant SDH enzyme itself, in contrast to the enzyme in other organisms.

  18. Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization.

    Directory of Open Access Journals (Sweden)

    Suwan Myung

    Full Text Available Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM from Thermus thermophiles, fructose bisphosphate aldolase (ALD from Thermotoga maritima, fructose bisphosphatase (FBP from T. maritima, and phosphoglucose isomerase (PGI from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9 mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.

  19. The genome sequences of Cellulomonas fimi and "Cellvibrio gilvus" reveal the cellulolytic strategies of two facultative anaerobes, transfer of "Cellvibrio gilvus" to the genus Cellulomonas, and proposal of Cellulomonas gilvus sp. nov.

    Directory of Open Access Journals (Sweden)

    Melissa R Christopherson

    Full Text Available Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484(T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic "Cellvibrio gilvus" ATCC 13127(T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that "Cellvibrio gilvus" belongs to the genus Cellulomonas. We thus propose to assign "Cellvibrio gilvus" to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482(T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.

  20. Effects of dietary supplementation of multi-enzyme complex on the ...

    African Journals Online (AJOL)

    Jane

    2011-07-25

    Jul 25, 2011 ... and protein escape digestion, reach the midgut and undergo fermentation ... soybean broiler starter diet with an enzyme preparation containing a mixture .... due to the rapid food rate and the deficiency of the necessary innate ...

  1. 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.

  2. Cellulose hydrolysis by fungi. 1. Screening of cellulolytic strains

    Energy Technology Data Exchange (ETDEWEB)

    Roussos, S.; Raimbault, M. (Laboratoire de Microbiologie ORSTOM, Centre de Recherche IRCHA, 91 - Vert-le-Petit (France))

    Trichoderma harzianum was selected from 30 strains of cellulolytic fungi with the aim of producing cellulases by solid state fermentation of lignocellulosic substrates. Special attention was paid to cellulase production (i. e. carboxymethylcellulase and filter paper activity), apical growth and conidia production. Under the conditions of our experiments, T. harzianum exhibited the highest cellulasic activities with 1,315 IU/I of carboxymethyl cellulose and 80 IU/l of filter paper activity. Apical growth (1 mm/h) and yield of conidial production (3.25 X 10/sup 10/ conidia/g of substrate dry weight) were also valuable characteristics of this strain in the use of solid state fermentation.

  3. Isolation and characterization of a cellulolytic actinomycete Microbispora bispora

    Energy Technology Data Exchange (ETDEWEB)

    Waldron, Jr, C R; Becker-Vallone, C A; Eveleigh, D E

    1986-09-01

    Protocols for the isolation of cellulolytic actinomycetes are described, and their use illustrated in the selection of thermophilic bacteria from soil. One isolate, Microbispora bispora, was selected for further study. It grew readily at 55/sup 0/C, produced an extracellular cellulase in good yield (endoglucanase, 5.9 U/ml) that had a broad pH range (pH 5.5 - 7.2) and was thermally stable. Its aryl-..beta..-glucosidase was cell-associated and was relatively resistant to end-product inhibition.

  4. Cellulolytic properties of an extremely thermophilic anaerobe

    Energy Technology Data Exchange (ETDEWEB)

    Hudson, J A; Morgan, H W; Daniel, R M [Waikato Univ., Hamilton (New Zealand). Microbial Biochemistry and Biotechnology Unit

    1990-09-01

    An extremely thermophilic anaerobe was isolated from a New Zealand hot spring by incubating bacterial mat strands in a medium containing xylan. The Gramreaction-negative organism that was subsequently purified had a temperature optimum of 70deg C and a pH optimum of 7.0. The isolate, designated strain H173, grew on a restricted range of carbon sources. In batch culture H173 could degrade Avicel completely when supplied at 5 or 10 g l{sup -1}. There was an initial growth phase, during which a cellulase complex was produced and carbohydrates fermented to form acetic and lactic acids, followed by a phase where cells were not metabolising but the cellulase complex actively converted cellulose to glucose. When co-cultered with strain Rt8.B1, an ethanologenic extreme thermophile, glucose was fermented to ethanol and acetate, and no reducing sugars accumulated in the medium. In pH controlled batch culture H173 produced an increased amount of lactate and acetate but there was again a phase when reducing sugars accumulated in the medium, and these were converted to ethanol by co-culture with Rt8.B1. (orig.).

  5. Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica

    Directory of Open Access Journals (Sweden)

    Guilherme L. Pinheiro

    2015-08-01

    Full Text Available The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC, p-nitrophenyl-b-D-glucopyranoside (pNPG, p-nitrophenyl-b-D-cellobioside (pNPC, 4-methylumbelliferyl-b-D-glucopyranoside (MUG, 4-methylumbelliferyl-b-D-cellobioside (MUC and 4-methylumbelliferyl-b-D-xylopyranoside (MUX. Our results indicate that the snail Achatina fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

  6. Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica.

    Science.gov (United States)

    Pinheiro, Guilherme L; Correa, Raquel F; Cunha, Raquel S; Cardoso, Alexander M; Chaia, Catia; Clementino, Maysa M; Garcia, Eloi S; de Souza, Wanderley; Frasés, Susana

    2015-01-01

    The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-β-D-cellobioside (MUC), and 4-methylumbelliferyl-β-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

  7. Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

    International Nuclear Information System (INIS)

    Pinak, Miroslav

    2000-02-01

    An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

  8. Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2000-02-01

    An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

  9. Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

    DEFF Research Database (Denmark)

    Terp, G E; Christensen, I T; Jørgensen, Flemming Steen

    2000-01-01

    Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach...... to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions...... in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural...

  10. Dynamics of complex microbiota and enzymes in Divle Cave cheese and their biochemical consequences

    NARCIS (Netherlands)

    Ozturkoglu Budak, S.

    2016-01-01

    Divle Cave cheese is a raw ewe’s milk cheese ripened with the aid of a rich microbiota and a wide range of protease and lipase enzymes secreted by individual strains belong to this microbial community. The study presented in this thesis mainly aims to define the diversity and evolution of the

  11. Effects of dietary supplementation of multi-enzyme complex on the ...

    African Journals Online (AJOL)

    Two experiments were conducted to determine the effects of dietary supplementation of REAP® enzyme into corn-soybean diet on the energy utilization in poultry and performance of broiler chicks. In the first experiment, a total of 16 50 weeks adult roosters (ISA-Brown) were divided into 4 groups with 4 birds per replicate ...

  12. 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.

  13. The total quasi-steady-state approximation for complex enzyme reactions

    DEFF Research Database (Denmark)

    Pedersen, Morten Gram; Bersani, A. M.; Bersani, E.

    2008-01-01

    ) approximation (or standard quasi-steady-state approximation (sQSSA)), which is valid when the enzyme concentration is sufficiently small. This condition is usually fulfilled for in vitro experiments, but often breaks down in vivo. The total QSSA (tQSSA), which is valid for a broader range of parameters covering...

  14. Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid.

    Science.gov (United States)

    Tabaei, Seyed R; Rabe, Michael; Zetterberg, Henrik; Zhdanov, Vladimir P; Höök, Fredrik

    2013-09-25

    Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

  15. The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

    Science.gov (United States)

    Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

    2018-01-01

    Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono

  16. Caseoperoxidase, mixed β-casein-SDS-hemin-imidazole complex: a nano artificial enzyme.

    Science.gov (United States)

    Moosavi-Movahedi, Zainab; Gharibi, Hussein; Hadi-Alijanvand, Hamid; Akbarzadeh, Mohammad; Esmaili, Mansoore; Atri, Maliheh S; Sefidbakht, Yahya; Bohlooli, Mousa; Nazari, Khodadad; Javadian, Soheila; Hong, Jun; Saboury, Ali A; Sheibani, Nader; Moosavi-Movahedi, Ali A

    2015-01-01

    A novel peroxidase-like artificial enzyme, named "caseoperoxidase", was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and k(cat) performance toward the native horseradish peroxidase demonstrated by the steady state kinetics using UV-vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein) was selected as an appropriate apo-protein for the heme active site because of its innate flexibility and exalted hydrophobicity. This selection was confirmed by homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation and provided a suitable protective role for the heme active-site. Additional experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.

  17. Enzyme Mechanism and Slow-Onset Inhibition of Plasmodium falciparum Enoyl-Acyl Carrier Protein Reductase by an Inorganic Complex

    Science.gov (United States)

    de Medeiros, Patrícia Soares de Maria; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; da Silva, Luiz Hildebrando Pereira

    2011-01-01

    Malaria continues to be a major cause of children's morbidity and mortality worldwide, causing nearly one million deaths annually. The human malaria parasite, Plasmodium falciparum, synthesizes fatty acids employing the Type II fatty acid biosynthesis system (FAS II), unlike humans that rely on the Type I (FAS I) pathway. The FAS II system elongates acyl fatty acid precursors of the cell membrane in Plasmodium. Enoyl reductase (ENR) enzyme is a member of the FAS II system. Here we present steady-state kinetics, pre-steady-state kinetics, and equilibrium fluorescence spectroscopy data that allowed proposal of P. falciparum ENR (PfENR) enzyme mechanism. Moreover, building on previous results, the present study also evaluates the PfENR inhibition by the pentacyano(isoniazid)ferrateII compound. This inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of PfENR. PMID:21603269

  18. The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist.

    Directory of Open Access Journals (Sweden)

    Garret Suen

    Full Text Available Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs, carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.

  19. A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate.

    Science.gov (United States)

    Fokina, K V; Dainyak, M B; Nagradova, N K; Muronetz, V I

    1997-09-15

    The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

  20. Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

    Science.gov (United States)

    Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

    2012-01-01

    Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

  1. Optimization of enzyme complexes for efficient hydrolysis of corn stover to produce glucose.

    Science.gov (United States)

    Yu, Xiaoxiao; Liu, Yan; Meng, Jiatong; Cheng, Qiyue; Zhang, Zaixiao; Cui, Yuxiao; Liu, Jiajing; Teng, Lirong; Lu, Jiahui; Meng, Qingfan; Ren, Xiaodong

    2015-05-01

    Hydrolysis of cellulose to glucose is the critical step for transferring the lignocellulose to the industrial chemicals. For improving the conversion rate of cellulose of corn stover to glucose, the cocktail of celllulase with other auxiliary enzymes and chemicals was studied in this work. Single factor tests and Response Surface Methodology (RSM) were applied to optimize the enzyme mixture, targeting maximum glucose release from corn stover. The increasing rate of glucan-to-glucose conversion got the higher levels while the cellulase was added 1.7μl tween-80/g cellulose, 300μg β-glucosidase/g cellulose, 400μg pectinase/g cellulose and 0.75mg/ml sodium thiosulphate separately in single factor tests. To improve the glucan conversion, the β-glucosidase, pectinase and sodium thiosulphate were selected for next step optimization with RSM. It is showed that the maximum increasing yield was 45.8% at 377μg/g cellulose Novozyme 188, 171μg/g cellulose pectinase and 1mg/ml sodium thiosulphate.

  2. UV inactivation of enzymes in supramolecular complexes of biological membranes. The phenomenon of photochemical allotropy

    International Nuclear Information System (INIS)

    Konev, S.V.; Volotovskij, I.D.; Sheiko, L.M.

    1978-01-01

    The photosensitivity of erythrocyte acetylcholinesterase (AChE) is different in its free and membrane-bound states. The modification of the structure of membraneous lipids by phospholipases A 2 , C and D or by cholesterol depletion is accompanied by a change in AChE photosensitivity. UV light was demonstrated to induce cooperative structural transitions in the erythrocyte membrane. This follows from the data obtained by circular dichroism and solubilization in detergents. In contrast to free AChE, UV light acts on the membraneous enzyme as a mixed inhibitor (simultaneous change in Vsub(max) and Ksub(m)). The anomalous behaviour of membrane-bound enzyme, termed the phenomenon of photochemical allotropy, is associated with a modification of the structure within the microenvironment of the residual AChE. The phenomenon depends on membrane integrity, and disappears after treatment of erythrocyte ghosts with ultrasound, trypsin, phospholipases and neuraminidase and remains unchanged in cholesterol-depleted membranes. The nature and localization of events responsible for this phenomenon are discussed. (author)

  3. Magnesium requirement of some of the principal rumen cellulolytic bacteria.

    Science.gov (United States)

    Morales, M S; Dehority, B A

    2014-09-01

    Information available on the role of Mg for growth and cellulose degradation by rumen bacteria is both limited and inconsistent. In this study, the Mg requirements for two strains each of the cellulolytic rumen species Fibrobacter succinogenes (A3c and S85), Ruminococcus albus (7 and 8) and Ruminococcus flavefaciens (B34b and C94) were investigated. Maximum growth, rate of growth and lag time were all measured using a complete factorial design, 2(3)×6; factors were: strains (2), within species (3) and Mg concentrations (6). R. flavefaciens was the only species that did not grow when Mg was singly deleted from the media, and both strains exhibited a linear growth response to increasing Mg concentrations (PR. flavefaciens B34b was estimated as 0.54 mM; whereas the requirement for R. flavefaciens C94 was >0.82 as there was no plateau in growth. Although not an absolute requirement for growth, strains of the two other species of cellulolytic bacteria all responded to increasing Mg concentrations. For F. succinogenes S85, R. albus 7 and R. albus 8, their requirement estimated from maximum growth was 0.56, 0.52 and 0.51, respectively. A requirement for F. succinogenes A3c could not be calculated because there was no solution for contrasts. Whether R. flavefaciens had a Mg requirement for cellulose degradation was determined in NH3-free cellulose media, using a 2×4 factorial design, 2 strains and 4 treatments. Both strains of R. flavefaciens were found to have an absolute Mg requirement for cellulose degradation. Based on reported concentrations of Mg in the rumen, 1.0 to 10.1 mM, it seems unlikely that an in vivo deficiency of this element would occur.

  4. Screening of physiologically active strain of the filamentous fungi - a producer of a complex of lytic enzymes

    International Nuclear Information System (INIS)

    Kurbatova, E.I.; Sokolova, E.N.; Borshcheva, Yu.A.; Alsivar, S.K.A.; Rimareva, L.V.

    2014-01-01

    Filamentous Aspergillus fungi were studied to obtain a producer of a complex of the enzymes specific to biodegradation of polymers of cellular walls of vegetable and microbic biomass. Strains were selected by the increased biosynthetic ability in relation to the beta-glucanase (BG), chitinase (CT), mannanase (MN), proteases and pectinases. It was estimated during deep cultivation in the environment containing wheat bran. The fullest complex of hydrolytic enzymes (glucanase, MN, CT, protease and a polygalacturonase (PG)), and also the level of enzymatic activities was in the culture liquid obtained as a result of biosynthesis of Aspergillus foetidus 37-4 (S 37-4) strain. For its cultivation the medium containing salts like potassium dihydrogen phosphate, magnesium sulfate and ammonium sulfate in optimum concentration, and also dioses (maltose, sucrose) and polysaccharides (starch, chitin, pectin) was chosen. The greatest zones of hydrolysis are traced during planting S 37-4 in agar medium containing maltose and low methoxyl citrus pectin. As the synthesis inductor of hemicellulase, MN and CT malt sprouts were used, and of PG - not clarified beet bin fibers. Cultivation was carried out on a thermostatically controlled shaker at 30 deg. C for 120 h. Increase of activity of synthesizable enzymes when using low methoxyl citrus pectin as a media part equaled for BG 5-19%, for PG - 25%, when using a maltose for CT - 100%, MN - 29%. To increase biosynthetic ability of S 37-4 as a mutagen 3-staged ultra-violet radiation (wavelength is 265 nanometers) was applied. The obtained 379-K-5 strain surpassed in activity level a parental strain BG - by 84.8%, CT - by 45.0%, MN - by 62.9%, PG - by 89.0%. The following (4th) stage of radiation led to death of the strain. In comparison with a parental S 37-4 the colony of a mutant strain possessed the bigger size and plentiful formation of an air mycelium, ability to sporogenesis was less expressed

  5. Naked mole-rats maintain healthy skeletal muscle and Complex IV mitochondrial enzyme function into old age.

    Science.gov (United States)

    Stoll, Elizabeth A; Karapavlovic, Nevena; Rosa, Hannah; Woodmass, Michael; Rygiel, Karolina; White, Kathryn; Turnbull, Douglass M; Faulkes, Chris G

    2016-12-19

    The naked mole-rat (NMR) Heterocephalus glaber is an exceptionally long-lived rodent, living up to 32 years in captivity. This extended lifespan is accompanied by a phenotype of negligible senescence, a phenomenon of very slow changes in the expected physiological characteristics with age. One of the many consequences of normal aging in mammals is the devastating and progressive loss of skeletal muscle, termed sarcopenia, caused in part by respiratory enzyme dysfunction within the mitochondria of skeletal muscle fibers. Here we report that NMRs avoid sarcopenia for decades. Muscle fiber integrity and mitochondrial ultrastructure are largely maintained in aged animals. While mitochondrial Complex IV expression and activity remains stable, Complex I expression is significantly decreased. We show that aged naked mole-rat skeletal muscle tissue contains some mitochondrial DNA rearrangements, although the common mitochondrial DNA deletions associated with aging in human and other rodent skeletal muscles are not present. Interestingly, NMR skeletal muscle fibers demonstrate a significant increase in mitochondrial DNA copy number. These results have intriguing implications for the role of mitochondria in aging, suggesting Complex IV, but not Complex I, function is maintained in the long-lived naked mole rat, where sarcopenia is avoided and healthy muscle function is maintained for decades.

  6. Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation.

    Science.gov (United States)

    Zhu, Shaotong; Vik, Steven B

    2015-08-21

    Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Alcohol--Induced Polyelectrolyte-Surfactant Complex Coacervate Systems: Characterization and Applications in Enzyme and Protein Extraction

    Science.gov (United States)

    Nejati Moshtaghin, Mahboubeh

    The focus of this thesis is to achieve a better understanding of the newly discovered surfactant-polyelectrolyte complex coacervate (SPCC) systems induced by fluoroalcohol/acid as well as short chain aliphatic alcohol; and to elucidate their applications in extraction and enrichment of proteins and enzyme. We have discovered that fluoroalcohols and --acids induce complex coacervation and phase separation in the aqueous mixtures of oppositely charged anionic polyelectrolytes; specifically, sodium salts of polyacrylic acid and polymethacrylic acid and cationic surfactant (cetyltrimethylammonium bromide, CTAB) over a broad range of concentrations of mole fractions of the oppositely charged amphiphiles. Accordingly, these new classes of coacervators will significantly broaden the scope and facilitate engineering of new coacervate phases. Toward these goals, we have inspected the formation of surfactant-polyelectrolyte complex coacervates in the presence of fluoroalcohols namely hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE). Furthermore, the extent of coacervation as a function of concentrations the system components, and charge ratios of the oppositely charged amphiphiles has been investigated. Polyelectrolytes are considered to be milder reagents, as compared to surfactants, regarding proteins denaturation. This highlights the importance of a detailed investigation of the efficiency of our coacervate systems for extraction and preconcentration of proteins and enzymes, especially, when the biological activity of the extracted proteins needs to be maintained based on the objectives mentioned above, the results of the investigations have been organized in four chapters. In Chapter II, the phase behavior of the FA-SPCC will be investigated. The objective is to examine the phase behavior and phase properties with respect to the extent of coacervation in different solution conditions. In particular, the effects of different solution variables such as concentration

  8. Enzyme-like catalysis via ternary complex mechanism: alkoxy-bridged dinuclear cobalt complex mediates chemoselective O-esterification over N-amidation.

    Science.gov (United States)

    Hayashi, Yukiko; Santoro, Stefano; Azuma, Yuki; Himo, Fahmi; Ohshima, Takashi; Mashima, Kazushi

    2013-04-24

    Hydroxy group-selective acylation in the presence of more nucleophilic amines was achieved using acetates of first-row late transition metals, such as Mn, Fe, Co, Cu, and Zn. Among them, cobalt(II) acetate was the best catalyst in terms of reactivity and selectivity. The combination of an octanuclear cobalt carboxylate cluster [Co4(OCOR)6O]2 (2a: R = CF3, 2b: R = CH3, 2c: R = (t)Bu) with nitrogen-containing ligands, such as 2,2'-bipyridine, provided an efficient catalytic system for transesterification, in which an alkoxide-bridged dinuclear complex, Co2(OCO(t)Bu)2(bpy)2(μ2-OCH2-C6H4-4-CH3)2 (10), was successfully isolated as a key intermediate. Kinetic studies and density functional theory calculations revealed Michaelis-Menten behavior of the complex 10 through an ordered ternary complex mechanism similar to dinuclear metallo-enzymes, suggesting the formation of alkoxides followed by coordination of the ester.

  9. Isolation and Screening of Potential Cellulolytic and Xylanolytic Bacteria from Soil Sample for Degradation of Lignocellulosic Biomass

    Directory of Open Access Journals (Sweden)

    Bhupal Govinda Shrestha

    2016-11-01

    them with the aptitude to produce stable enzymes, little emphasis has been given to cellulose/xylanase production from bacteria. Seven soil samples were collected from eastern hilly districts of Nepal viz. Taplejung, Panchthar and Sankhuwasabha districts, from soil surface and at depth of 10cm to 20cm, and were isolated separately. From the seven soil samples, four bacterial isolates were obtained. Isolates (PSS, P1D, TLC, SNK were then screened for cellulolytic/xylanolytic activity using Congo red assay on Carboxymethylcellulose (CMC/xylan agar plates. The enzyme activity obtained from isolates was dependent on substrate concentration. The activity of enzymes produced by isolates were also measured and compared on pretreated sugarcane bagasse. Among those samples, the greatest zone of inhibition in both CMC (1.3 cm and xylan (1.0 cm agar media was seen in isolate P1D. It also produced the highest activity of endoglucanase and xylanase i.e. activity 0.035 U/mL and 0.050 U/mL respectively at 0.010 mg mL-1 standard substrate concentration of CMC and xylan.

  10. The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function

    DEFF Research Database (Denmark)

    Rowland, Paul; Bjørnberg, Olof; Nielsen, Finn S.

    1998-01-01

    Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA......) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex...

  11. Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

    Science.gov (United States)

    Zhang, Jun; Zhang, Lei; Geng, Alei; Liu, Fanghua; Zhao, Guoping; Wang, Shengyue; Zhou, Zhihua; Yan, Xing

    2015-01-01

    Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future. PMID:26070087

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

    Directory of Open Access Journals (Sweden)

    P. Kang

    2013-02-01

    Full Text Available Paddy rice is rarely used as a feed because of its high fiber content. In this study, two experiments were conducted to study the effects of supplementing an enzyme complex consisting of xylanase, beta-glucanase and cellulase, to paddy-based diets on the performance and nutrient digestibility in meat-type ducks. In the both experiments, meat-type ducks (Cherry Valley were randomly assigned to four treatments. Treatment 1 was a basal diet of corn-soybean; treatment 2 was a basal diet of corn-paddy-soybean; treatment 3, had enzyme complex added to the corn-paddy-soybean basal diet at levels of 0.5 g/kg diet; and treatment 4, had enzyme complex added to the corn-paddy-soybean diet at levels of 1.0 g/kg diet. The results showed that the enzyme complex increased the ADG, and decreased the ADFI and F/G significantly (p0.05. The outcome of this research indicates that the application of enzyme complex made up of xylanase, beta-glucanase, and cellulase, in the corn-paddy-soybean diet, can improve performance and nutrition digestibility in meat-type ducks.

  13. A Rhodium(III) Complex as an Inhibitor of Neural Precursor Cell Expressed, Developmentally Down-Regulated 8-Activating Enzyme with in Vivo Activity against Inflammatory Bowel Disease.

    Science.gov (United States)

    Zhong, Hai-Jing; Wang, Wanhe; Kang, Tian-Shu; Yan, Hui; Yang, Yali; Xu, Lipeng; Wang, Yuqiang; Ma, Dik-Lung; Leung, Chung-Hang

    2017-01-12

    We report herein the identification of the rhodium(III) complex [Rh(phq) 2 (MOPIP)] + (1) as a potent and selective ATP-competitive neural precursor cell expressed, developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) inhibitor. Structure-activity relationship analysis indicated that the overall organometallic design of complex 1 was important for anti-inflammatory activity. Complex 1 showed promising anti-inflammatory activity in vivo for the potential treatment of inflammatory bowel disease.

  14. Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by family 63 inverting α-glycosidase.

    Science.gov (United States)

    Miyazaki, Takatsugu; Nishikawa, Atsushi; Tonozuka, Takashi

    2016-12-01

    Glycoside hydrolases are divided into two groups, known as inverting and retaining enzymes, based on their hydrolytic mechanisms. Glycoside hydrolase family 63 (GH63) is composed of inverting α-glycosidases, which act mainly on α-glucosides. We previously found that Escherichia coli GH63 enzyme, YgjK, can hydrolyze 2-O-α-d-glucosyl-d-galactose. Two constructed glycosynthase mutants, D324N and E727A, which catalyze the transfer of a β-glucosyl fluoride donor to galactose, lactose, and melibiose. Here, we determined the crystal structures of D324N and E727A soaked with a mixture of glucose and lactose at 1.8- and 2.1-Å resolutions, respectively. Because glucose and lactose molecules are found at the active sites in both structures, it is possible that these structures mimic the enzyme-product complex of YgjK. A glucose molecule found at subsite -1 in both structures adopts an unusual 1 S 3 skew-boat conformation. Comparison between these structures and the previously determined enzyme-substrate complex structure reveals that the glucose pyranose ring might be distorted immediately after nucleophilic attack by a water molecule. These structures represent the first enzyme-product complex for the GH63 family, as well as the structurally-related glycosidases, and it may provide insight into the catalytic mechanism of these enzymes. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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...

  16. Technique for preparation of anaerobic microbes: Rodshaped cellulolytic bacteria

    Directory of Open Access Journals (Sweden)

    Amlius Thalib

    2001-10-01

    Full Text Available Preparation of anaerobic-rod cellulolytic bacteria with coating technique has been conducted. Steps of the processes involved were cultivation, coating, evaporation, and drying. Coating agent used was Gum Arabic, and drying techniquesconducted were freeze drying and sun drying. pH of culture media was firstly optimized to obtain the maximal population ofbacteria. Both coated and uncoated preparates were subjected to drying. Morphological and Gram type identifications showed that uncoated preparate dried with freeze drying is not contaminated (ie. all bacteria are rod shape with Gram-negative type while the one dried with sun drying is not morphologically pure (ie. containing of both rod and coccus shapes with Gram negative and positive. The coated preparates dried by both freeze and sun drying, were not contaminated (ie. all are rods with Gram-negative. The coating and drying processes decreased viability of preparates significantly. However, the decreasing of viability of coated preparate are lower than uncoated preparate (ie. 89 vs. 97%. Total count of bacteria in sun-drying coated preparate are higher (P<0.05 than the uncoated preparate (ie. 3.38 x 1010 vs. 1.97 x 1010 colony/g DM. Activity of sun-drying coated preparate to digest elephant grass and rice straw was higher (P<0.01 than the sun-drying uncoated preparate with the in vitro DMD values were 42.7 vs. 35.5% for elephant grass substrate and 29.3 vs. 24.6% for rice straw substrate. Therefore, it is concluded that coating technique has a positive effects on the preparation of rumen bacteria.

  17. Enzyme-polyelectrolyte complexes in water-ethanol mixtures: negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects.

    Science.gov (United States)

    Kudryashova, E V; Gladilin, A K; Vakurov, A V; Heitz, F; Levashov, A V; Mozhaev, V V

    1997-07-20

    Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

  18. Isolation and Identification of cellulolytic bacteria from mangrove sediment in Bangka Island

    Science.gov (United States)

    Kurniawan, A.; Prihanto, A. A.; Sari, S. P.; Febriyanti, D.; Kurniawan, A.; Sambah, A. B.; Asriani, E.

    2018-04-01

    Cellulolytic bacteria is bacteria which hydrolyze cellulose to reducing sugars. This research aims to obtain cellulolytic bacteria from the sediment of mangroves in Bangka island. Reasearch was conducted from March to August 2017. Sampling was conducted at Sungailiat, and Tukak Sadai, South of Bangka. Bacteria was isolated using 1% Carboxymetyl Cellulosa (CMC). The isolation resulted in four isolates from Sungailiat and nine isolates from Tukak Sadai. Total five isolates, namely Bacillus pumilus, Pseudomonas sp., Bacillus amyloliquefacien, Bacillus alvei, Bacillus coagulant were identified. The best isolates that produced cellulose was Pseudomonas aeruginosa.

  19. Enzymatic degradation of cellulose for thermophilic actinomycete: isolation, characterization and cellulolytic activity determination

    Directory of Open Access Journals (Sweden)

    Pablo Ramírez

    2013-06-01

    Full Text Available One hundred and forty five cellulolytic thermophilic actinomycete strains were isolated from 71 compost, soil, hay and dung samples. Streptomyces sp. (50,63%, Thermomonospora curvata (15,82%, T. chromogena (13,92%, and other species were identified. Endoglucanase, exoglucanase and β-glucosidase activities were evaluated from 10 cellulolytic actinomycete strains. Among these the Streptomyces sp. 7CMC10 strain showed the biggest activity levels corresponding to 20,14; 2,61 and 5,40 UI/mg of protein, respectively.

  20. The AMPK enzyme-complex: From the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders

    NARCIS (Netherlands)

    Antonioli, Luca; Colucci, Rocchina; Pellegrini, Carolina; Giustarini, Giulio; Sacco, Deborah; Tirotta, Erika; Caputi, Valentina; Marsilio, Ilaria; Giron, Maria Cecilia; Németh, Zoltán H; Blandizzi, Corrado; Fornai, Matteo

    2016-01-01

    Introduction: Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular

  1. Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex

    NARCIS (Netherlands)

    Braun, P; Bitter, W; Tommassen, J

    2000-01-01

    The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes

  2. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    Science.gov (United States)

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-09

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

  3. Isolation and characterization of Caldicellulosiruptor lactoaceticus sp. nov., an extremely thermophilic, cellulolytic, anaerobic bacterium

    DEFF Research Database (Denmark)

    Mladenovska, Zuzana; Mathrani, Indra M.; Ahring, Birgitte Kiær

    1995-01-01

    An anaerobic, extremely thermophilic, cellulolytic, non-spore-forming bacterium, strain 6A, was isolated from an alkaline hot spring in Hverageroi, Iceland. The bacterium was non-motile, rod-shaped (1.5-3.5 x 0.7 mu m) and occurred singly, in pairs or in chains and stained gram-negative. The growth...

  4. Sample handling factors affecting the enumeration of lactobacilli and cellulolytic bacteria in equine feces

    Science.gov (United States)

    The objectives were to compare media types and evaluate the effects of fecal storage time and temperature on the enumeration of cellulolytic bacteria and lactobacilli from horses. Fecal samples were collected from horses (n = 3) and transported to the lab (CO2, 37 ºC, 0.5 h). The samples were assign...

  5. High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors and antihypertensive drugs.

    Science.gov (United States)

    Akif, Mohd; Georgiadis, Dimitris; Mahajan, Aman; Dive, Vincent; Sturrock, Edward D; Isaac, R Elwyn; Acharya, K Ravi

    2010-07-16

    Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE. 2010 Elsevier Ltd. All rights reserved.

  6. Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity

    Directory of Open Access Journals (Sweden)

    Wei Hui

    2012-04-01

    Full Text Available Abstract Background Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. Results In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera wood-chips and mown lawn grass clippings (85:15 in dry-weight and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. Conclusion The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP and solid-state fermentation for the production of cellulolytic enzymes and biofuels.

  7. Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity

    Science.gov (United States)

    2012-01-01

    Background Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. Results In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. Conclusion The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels. PMID:22490508

  8. In vitro antioxidant activity, enzyme kinetics, biostability and cellular SOD mimicking ability of 1:1 curcumin-copper (II) complex

    International Nuclear Information System (INIS)

    Kunwar, A.; Mishra, B.; Barik, A.; Priyadarsini, K.I.; Narang, H.; Krishna, M.

    2008-01-01

    In vitro antioxidant activity of 1:1 curcumin copper (II) complex was evaluated by following the inhibition of γ-radiation induced lipid peroxidation and protein oxidation in model systems. The SOD enzyme kinetic parameters K m and V max values and the turn over number of the complex were determined. The complex is stable in bio-fluids and prevents oxidation of lipid and protein solution in presence of H 2 O 2 and showed reduction in MnSOD level in spleen cells without having any effect on cell viability. (author)

  9. In vitro antioxidant activity, enzyme kinetics, biostability and cellular SOD mimicking ability of 1:1 curcumin-copper (II) complex

    Energy Technology Data Exchange (ETDEWEB)

    Kunwar, A; Mishra, B; Barik, A; Priyadarsini, K I [Radiation and Photochemistry Div., Bhabha Atomic Research Centre, Mumbai (India); Narang, H; Krishna, M [Radiation Biology and Health Sciences Div., Bhabha Atomic Research Centre, Mumbai (India)

    2008-01-15

    In vitro antioxidant activity of 1:1 curcumin copper (II) complex was evaluated by following the inhibition of {gamma}-radiation induced lipid peroxidation and protein oxidation in model systems. The SOD enzyme kinetic parameters K{sub m} and V{sub max} values and the turn over number of the complex were determined. The complex is stable in bio-fluids and prevents oxidation of lipid and protein solution in presence of H{sub 2}O{sub 2} and showed reduction in MnSOD level in spleen cells without having any effect on cell viability. (author)

  10. Cellulolytic activity of gut extract of subterranean termite ...

    African Journals Online (AJOL)

    Lignocellulosic biomass is a chief and cheap raw material for bioethanol production. However, pretreatment is a critical and most expensive step in lignocellulosic ... alternative for lignocellulosic biomass conversion using enzyme hydrolysis.

  11. FERROFLUIDS INFLUENCE ON DEHYDROGENASES ACTIVITY IN CELLULOLYTIC FUNGUS CHAETOMIUM GLOBOSUM

    Directory of Open Access Journals (Sweden)

    Alexandru Manoliu

    2003-08-01

    Different results were noticed for different ferrofluids concentrations: 20, 40, 60, 80 and 100 μl/L. Inhibitory or stimulatory ferrofluids effect was obtained depending on the nature of the investigated enzyme.

  12. Development of a commercial enzymes system for lignocellulosic biomass saccharification

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj

    2012-12-20

    DSM Innovation Inc., in its four year effort was able to evaluate and develop its in-house DSM fungal cellulolytic enzymes system to reach enzyme efficiency mandates set by DoE Biomass program MYPP goals. DSM enzyme cocktail is uniquely active at high temperature and acidic pH, offering many benefits and product differentiation in 2G bioethanol production. Under this project, strain and process development, ratio optimization of enzymes, protein and genetic engineering has led to multitudes of improvement in productivity and efficiency making development of a commercial enzyme system for lignocellulosic biomass saccharification viable. DSM is continuing further improvement by additional biodiversity screening, protein engineering and overexpression of enzymes to continue to further lower the cost of enzymes for saccharification of biomass.

  13. Effect of curcumin and curcumin copper complex (1:1) on radiation-induced changes of anti-oxidant enzymes levels in the livers of Swiss albino mice

    Energy Technology Data Exchange (ETDEWEB)

    Koiram, P R; Veerapur, V P; Mazhuvancherry, U K [Manipal Coll. of Pharmaceutical Sciences, Manipal (India); Kunwar, A; Mishra, B; Barik, A; Priyadarsini, I K [Bhabha Atomic Research Center, Mumbai (India)

    2007-05-15

    The effect of mononuclear copper (II) complex of curcumin in 1:1 stoichiometry (hereafter referred to as complex) administered 30 mim before {gamma}-irradiation (4.5 Gy) on alterations in antioxidant and Thiobarbituric acid reactive substances (TBARS) levels in livers was studied in comparison to curcumin at a dose of 50 mg/kg. The different antioxidants like glutathione (GSH), glutathione-S-transferase (GST), catalase, superoxide dismuatase (SOD), TBARS and total thiols were estimated in the liver homogenates excised at different time intervals (1, 2 and 4 h) post irradiation using colorimetric methods. There was a radiation-induced decrease in the levels of all the studied enzymes at 1 h post irradiation, while an increase was observed at later time points. Both curcumin and complex treatment in sham-irradiated mice decreased the levels of GSH and total thiols, whereas there was an increase in the levels of catalase, GST and SOD compared to normal control. Under the influence of irradiation, both curcumin and complex treatment protected the decline in the levels of GSH, GST, SOD, catalase and total thiols, and inhibited radiation-induced lipid peroxidation. Further, the complex was found to be more effective in protecting the enzymes at 1 h post irradiation compared to curcumin treated group. This may be due to the higher rate constants of the complex compared to curcumin for their reactions with various free radicals. (author)

  14. Effect of curcumin and curcumin copper complex (1:1) on radiation-induced changes of anti-oxidant enzymes levels in the livers of Swiss albino mice

    International Nuclear Information System (INIS)

    Koiram, P.R.; Veerapur, V.P.; Mazhuvancherry, U.K.; Kunwar, A.; Mishra, B.; Barik, A.; Priyadarsini, I.K.

    2007-01-01

    The effect of mononuclear copper (II) complex of curcumin in 1:1 stoichiometry (hereafter referred to as complex) administered 30 mim before γ-irradiation (4.5 Gy) on alterations in antioxidant and Thiobarbituric acid reactive substances (TBARS) levels in livers was studied in comparison to curcumin at a dose of 50 mg/kg. The different antioxidants like glutathione (GSH), glutathione-S-transferase (GST), catalase, superoxide dismuatase (SOD), TBARS and total thiols were estimated in the liver homogenates excised at different time intervals (1, 2 and 4 h) post irradiation using colorimetric methods. There was a radiation-induced decrease in the levels of all the studied enzymes at 1 h post irradiation, while an increase was observed at later time points. Both curcumin and complex treatment in sham-irradiated mice decreased the levels of GSH and total thiols, whereas there was an increase in the levels of catalase, GST and SOD compared to normal control. Under the influence of irradiation, both curcumin and complex treatment protected the decline in the levels of GSH, GST, SOD, catalase and total thiols, and inhibited radiation-induced lipid peroxidation. Further, the complex was found to be more effective in protecting the enzymes at 1 h post irradiation compared to curcumin treated group. This may be due to the higher rate constants of the complex compared to curcumin for their reactions with various free radicals. (author)

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

    Directory of Open Access Journals (Sweden)

    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

  16. Selection of cellulolytic fungi isolated from diverse substrates

    Directory of Open Access Journals (Sweden)

    Mônica Caramez Triches Damaso

    2012-08-01

    Full Text Available The aim of the present work was to select filamentous fungi isolated from diverse substrates to obtain the strains with potential to produce the hydrolytic enzymes. From a total of 215 strains, seven strains from the soils, six from the plants and one from sugarcane bagasse were selected and identified as belonging to the Trichoderma, Penicillium and Aspergillus genera. The best hydrolytic activities obtained by semi-solid fermentation using these strains were approximately: 35; 1; 160; 170 and 120 U/gdm (CMCase, FPase, β-glucosidase, xylanase and polygalacturonase, respectively, demonstrating their potential to synthesize the enzymes compared with the results reported in the literature.

  17. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    Directory of Open Access Journals (Sweden)

    Chattopadhyay Debasish

    2009-02-01

    Full Text Available Abstract Background The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. Results We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Å resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in

  18. Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica

    OpenAIRE

    Guilherme L. Pinheiro; Guilherme L. Pinheiro; Raquel eCorrea; Raquel eSoares; Alexander eCardoso; Catia eChaia; Mayssa M Clementino; Eloi eGarcia; Wanderley eDe Souza; Wanderley eDe Souza; Susana eFrases; Susana eFrases

    2015-01-01

    The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic bacterial divers...

  19. Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood

    Energy Technology Data Exchange (ETDEWEB)

    Mes-Hartree, M.; Hogan, C.M.; Saddler, J.N.

    1987-09-01

    The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the recalcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original filter paper activity could be recovered. When steam-treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis process, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of cellulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc. (Refs. 14).

  20. Iron and Zinc Complexes of Bulky Bis-Imidazole Ligands : Enzyme Mimicry and Ligand-Centered Redox Activity

    NARCIS (Netherlands)

    Folkertsma, E.

    2016-01-01

    The research described in this thesis is directed to the development of cheap and non-toxic iron-based homogeneous catalysts, using enzyme models and redox non-innocent ligands. Inspired by nature, the first approach focuses on the synthesis of structural models of the active site of non-heme iron

  1. Enzyme Kinetics Experiment with the Multienzyme Complex Viscozyme L and Two Substrates for the Accurate Determination of Michaelian Parameters

    Science.gov (United States)

    Guerra, Nelson Pérez

    2017-01-01

    A laboratory experiment in which students study the kinetics of the Viscozyme-L-catalyzed hydrolysis of cellulose and starch comparatively was designed for an upper-division biochemistry laboratory. The main objective of this experiment was to provide an opportunity to perform enhanced enzyme kinetics data analysis using appropriate informatics…

  2. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  3. Relationship between soil cellulolytic activity and suppression of seedling blight of barley in arable soils

    DEFF Research Database (Denmark)

    Rasmussen, Peter Have; Knudsen, I.; Elmholt, S.

    2002-01-01

    the Hanes-Wolf transformation of the Michaelis-Menten equation. Soil samples from 6 to 13 cm depth were collected in the early spring as undisturbed blocks from 10 arable soils with different physico-chemical properties and cultivation history. Significant correlations were found between soil suppresiveness......The objective was to investigate the relationship between soil suppression of seedling blight of barley caused by Fusarium culmorum (W.G. Smith) Sacc. and the soil cellulolytic activity of beta-glucosidase, cellobiohydrolase and endocellulase. Disease suppression was investigated in bioassays...... with test soils mixed with sand, and barley seeds inoculated with F. culmorum. After 19 days, disease severity was evaluated on the barley seedlings. Soil cellulolytic activities were measured using 4-methylumbelliferyl-labelled fluorogenic substrates, and were expressed as V-max values obtained by using...

  4. Enrichment and identification of cellulolytic bacteria from the gastrointestinal tract of Giant African snail, Achatina fulica.

    Science.gov (United States)

    Pawar, Kiran D; Dar, Mudasir A; Rajput, Bharati P; Kulkarni, Girish J

    2015-02-01

    The cellulolytic bacterial community structure in gastrointestinal (GI) tract of Achatina fulica was studied using culture-independent and -dependent methods by enrichment in carboxymethyl cellulose (CMC). Culture-dependent method indicated that GI tract of snail was dominated by Enterobacteriaceae members. When tested for cellulase activities, all isolates obtained by culture-dependent method showed both or either of CMCase or avicelase activity. Isolate identified as Citrobacter freundii showed highest CMCase and medium avicelase activity. Sequencing of clones from the 16S rRNA gene clone library identified ten operational taxonomic units (OTUs), which were affiliated to Enterobacteriaceae of phylum Gammaproteobacteria. Of these ten OTUs, eight OTUs closely matched with Enterobacter and Klebsiella genera. The most abundant OTU allied to Klebsiella oxytoca accounted for 70 % of the total sequences. The members of Klebsiella and Enterobacter were observed by both methods indicating their dominance among the cellulolytic bacterial community in the GI tract of the snail.

  5. Prompt and easy activation by specific thioredoxins of calvin cycle enzymes of Arabidopsis thaliana associated in the GAPDH/CP12/PRK supramolecular complex.

    Science.gov (United States)

    Marri, Lucia; Zaffagnini, Mirko; Collin, Valérie; Issakidis-Bourguet, Emmanuelle; Lemaire, Stéphane D; Pupillo, Paolo; Sparla, Francesca; Miginiac-Maslow, Myroslawa; Trost, Paolo

    2009-03-01

    The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

  6. Mesoporous silica nanoparticles supported copper(II) and nickel(II) Schiff base complexes: Synthesis, characterization, antibacterial activity and enzyme immobilization

    Science.gov (United States)

    Tahmasbi, Leila; Sedaghat, Tahereh; Motamedi, Hossein; Kooti, Mohammad

    2018-02-01

    Mesoporous silica nanoparticles (MSNs) were prepared by sol-gel method and functionalized with 3-aminopropyltriethoxysilane. Schiff base grafted mesoporous silica nanoparticle was synthesized by the condensation of 2-hydroxy-3-methoxybenzaldehyde and amine-functionalized MSNs. The latter material was then treated with Cu(II) and Ni(II) salts separately to obtain copper and nickel complexes anchored mesoporous composites. The newly prepared hybrid organic-inorganic nanocomposites have been characterized by several techniques such as FT-IR, LA-XRD, FE-SEM, TEM, EDS, BET and TGA. The results showed all samples have MCM-41 type ordered mesoporous structure and functionalization occurs mainly inside the mesopore channel. The presence of all elements in synthesized nanocomposites and the coordination of Schiff base via imine nitrogen and phenolate oxygen were confirmed. MSNs and all functionalized MSNs have uniform spherical nanoparticles with a mean diameter less than 100 nm. The as-synthesized mesoporous nanocomposites were investigated for antibacterial activity against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria, as carrier for gentamicin and also for immobilization of DNase, coagulase and amylase enzymes. MSN-SB-Ni indicated bacteriocidal effect against S.aureus and all compounds were found to be good carrier for gentamicin. Results of enzyme immobilization for DNase and coagulase and α-amylase revealed that supported metal complexes efficiently immobilized enzymes.

  7. Isolation and identification of cellulolytic bacteria from termites gut (Cryptotermes sp.)

    Science.gov (United States)

    Peristiwati; Natamihardja, Y. S.; Herlini, H.

    2018-05-01

    The energy and environmental crises developed due to a huge amount of cellulosic materials are disposed of as “waste.” Cellulose is the most abundant biopolymer on Earth. The hydrolysis of cellulose to glucose and soluble sugars has thus become a subject of intense research. Termites are one of the most important soil insects that efficiently decompose lignocelluloses with the aid of their associated microbial symbionts to a simpler form of sugars. The steps of this study consisted of cellulose isolation, cellulolytic bacteria isolation and identification. Cellulose degrading bacteria from termite (Cryptotermes sp.) gut flora were isolated, screened and their identification was studied which showed halo zones due to CMC agar. Among 12 isolates of bacteria, six isolates were cellulolytic. MLC-A isolate had shown a maximum in a cellulolytic index (1.32). Each isolate was identified based on standard physical and biochemical tests. Three isolates were identified in the genus of Clostridium, one isolate be placed in the group of Mycobacteriaceae, Lactobacillaceae or Coryneform and the last one in the genus Proteus.

  8. Evaluation in Cellulolytic Activity of Stenotrophomonas sp. in Cellulose Nitrogen Free Mineral Medium

    International Nuclear Information System (INIS)

    Honey Thet Paing Htway; San San Yu; Zaw Ko Latt

    2011-12-01

    Three bacterial strains were isolated from rice rhizospheric soil and their nitrogen fixing activity was determined in nitrogen free mineral medium and broth with glucose and cellulose as carbon sources and they produced ammonium concentration (above 3ppm) in G-NFFMM and (2-3ppm) in C-NFMM. Moreover, their cellulolytic activity was determined by DNS mothod and strain H3 having the cellulolytic activity was selected. Then, cellulose, carboxymethyl cellulose, baggasse, pea haulm, corn stem, rice straw were used as substrates and determined its reducing sugar concentration. After detection of the cellulolytic activity, the bacteria produced the highest concentration of reducing sugar on cellulose substrate at 12 day incubation period with the reducing sugar amount of 0.12mg/ml and 0.298mg/ml on CMC substrates. In the study of argicultral wastes as substrates, the selected strain, H3, produced in the reducing sugar concentration with 0.12, 0.116,0.103 and 0.098mg/ml respectively. The selected strain was identified by biochemical characterists and 16s ribosomal DNA analysis and it was Stenotrophomonas sp.

  9. Use of Cellulolytic Marine Bacteria for Enzymatic Pretreatment in Microalgal Biogas Production

    Science.gov (United States)

    Muñoz, Camilo; Hidalgo, Catalina; Zapata, Manuel; Jeison, David; Riquelme, Carlos

    2014-01-01

    In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis, a Chilean mussel, and four strains from Mesodesma donacium, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with “whole-cell” cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii, and R. ornithinolytica MC3 and MA5 degraded N. gaditana. In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production. PMID:24795376

  10. Cellulolytic potential under environmental changes in microbial communities from grassland litter

    Directory of Open Access Journals (Sweden)

    Renaud eBerlemont

    2014-11-01

    Full Text Available In many ecosystems, global changes are likely to profoundly affect microorganisms. In Southern California, changes in precipitation and nitrogen deposition may influence the composition and functional potential of microbial communities and their resulting ability to degrade plant material. To test whether environmental changes impact the distribution of functional groups involved in leaf litter degradation, we determined how the genomic diversity of microbial communities in a semi-arid grassland ecosystem changed under reduced precipitation or increased N deposition. We monitored communities seasonally over a period of two years to place environmental change responses into the context of natural variation. Fungal and bacterial communities displayed strong seasonal patterns, Fungi being mostly detected during the dry season whereas Bacteria were common during wet periods. Most putative cellulose degraders were associated with 33 bacterial genera and constituted ~18.2% of the microbial community. Precipitation reduction reduced bacterial abundance and cellulolytic potential whereas nitrogen addition did not affect the cellulolytic potential of the microbial community. Finally, we detected a strong correlation between the frequencies of genera putative cellulose degraders and cellulase genes. Thus, microbial taxonomic composition was predictive of cellulolytic potential. This work provides a framework for how environmental changes affect microorganisms responsible for plant litter deconstruction.

  11. MOLECULAR MODELLING OF HUMAN ALDEHYDE OXIDASE AND IDENTIFICATION OF THE KEY INTERACTIONS IN THE ENZYME-SUBSTRATE COMPLEX

    Directory of Open Access Journals (Sweden)

    Siavoush Dastmalchi

    2005-05-01

    Full Text Available Aldehyde oxidase (EC 1.2.3.1, a cytosolic enzyme containing FAD, molybdenum and iron-sulphur cluster, is a member of non-cytochrome P-450 enzymes called molybdenum hydroxylases which is involved in the metabolism of a wide range of endogenous compounds and many drug substances. Drug metabolism is one of the important characteristics which influences many aspects of a therapeutic agent such as routes of administration, drug interaction and toxicity and therefore, characterisation of the key interactions between enzymes and substrates is very important from drug development point of view. The aim of this study was to generate a three-dimensional model of human aldehyde oxidase (AO in order to assist us to identify the mode of interaction between enzyme and a set of phethalazine/quinazoline derivatives. Both sequence-based (BLAST and inverse protein fold recognition methods (THREADER were used to identify the crystal structure of bovine xanthine dehydrogenase (pdb code of 1FO4 as the suitable template for comparative modelling of human AO. Model structure was generated by aligning and then threading the sequence of human AO onto the template structure, incorporating the associated cofactors, and molecular dynamics simulations and energy minimization using GROMACS program. Different criteria which were measured by the PROCHECK, QPACK, VERIFY-3D were indicative of a proper fold for the predicted structural model of human AO. For example, 97.9 percentages of phi and psi angles were in the favoured and most favoured regions in the ramachandran plot, and all residues in the model are assigned environmentally positive compatibility scores. Further evaluation on the model quality was performed by investigation of AO-mediated oxidation of a set of phthalazine/quinazoline derivatives to develop QSAR model capable of describing the extent of the oxidation. Substrates were aligned by docking onto the active site of the enzyme using GOLD technology and then

  12. Host-Symbiont Cospeciation of Termite-Gut Cellulolytic Protists of the Genera Teranympha and Eucomonympha and their Treponema Endosymbionts.

    Science.gov (United States)

    Noda, Satoko; Shimizu, Daichi; Yuki, Masahiro; Kitade, Osamu; Ohkuma, Moriya

    2018-03-29

    Cellulolytic flagellated protists inhabit the hindgut of termites. They are unique and essential to termites and related wood-feeding cockroaches, enabling host feeding on cellulosic matter. Protists of two genera in the family Teranymphidae (phylum Parabasalia), Eucomonympha and Teranympha, are phylogenetically closely related and harbor intracellular endosymbiotic bacteria from the genus Treponema. In order to obtain a clearer understanding of the evolutionary history of this triplex symbiotic relationship, the molecular phylogenies of the three symbiotic partners, the Teranymphidae protists, their Treponema endosymbionts, and their host termites, were inferred and compared. Strong congruence was observed in the tree topologies of all interacting partners, implying their cospeciating relationships. In contrast, the coevolutionary relationship between the Eucomonympha protists and their endosymbionts was more complex, and evidence of incongruence against cospeciating relationships suggested frequent host switches of the endosymbionts, possibly because multiple Eucomonympha species are present in the same gut community. Similarities in the 16S rRNA and gyrB gene sequences of the endosymbionts were higher among Teranympha spp. (>99.25% and >97.2%, respectively), whereas those between Teranympha and Eucomonympha were lower (<97.1% and <91.9%, respectively). In addition, the endosymbionts of Teranympha spp. formed a phylogenetic clade distinct from those of Eucomonympha spp. Therefore, the endosymbiont species of Teranympha spp., designated here as "Candidatus Treponema teratonymphae", needs to be classified as a species distinct from the endosymbiont species of Eucomonympha spp.

  13. Optimization of parameters for enhanced oil recovery from enzyme treated wild apricot kernels.

    Science.gov (United States)

    Rajaram, Mahatre R; Kumbhar, Baburao K; Singh, Anupama; Lohani, Umesh Chandra; Shahi, Navin C

    2012-08-01

    Present investigation was undertaken with the overall objective of optimizing the enzymatic parameters i.e. moisture content during hydrolysis, enzyme concentration, enzyme ratio and incubation period on wild apricot kernel processing for better oil extractability and increased oil recovery. Response surface methodology was adopted in the experimental design. A central composite rotatable design of four variables at five levels was chosen. The parameters and their range for the experiments were moisture content during hydrolysis (20-32%, w.b.), enzyme concentration (12-16% v/w of sample), combination of pectolytic and cellulolytic enzyme i.e. enzyme ratio (30:70-70:30) and incubation period (12-16 h). Aspergillus foetidus and Trichoderma viride was used for production of crude enzyme i.e. pectolytic and cellulolytic enzyme respectively. A complete second order model for increased oil recovery as the function of enzymatic parameters fitted the data well. The best fit model for oil recovery was also developed. The effect of various parameters on increased oil recovery was determined at linear, quadric and interaction level. The increased oil recovery ranged from 0.14 to 2.53%. The corresponding conditions for maximum oil recovery were 23% (w.b.), 15 v/w of the sample, 60:40 (pectolytic:cellulolytic), 13 h. Results of the study indicated that incubation period during enzymatic hydrolysis is the most important factor affecting oil yield followed by enzyme ratio, moisture content and enzyme concentration in the decreasing order. Enzyme ratio, incubation period and moisture content had insignificant effect on oil recovery. Second order model for increased oil recovery as a function of enzymatic hydrolysis parameters predicted the data adequately.

  14. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: Implications for the enzyme mechanism‡

    OpenAIRE

    Ogura, Hiroshi; Evans, John P.; Peng, Dungeng; Satterlee, James D.; de Montellano, Paul R. Ortiz; Mar, Gerd N. La

    2009-01-01

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase-1, (hHO) has been investigated by 1H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. 2D 1H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts, that places the lone iron π-spin in the dxz orbital, rather than the dyz orbital found in the cya...

  15. The crystal structure of an intermediate dimer of aspergilloglutamic peptidase that mimics the enzyme-activation product complex produced upon autoproteolysis.

    Science.gov (United States)

    Sasaki, Hiroshi; Kubota, Keiko; Lee, Woo C; Ohtsuka, Jun; Kojima, Masaki; Iwata, So; Nakagawa, Atsushi; Takahashi, Kenji; Tanokura, Masaru

    2012-07-01

    Aspergilloglutamic peptidase from Aspergillus niger var. macrosporus (AGP) is one of the so-called pepstatin-insensitive acid endopeptidases, which are distinct from the well-studied aspartic peptidases. Among the known homologues of the glutamic peptidases, AGP is a unique two-chain enzyme with a light chain and a heavy chain bound non-covalently with each other, and thus is an interesting target for protein structure-function relationship studies. In this article, we report the crystal structure of a dimeric form of the enzyme at a resolution of 1.6 Å. This form has a unique structure in which the C-terminal region of the light chain of one of the molecules binds to the active site cleft of the other molecule like a part of a substrate. This form mimics the enzyme-activation product complex produced upon autoproteolysis, and provides a structural clue that could help to clarify the activation mechanism. This type of dimeric structure of a peptidase is here reported for the first time.

  16. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Gary [Los Alamos National Laboratory; Detter, Chris [Los Alamos National Laboratory; Bruce, David [Los Alamos National Laboratory; Challacome, Jean F [Los Alamos National Laboratory; Brettin, Thomas S [Los Alamos National Laboratory; Barabote, Ravi D [UC DAVIS; Leu, David [UC DAVIS; Normand, Philippe [CNRS, UNIV LYON; Necsula, Anamaria [CNRS, UNIV LYON; Daubin, Vincent [CNRS, UNIV LYON; Medigue, Claudine [CNRS/GENOSCOPE; Adney, William S [NREL; Xu, Xin C [UC DAVIS; Lapidus, Alla [DOE JOINT GENOME INST.; Pujic, Pierre [CNRS, UNIV LYON; Richardson, Paul [DOE JOINT GENOME INST; Berry, Alison M [UC DAVIS

    2008-01-01

    We present here the complete 2.4 MB genome of the actinobacterial thermophile, Acidothermus cellulolyticus lIB, that surprisingly reveals thermophilic amino acid usage in only the cytosolic subproteome rather than its whole proteome. Thermophilic amino acid usage in the partial proteome implies a recent, ongoing evolution of the A. cellulolyticus genome since its divergence about 200-250 million years ago from its closest phylogenetic neighbor Frankia, a mesophilic plant symbiont. Differential amino acid usage in the predicted subproteomes of A. cellulolyticus likely reflects a stepwise evolutionary process of modern thermophiles in general. An unusual occurrence of higher G+C in the non-coding DNA than in the transcribed genome reinforces a late evolution from a higher G+C common ancestor. Comparative analyses of the A. cellulolyticus genome with those of Frankia and other closely-related actinobacteria revealed that A. cellulolyticus genes exhibit reciprocal purine preferences at the first and third codon positions, perhaps reflecting a subtle preference for the dinucleotide AG in its mRNAs, a possible adaptation to a thermophilic environment. Other interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote reveal streamlining for adaptation to its specialized ecological niche. These include a low occurrence of pseudogenes or mobile genetic elements, a flagellar gene complement previously unknown in this organism, and presence of laterally-acquired genomic islands of likely ecophysiological value. New glycoside hydrolases relevant for lignocellulosic biomass deconstruction were identified in the genome, indicating a diverse biomass-degrading enzyme repertoire several-fold greater than previously characterized, and significantly elevating the industrial value of this organism.

  17. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Gary [Los Alamos National Laboratory; Detter, John C [Los Alamos National Laboratory; Bruce, David C [Los Alamos National Laboratory; Challacombe, Jean F [Los Alamos National Laboratory; Brettin, Thomas S [Los Alamos National Laboratory; Necsulea, Anamaria [UNIV LYON; Daubin, Vincent [UNIV LYON; Medigue, Claudine [GENOSCOPE; Adney, William S [NREL; Xu, Xin C [UC DAVIS; Lapidus, Alla [JGI; Pujic, Pierre [UNIV LYON; Berry, Alison M [UC DAVIS; Barabote, Ravi D [UC DAVIS; Leu, David [UC DAVIS; Normand, Phillipe [UNIV LYON

    2009-01-01

    We present here the complete 2.4 MB genome of the actinobacterial thermophile, Acidothermus cellulolyticus 11B, that surprisingly reveals thermophilic amino acid usage in only the cytosolic subproteome rather than its whole proteome. Thermophilic amino acid usage in the partial proteome implies a recent, ongoing evolution of the A. cellulolyticus genome since its divergence about 200-250 million years ago from its closest phylogenetic neighbor Frankia, a mesophilic plant symbiont. Differential amino acid usage in the predicted subproteomes of A. cellulolyticus likely reflects a stepwise evolutionary process of modern thermophiles in general. An unusual occurrence of higher G+C in the non-coding DNA than in the transcribed genome reinforces a late evolution from a higher G+C common ancestor. Comparative analyses of the A. cellulolyticus genome with those of Frankia and other closely-related actinobacteria revealed that A. cellulolyticus genes exhibit reciprocal purine preferences at the first and third codon positions, perhaps reflecting a subtle preference for the dinucleotide AG in its mRNAs, a possible adaptation to a thermophilic environment. Other interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote reveal streamlining for adaptation to its specialized ecological niche. These include a low occurrence of pseudo genes or mobile genetic elements, a flagellar gene complement previously unknown in this organism, and presence of laterally-acquired genomic islands of likely ecophysiological value. New glycoside hydrolases relevant for lignocellulosic biomass deconstruction were identified in the genome, indicating a diverse biomass-degrading enzyme repertoire several-fold greater than previously characterized, and significantly elevating the industrial value of this organism.

  18. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: Implications for the enzyme mechanism‡

    Science.gov (United States)

    Ogura, Hiroshi; Evans, John P.; Peng, Dungeng; Satterlee, James D.; de Montellano, Paul R. Ortiz; Mar, Gerd N. La

    2009-01-01

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase-1, (hHO) has been investigated by 1H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. 2D 1H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts, that places the lone iron π-spin in the dxz orbital, rather than the dyz orbital found in the cyanide complex. Comparison of iron spin relaxivity, magnetic anisotropy and magnetic susceptibilities argues for a low-spin, (dxy)2(dyz,dxz)3, ground state in both azide and cyanide complexes. The switch from singly-occupied dyz for the cyanide to dxz for the azide complex of hHO is shown to be consistent with the orbital hole determined by the azide π-plane in the latter complex, which is ∼90° in-plane rotated from that of the imidazole π-plane. The induction of the altered orbital ground state in the azide relative to the cyanide hHO complex, as well as the mean low-field bias of methyl hyperfine shifts and their paramagnetic relaxivity relative to those in globins, indicate that azide exerts a stronger ligand field in hHO than in the globins, or that the distal H-bonding to azide is weaker in hHO than in globins. The Asp140 → Ala hHO mutant that abolishes activity retains the unusual WT azide complex spin/orbital ground state. The relevance of our findings for other HO complexes and the HO mechanism is discussed. PMID:19243105

  19. The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: implications for the enzyme mechanism.

    Science.gov (United States)

    Ogura, Hiroshi; Evans, John P; Peng, Dungeng; Satterlee, James D; Ortiz de Montellano, Paul R; La Mar, Gerd N

    2009-04-14

    The active site electronic structure of the azide complex of substrate-bound human heme oxygenase 1 (hHO) has been investigated by (1)H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. Two-dimensional (1)H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts that places the lone iron pi-spin in the d(xz) orbital, rather than the d(yz) orbital found in the cyanide complex. Comparison of iron spin relaxivity, magnetic anisotropy, and magnetic susceptibilities argues for a low-spin, (d(xy))(2)(d(yz),d(xz))(3), ground state in both azide and cyanide complexes. The switch from singly occupied d(yz) for the cyanide to d(xz) for the azide complex of hHO is shown to be consistent with the orbital hole determined by the azide pi-plane in the latter complex, which is approximately 90 degrees in-plane rotated from that of the imidazole pi-plane. The induction of the altered orbital ground state in the azide relative to the cyanide hHO complex, as well as the mean low-field bias of methyl hyperfine shifts and their paramagnetic relaxivity relative to those in globins, indicates that azide exerts a stronger ligand field in hHO than in the globins, or that the distal H-bonding to azide is weaker in hHO than in globins. The Asp140 --> Ala hHO mutant that abolishes activity retains the unusual WT azide complex spin/orbital ground state. The relevance of our findings for other HO complexes and the HO mechanism is discussed.

  20. An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish; (UAB)

    2009-06-08

    The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate

  1. Isolation and characterization of efficient cellulolytic fungi from ...

    African Journals Online (AJOL)

    user

    potentially sustainable approach to develop novel bioprocesses and products. Microbial cellulases have become the focal biocatalysts due to their complex nature and wide spread industrial applications (Kuhad et al.,. 2011). The search for efficient microorganisms which can produce all the three types of cellulases that can ...

  2. Production and characterization of cellulolytic activities produced by ...

    African Journals Online (AJOL)

    Hind

    2013-01-30

    Jan 30, 2013 ... These activities were stable at 50°C after 5 h incubation in a pH ... chemical methods for environmental reasons. ... components in a complex in which the product of one ... high yielding parent strain of many commercially strains .... variance (ANOVA) and expressed as mean ± standard deviation (SD).

  3. Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts.

    Science.gov (United States)

    Liwak-Muir, Urszula; Mamady, Hapsatou; Naas, Turaya; Wylie, Quinlan; McBride, Skye; Lines, Matthew; Michaud, Jean; Baird, Stephen D; Chakraborty, Pranesh K; Holcik, Martin

    2016-06-18

    SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known. We show that the disease causing mutations in patient-derived fibroblasts do not affect subcellular localization of TRNT1 and show no gross morphological differences when compared to control cells. Analysis of cellular respiration and oxidative phosphorylation (OXPHOS) complexes demonstrates that both basal and maximal respiration rates are decreased in patient cells, which may be attributed to an observed decrease in the abundance of select proteins of the OXPHOS complexes. Our data provides further insight into cellular pathophysiology of SIFD.

  4. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

    Science.gov (United States)

    Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

    2009-06-01

    Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a

  5. Biomass production and secretion of hydrolytic enzymes are influenced by the structural complexity of the nitrogen source in Fusarium oxysporum and Aspergillus nidulans.

    Science.gov (United States)

    da Silva, M C; Bertolini, M C; Ernandes, J R

    2001-01-01

    The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.

  6. Evaluation of liver enzyme levels in workers exposed to vinyl chloride vapors in a petrochemical complex: a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Dolati Mandana

    2007-08-01

    Full Text Available Abstract Background Polyvinyl chloride is used in production and manufacturing of many essential tools (e.g. plastic pipes, photography films, etc.. Its production is impossible without the use of vinyl chloride monomer (VCM, which can cause liver damage in long-term. In this study we intend to assess the effects of mild to moderate long term exposure to VCM on liver and to assess the importance of liver enzyme measurements as a screening tool. Methods In this study, liver enzyme levels of 52 workers were compared to 48 control workers using the T-test. The cases all worked in a PVC production unit in a petrochemical complex and the controls were randomly selected from office personnel of the same complex. A questionnaire was also filled in about information such as age, weight, work history, etc. in both groups. Results Mean comparisons for ALP and GGT using T-test showed statistically significant differences between the two groups. For AST, ALT and bilirubin (total, direct the mean was higher in the case group but this difference was not statistically significant. Discussion This study showed that mild exposure to VCM can cause mild liver cholestasis. So, using cholestasis assessment tests such as ALP and GGT should be considered in periodic assessment of liver function in PVC producing units.

  7. Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase.adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

    International Nuclear Information System (INIS)

    Renosto, F.; Martin, R.L.; Segel, I.H.

    1989-01-01

    At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no ''substrate channeling'' of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [ 35 S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a ''3'-phosphoadenosine 5'-phosphosulfate synthetase'' complex

  8. Anticancer efficacy of a supramolecular complex of a 2-diethylaminoethyl–dextran–MMA graft copolymer and paclitaxel used as an artificial enzyme

    Directory of Open Access Journals (Sweden)

    Yasuhiko Onishi

    2014-12-01

    Full Text Available The anticancer efficacy of a supramolecular complex that was used as an artificial enzyme against multi-drug-resistant cancer cells was confirmed. A complex of diethylaminoethyl–dextran–methacrylic acid methylester copolymer (DDMC/paclitaxel (PTX, obtained with PTX as the guest and DDMC as the host, formed a nanoparticle 50–300 nm in size. This complex is considered to be useful as a drug delivery system (DDS for anticancer compounds since it formed a stable polymeric micelle in water. The resistance of B16F10 melanoma cells to PTX was shown clearly through a maximum survival curve. Conversely, the DDMC/PTX complex showed a superior anticancer efficacy and cell killing rate, as determined through a Michaelis–Menten-type equation, which may promote an allosteric supramolecular reaction to tubulin, in the same manner as an enzymatic reaction. The DDMC/PTX complex showed significantly higher anticancer activity compared to PTX alone in mouse skin in vivo. The median survival times of the saline, PTX, DDMC/PTX4 (particle size 50 nm, and DDMC/PTX5 (particle size 290 nm groups were 120 h (treatment (T/control (C, 1.0, 176 h (T/C, 1.46, 328 h (T/C, 2.73, and 280 h (T/C, 2.33, respectively. The supramolecular DDMC/PTX complex showed twice the effectiveness of PTX alone (p < 0.036. Above all, the DDMC/PTX complex is not degraded in cells and acts as an intact supramolecular assembly, which adds a new species to the range of DDS.

  9. Crystal structure of an essential enzyme in seed starch degradation - barley limit dextrinase in complex with cyclodextrins

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Abou Hachem, Maher; Svensson, Birte

    2010-01-01

    LD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight...

  10. Identification and functional analysis of the L-ascorbate-specific enzyme II complex of the phosphotransferase system in Streptococcus mutans.

    Science.gov (United States)

    Wu, Xinyu; Hou, Jin; Chen, Xiaodan; Chen, Xuan; Zhao, Wanghong

    2016-03-22

    Streptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic acids that cause enamel demineralization. Phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays an important role in carbohydrates uptake of S. mutans. The ptxA and ptxB genes in S. mutans encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS. The aim of this study was to analyze the function of these proteins and understand the transcriptional regulatory mechanism. ptxA (-), ptxB (-), as well as ptxA (-) , ptxB (-) double-deletion mutants all had more extended lag phase and lower growth yield than wild-type strain UA159 when grown in the medium using L-ascorbate as the sole carbon source. Acid production and acid killing assays showed that the absence of the ptxA and ptxB genes resulted in a reduction in the capacity for acidogenesis, and all three mutant strains did not survive an acid shock. According to biofilm and extracellular polysaccharides (EPS) formation analysis, all the mutant strains formed much less prolific biofilms with small amounts of EPS than wild-type UA159 when using L-ascorbate as the sole carbon source. Moreover, PCR analysis and quantitative real-time PCR revealed that sgaT, ptxA, ptxB, SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon. The transcription levels of these genes were all elevated in the presence of L-ascorbate, and the expression of ptxA gene decreased significantly once ptxB gene was knockout. The ptxA and ptxB genes are involved in the growth, aciduricity, acidogenesis, and formation of biofilms and EPS of S. mutans when L-ascorbate is the sole carbon source. In addition, the expression of ptxA is regulated by ptxB. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon, and L-ascorbate is a potential inducer of the operon.

  11. Isolation, screening, and identification of potential cellulolytic and xylanolytic producers for biodegradation of untreated oil palm trunk and its application in saccharification of lemongrass leaves.

    Science.gov (United States)

    Ang, S K; Yahya, Adibah; Abd Aziz, Suraini; Md Salleh, Madihah

    2015-01-01

    This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g(-1)), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g(-1)) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid).

  12. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

    2015-11-04

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  13. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2015-09-08

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  14. Characterization of a chitinase from the cellulolytic actinomycete Thermobifida fusca.

    Science.gov (United States)

    Gaber, Yasser; Mekasha, Sophanit; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H; Fraaije, Marco W

    2016-09-01

    Thermobifida fusca is a well-known cellulose-degrading actinomycete, which produces various glycoside hydrolases for this purpose. However, despite the presence of putative chitinase genes in its genome, T. fusca has not been reported to grow on chitin as sole carbon source. In this study, a gene encoding a putative membrane-anchored GH18 chitinase (Tfu0868) from T. fusca has been cloned and overexpressed in Escherichia coli. The protein was produced as SUMO fusion protein and, upon removal of the SUMO domain, soluble pure TfChi18A was obtained with yields typically amounting to 150mg per litre of culture. The enzyme was found to be relatively thermostable (apparent Tm=57.5°C) but not particularly thermoactive, the optimum temperature being 40-45°C. TfChi18A bound to α- and β-chitin and degraded both these substrates. Interestingly, activity towards colloidal chitin was minimal and in this case, substrate inhibition was observed. TfChi18A also cleaved soluble chito-oligosaccharides and showed a clear preference for substrates having five sugars or more. While these results show that TfChi18A is a catalytically competent GH18 chitinase, the observed catalytic rates were low compared to those of well-studied GH18 chitinases. This suggests that TfChi18A is not a true chitinase and not likely to endow T. fusca with the ability to grow on chitin. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Cellulolytic and Butyrivibrio fibrisolvens bacteria population density, after supplementing fodder diets (Pennisetum clandestinum

    Directory of Open Access Journals (Sweden)

    Licet Molina G

    2015-11-01

    Full Text Available Objective. Determine the population density of cellulolytic bacteria, Butyrivibrio fibrisolvens and the concentration of vaccenic acid, by supplementing diets consisting of kikuyu grass (Pennisetum clandestinum Hoechst. Ex Chiov. as base ingredient, together with cassava flour and biomass (effluent from ethanol production in rumen simulator-Rusitec. Materials and methods. Four treatments (T were evaluated, these were composed as: T1/Control 1: 100% kikuyu grass with a total protein intake of 23.9%, T2: a mixture of 70% kikuyu grass, 20% biomass and 10% cassava flour with a total protein intake of 19.4%; T3/Control 2: 100% kikuyu grass, with a 17.8% protein intake and T4: 70% kikuyu grass, 20% biomass and 10% cassava flour with a 15.3% protein intake. One and two-way variance analysis was made and the Pearson correlation coefficient was determined. Results. An increase was observed in the population density of viable cellulolytic bacteria (CFU/ml and B. fibrisolvens statistically significant (p<0.005 with treatment T2, in contrast to T1, T3 and T4 treatments. In addition, there was a significant increase in the concentration of vaccenic acid (mg/L in the ruminal content in Rusitec with the same treatment (T2. Conclusions. Results obtained in this ruminal simulation study are evidence to the benefits of kikuyu grass together with cassava flour and biomass diet implementation on the growth of ruminal cellulolytic and B. fibrisolvens bacteria, as well as on the production of vaccenic acid. The study also suggests the nutritional potential that such supplements could provide to grazing bovine feeding.

  16. Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

    Science.gov (United States)

    Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

    2017-10-27

    G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Pancreatic Enzymes

    Science.gov (United States)

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  18. Photoactive protochlorophyllide-enzyme complexes reconstituted with PORA, PORB and PORC proteins of A. thaliana: fluorescence and catalytic properties.

    Directory of Open Access Journals (Sweden)

    Michał Gabruk

    Full Text Available Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide and recombinant light-dependent protochlorophyllide oxidoreductase (POR proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.

  19. Cytokinin oxidase from Phaseolus vulgaris callus tissues. Enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes

    International Nuclear Information System (INIS)

    Chatfield, J.M.; Armstrong, D.J.

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N 6 -(Δ 2 -isopentenyl)-adenine-2,8- 3 H (i 6 Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N 6 -side chain of i 6 Ade

  20. EFFECT OF POLYPHENOLIC COMPLEX FROM WINE ON RATS ANTIOXIDANT ENZYMES ACTIVITY AT X-RAY IRRADIATION LOW DOSES

    Directory of Open Access Journals (Sweden)

    U. V. Datsyuk

    2014-04-01

    Full Text Available It is shown that the consumption of natural polyphenolic complex from grape wine in drinking water in the daily dose 2.5 ± 1.1 mg polyphenols/kg body mass of rats during the 10 day before exposure to radiation leads to increased of superoxide dismutase and gluthathione reductase activities in peripheral blood on 24 and 48 hours after full body X-ray irradiation (30 cGy. The of catalase, gluthathione peroxidase activities and the of the reactive thiobarbituric acid substances content in total lysates of peripheral blood within 72 hours after exposure are comparable to those in control rats. Marked decreased of catalase and superoxide dismutase activities at 24, 48 and 24 hours, respectively, was observed after exposure to ionizing radiation and increased content of lipid peroxidation products in all above mentioned time points. The decreased of superoxide dismutase and gluthathione peroxidase activities in lysates of rats aorta at 48 hour and increased content of the reactive thiobarbituric acid substances during 72 hours after radiation exposure were observed. The consumption of polyphenolic complex from wine did not change the superoxide dismutase and catalase activities in lysates of aorta rats treated with ionizing radiation, whereas gluthathione reductase and gluthathione peroxidase activities was increased during 72 hours after radiation influence. The content of TBA reactive substances was significantly decreased in lysates of aorta rats that were exposed to radiation and polyphenols of grape wine, compared with those of animals that were exposed to radiation alone.

  1. STAY-GREEN and Chlorophyll Catabolic Enzymes Interact at Light-Harvesting Complex II for Chlorophyll Detoxification during Leaf Senescence in Arabidopsis[C][W

    Science.gov (United States)

    Sakuraba, Yasuhito; Schelbert, Silvia; Park, So-Yon; Han, Su-Hyun; Lee, Byoung-Doo; Andrès, Céline Besagni; Kessler, Felix; Hörtensteiner, Stefan; Paek, Nam-Chon

    2012-01-01

    During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel’s green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC. PMID:22366162

  2. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test

    International Nuclear Information System (INIS)

    Casali, P.; Bossus, A.; Carpentier, N.A.; Lambert, P.H.

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample was incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes were detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test did not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG was 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the 125 I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, 125 I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application. (author)

  3. Hypothesized diprotomeric enzyme complex supported by stochastic modelling of palytoxin-induced Na/K pump channels.

    Science.gov (United States)

    Vilallonga, Gabriel D; de Almeida, Antônio-Carlos G; Ribeiro, Kelison T; Campos, Sergio V A; Rodrigues, Antônio M

    2018-03-01

    The sodium-potassium pump (Na + /K + pump) is crucial for cell physiology. Despite great advances in the understanding of this ionic pumping system, its mechanism is not completely understood. We propose the use of a statistical model checker to investigate palytoxin (PTX)-induced Na + /K + pump channels. We modelled a system of reactions representing transitions between the conformational substates of the channel with parameters, concentrations of the substates and reaction rates extracted from simulations reported in the literature, based on electrophysiological recordings in a whole-cell configuration. The model was implemented using the UPPAAL-SMC platform. Comparing simulations and probabilistic queries from stochastic system semantics with experimental data, it was possible to propose additional reactions to reproduce the single-channel dynamic. The probabilistic analyses and simulations suggest that the PTX-induced Na + /K + pump channel functions as a diprotomeric complex in which protein-protein interactions increase the affinity of the Na + /K + pump for PTX.

  4. In vitro Cellulose Rich Organic Material Degradation by Cellulolytic Streptomyces albospinus (MTCC 8768

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    Pinky Prasad

    2012-09-01

    Full Text Available Aims: Cellulosic biomass is the only foreseeable sustainable source of fuels and is also one of the dominating waste materials in nature resulting from human activities. Keeping in view the environmental problems like disposal of large volumes of cellulosic wastes and shortage of fossil fuel in the world, the main aim of the present investigation was to characterize and study the cellulolytic activity of Streptomyces albospinus (MTCC 8768, isolated from municipal wastes, on natural cellulosic substrates viz. straw powder, wood powder and finely grated vegetable peels.Methodology and Result: Stanier’s Basal broth with 100 mg of each of the substrates was inoculated separately with S. albospinus (MTCC No. 8768 and incubated at 37 °C for 8 days. The cellulosic substrates were re-weighed at an interval of 2 days and the difference between the initial weight and the final weight gave the amount of substratesdegraded by the isolate. It was observed that maximum degradation was observed in the grated vegetable peels (64 mg followed by straw powder (38 mg and wood powder (28 mg over a period of 8 days.Conclusion, significance and impact of study: By the selection of efficient cellulolytic microorganisms and cost-effective operational techniques, the production of useful end products from the biodegradation of the low cost enormous stock of cellulose in nature can be very beneficial.

  5. Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost.

    Science.gov (United States)

    Izquierdo, Javier A; Sizova, Maria V; Lynd, Lee R

    2010-06-01

    The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.

  6. Evaluation of a Cordia-IC enzyme-linked immunosorbent assay kit for the detection of circulating immune complexes.

    Science.gov (United States)

    Landoy, Z; West, T E; Vladutiu, A O; Fitzpatrick, J E

    1985-01-01

    A commercial kit (Cordia-IC) from Cordis Laboratory, Miami, Fla., was compared with the Raji cell radioimmunoassay for its ability to detect circulating immune complexes (CIC) in sera from 30 control subjects and 118 patients with infectious diseases. The 118 patients were categorized into the following groups: (i) 23 patients with bacterial endocarditis, (ii) 41 patients with bacteremia from an infected intravascular catheter or access device, and (iii) 54 patients with Staphylococcus aureus bacteremia related to a deep tissue infection. The Cordia-IC was comparable to the Raji cell radioimmunoassay in intraassay variability (4.0 versus 8.0%) and interassay reproducibility (8.7 versus 20.0%). Neither assay found CIC amounts above 12.5 micrograms equivalents (eq) of aggregated human gamma globulin (AHG) per ml in any of the 30 control individuals. In group 1, Cordia-IC detected 19 of 23 positives (mean, 73.6 micrograms eq of AHG per ml), whereas the Raji cell detected 16 of 23 positives (mean, 54.8 micrograms eq of AHG per ml). In group 2, Cordia-IC detected 19 of 41 positives (mean, 20.6 micrograms eq of AHG per ml), whereas the Raji cell detected 16 of 41 positives (mean, 15.1 micrograms eq of AHG per ml). In group 3, Cordia-IC found 38 of 54 positives (mean, 28.0 micrograms eq of AHG per ml), whereas the Raji cell found 32 of 54 positives (mean, 23.9 micrograms eq of AHG per ml). Statistically, these findings were not significantly different in any of the three patient groups (P> 0.15), and there was an overall good correlation between the results obtained by the two assays (r+0.64, PCordia-IC provided a suitable assay for the detection of CIC and might find application in routine clinical laboratories. PMID:3897269

  7. The improvement of eggs quality of Mojosari duck (Anas javanica with soybean husk fermentation using cellulolytic bacteria of Spodoptera litura

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    Sri Hidanah

    2018-05-01

    Full Text Available Aim: This study was aimed to improve the quality of the eggs of Mojosari duck (Anas javanica through complete feeding containing soybean husk was fermented using cellulolytic bacteria of Spodoptera litura. Materials and Methods: This study consisted of three stages: The first stages, isolation and identification of cellulolytic bacteria from S. litura; the second stage, the fermentation of soybean husk through the application of bacterial cellulolytic isolate from the first stage; and the third stage, the application of the best complete feed formulation from the second stage to Mojosari duck. Results: There are four dominant bacteria: Bacillus sp., Cellulomonas sp., Pseudomonas sp., and Cytophaga sp. Furthermore, the best reduction of the crude fiber of soybean husks is the use of Cellulomonas sp. bacteria. The final of the study, the quality of the eggs of Anas javanica, was improved, as indicated by cholesterol decrease from the yolk without the decrease of egg weight and eggshell thickness, although the decrease in egg yolk color was inevitable. Conclusion: Soy husk fermentation using cellulolytic bacteria of S. litura was added to complete feeding can be performed to improve the quality of the eggs of Mojosari duck.

  8. Effect of storage time and temperature of equine feces on the subsequent enumeration of lactobacilli and cellulolytic bacteria

    Science.gov (United States)

    Cellulolytic bacteria and lactobacilli are beneficial microbes in the equine hindgut. There are several existing methodologies for the enumeration of these bacteria, which vary based on selective and differential media and sample handling procedures including storage time and temperature. The object...

  9. Fermentation and complex enzyme hydrolysis for improving the total soluble phenolic contents, flavonoid aglycones contents and bio-activities of guava leaves tea.

    Science.gov (United States)

    Wang, Lu; Luo, You; Wu, Yanan; Liu, Yan; Wu, Zhenqiang

    2018-10-30

    There are both soluble and insoluble-bound forms of phenolics in tea-leaf products. In order to increase total soluble phenolics contents, guava leaves tea (GLT) was first fermented with Monascus anka and Saccharomyces cerevisiae, and then hydrolyzed with complex enzymes. The changes in phenolics profiles, antioxidant activities and inhibitory effect on α-glucosidase in processed GLT were investigated. Compared with the un-fermented GLT, fermentation and complex enzymatic processing (FE) significantly increased the total phenolics, total flavonoids, quercetin and kaempferol contents by 2.1, 2.0, 13.0 and 6.8 times, respectively. After the FE, a major proportion of phenolics existed in the soluble form. Quercetin was released in the highest amount among different phenolics. In addition, soluble phenolic extracts from GLT following FE exhibited a highest antioxidant activity and inhibitory effect on α-glucosidase. The paper suggested an improved method for processing GLT into high-value products rich in phenolics and flavonoids aglycones with enhanced health benefits. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1–FoxM1 complex

    Science.gov (United States)

    Yang, Jin; Feng, Xuhui; Zhou, Qiong; Cheng, Wei; Shang, Ching; Han, Pei; Lin, Chiou-Hong; Chen, Huei-Sheng Vincent; Quertermous, Thomas; Chang, Ching-Pin

    2016-01-01

    Genes encoding angiotensin-converting enzymes (Ace and Ace2) are essential for heart function regulation. Cardiac stress enhances Ace, but suppresses Ace2, expression in the heart, leading to a net production of angiotensin II that promotes cardiac hypertrophy and fibrosis. The regulatory mechanism that underlies the Ace2-to-Ace pathological switch, however, is unknown. Here we report that the Brahma-related gene-1 (Brg1) chromatin remodeler and forkhead box M1 (FoxM1) transcription factor cooperate within cardiac (coronary) endothelial cells of pathologically stressed hearts to trigger the Ace2-to-Ace enzyme switch, angiotensin I-to-II conversion, and cardiac hypertrophy. In mice, cardiac stress activates the expression of Brg1 and FoxM1 in endothelial cells. Once activated, Brg1 and FoxM1 form a protein complex on Ace and Ace2 promoters to concurrently activate Ace and repress Ace2, tipping the balance to Ace2 expression with enhanced angiotensin II production, leading to cardiac hypertrophy and fibrosis. Disruption of endothelial Brg1 or FoxM1 or chemical inhibition of FoxM1 abolishes the stress-induced Ace2-to-Ace switch and protects the heart from pathological hypertrophy. In human hypertrophic hearts, BRG1 and FOXM1 expression is also activated in endothelial cells; their expression levels correlate strongly with the ACE/ACE2 ratio, suggesting a conserved mechanism. Our studies demonstrate a molecular interaction of Brg1 and FoxM1 and an endothelial mechanism of modulating Ace/Ace2 ratio for heart failure therapy. PMID:27601681

  11. Identification and in vitro analysis of the GatD/MurT enzyme-complex catalyzing lipid II amidation in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Daniela Münch

    2012-01-01

    Full Text Available The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly₅. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly₅ may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

  12. Isolation, Screening, and Identification of Cellulolytic Bacteria from Natural Reserves in the Subtropical Region of China and Optimization of Cellulase Production by Paenibacillus terrae ME27-1

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    Yan-Ling Liang

    2014-01-01

    Full Text Available From different natural reserves in the subtropical region of China, a total of 245 aerobic bacterial strains were isolated on agar plates containing sugarcane bagasse pulp as the sole carbon source. Of the 245 strains, 22 showed hydrolyzing zones on agar plates containing carboxymethyl cellulose after Congo-red staining. Molecular identification showed that the 22 strains belonged to 10 different genera, with the Burkholderia genus exhibiting the highest strain diversity and accounting for 36.36% of all the 22 strains. Three isolates among the 22 strains showed higher carboxymethyl cellulase (CMCase activity, and isolate ME27-1 exhibited the highest CMCase activity in liquid culture. The strain ME27-1 was identified as Paenibacillus terrae on the basis of 16S rRNA gene sequence analysis as well as physiological and biochemical properties. The optimum pH and temperature for CMCase activity produced by the strain ME27-1 were 5.5 and 50°C, respectively, and the enzyme was stable at a wide pH range of 5.0–9.5. A 12-fold improvement in the CMCase activity (2.08 U/mL of ME27-1 was obtained under optimal conditions for CMCase production. Thus, this study provided further information about the diversity of cellulose-degrading bacteria in the subtropical region of China and found P. terrae ME27-1 to be highly cellulolytic.

  13. Bacillus coagulans MA-13: a promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate.

    Science.gov (United States)

    Aulitto, Martina; Fusco, Salvatore; Bartolucci, Simonetta; Franzén, Carl Johan; Contursi, Patrizia

    2017-01-01

    The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed. A thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans , named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 °C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-)hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 °C), still retaining an optimal operational activity at 50 °C. The above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto

  14. Microbial Consortium with High Cellulolytic Activity (MCHCA for enhanced biogas production.

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    Krzysztof ePoszytek

    2016-03-01

    Full Text Available The use of lignocellulosic biomass as a substrate in agricultural biogas plants is very popular and yields good results. However, the efficiency of anaerobic digestion, and thus biogas production, is not always satisfactory due to the slow or incomplete degradation (hydrolysis of plant matter. To enhance the solubilization of the lignocellulosic biomass various physical, chemical and biological pretreatment methods are used.The aim of this study was to select and characterize cellulose-degrading bacteria, and to construct a microbial consortium, dedicated for degradation of maize silage and enhancing biogas production from this substrate.Over one hundred strains of cellulose-degrading bacteria were isolated from: sewage sludge, hydrolyzer from an agricultural biogas plant, cattle slurry and manure. After physiological characterization of the isolates, sixteen strains (representatives of Bacillus, Providencia and Ochrobactrum genera were chosen for the construction of a Microbial Consortium with High Cellulolytic Activity, called MCHCA. The selected strains had a high endoglucanase activity (exceeding 0.21 IU/mL CMCase activity and a wide range of tolerance to various physical and chemical conditions. Lab-scale simulation of biogas production using the selected strains for degradation of maize silage was carried out in a two-bioreactor system, similar to those used in agricultural biogas plants.The obtained results showed that the constructed MCHCA consortium is capable of efficient hydrolysis of maize silage, and increases biogas production by even 38%, depending on the inoculum used for methane fermentation. The results in this work indicate that the mesophilic Microbial Consortium with High Cellulolytic Activity has a great potential for application on industrial scale in agricultural biogas plants.

  15. Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae.

    Science.gov (United States)

    Noguchi, Yuji; Sano, Motoaki; Kanamaru, Kyoko; Ko, Taro; Takeuchi, Michio; Kato, Masashi; Kobayashi, Tetsuo

    2009-11-01

    XlnR is a Zn(II)2Cys6 transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus. Overexpression of the aoxlnR gene in Aspergillus oryzae (A. oryzae xlnR gene) resulted in elevated xylanolytic and cellulolytic activities in the culture supernatant, in which nearly 40 secreted proteins were detected by two-dimensional electrophoresis. DNA microarray analysis to identify the transcriptional targets of AoXlnR led to the identification of 75 genes that showed more than fivefold increase in their expression in the AoXlnR overproducer than in the disruptant. Of these, 32 genes were predicted to encode a glycoside hydrolase, highlighting the biotechnological importance of AoXlnR in biomass degradation. The 75 genes included the genes previously identified as AoXlnR targets (xynF1, xynF3, xynG2, xylA, celA, celB, celC, and celD). Thirty-six genes were predicted to be extracellular, which was consistent with the number of proteins secreted, and 61 genes possessed putative XlnR-binding sites (5'-GGCTAA-3', 5'-GGCTAG-3', and 5'-GGCTGA-3') in their promoter regions. Functional annotation of the genes revealed that AoXlnR regulated the expression of hydrolytic genes for degradation of beta-1,4-xylan, arabinoxylan, cellulose, and xyloglucan and of catabolic genes for the conversion of D-xylose to xylulose-5-phosphate. In addition, genes encoding glucose-6-phosphate 1-dehydrogenase and L-arabinitol-4- dehydrogenase involved in D-glucose and L-arabinose catabolism also appeared to be targets of AoXlnR.

  16. Effect of bioaugmentation by cellulolytic bacteria enriched from sheep rumen on methane production from wheat straw.

    Science.gov (United States)

    Ozbayram, E Gozde; Kleinsteuber, Sabine; Nikolausz, Marcell; Ince, Bahar; Ince, Orhan

    2017-08-01

    The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mL N CH 4 g VS -1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Development of over-production strain of saccharification enzyme and biomass pretreatment by proton beam irradiation

    International Nuclear Information System (INIS)

    Kim, S. W.; Lee, J. Y.; Song, Y. S.; Lee, S. J.; Shin, H. Y.; Kim, S. B.

    2010-04-01

    When lignocellulosic biomass converts to ethanol, enzyme takes lots of part of whole cost. Therefore, cellulase production is one of the important processes for the successful enzymatic conversion of cellulosic biomass to ethanol. Among cellulolytic enzymes, cellulase is multi-complex enzyme containing endo-glucanase, exo-glucanase and β-glucosidase. Cellulolyticfungi, Trichodema reesei is well known to produce the highest yields of cellulase. Especially, suitable cellulase composition was important for the effective saccharification of lignocellulosic biomass and strain having high level production of cellulase should be developed for hydrolysis. For efficient ethanol production, hemicellullase of Aspergillus also develop to use xylose generated from saccharification of biomass. In this study, pretreatment process of rice straw using proton beam irradiation (PBI) was carried out for enhancement of enzyme digestibility at different proton beam doses. Also, PBI pretreatment on ammonia soaking treated (SAA, Soaking aqueous ammonia) rice straw was conducted to solve the problem that is micro-structural inhibition of rice straw. Optimal dosages of proton beam on rice straw and SAA treated rice straw for efficient recovery of sugar were 15 KGy and 3 KGy, respectively. Enzymatic saccharification of PBI treated rice straw and SAA rice straw was conducted for the guidance of NREL standard procedure. Analysis using X-ray diffractometry (XRD) for crystallinity index was carried out and CrI found to be 33.38% of control and 35.72% of 15 KGy. Also, CrI was determined to be 67.11% of control and approximately 65.58% of 3 kGy dose in PBI pretreatment on SAA treated rice straw. The result of sugar recovery of both was approximately 70 % and 91 % of theoretical glucose contents, respectively. The initial reaction rate was increased from 7.610 -4 g·l -1 ·s -1 of 15 KGy (PBI pretreated rice straw) to 9.710 -4 g·l -1 ·s -1 (3 KGy PBI pretreated SAA rice straw). The selection of

  18. The AMPK enzyme-complex: from the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders.

    Science.gov (United States)

    Antonioli, Luca; Colucci, Rocchina; Pellegrini, Carolina; Giustarini, Giulio; Sacco, Deborah; Tirotta, Erika; Caputi, Valentina; Marsilio, Ilaria; Giron, Maria Cecilia; Németh, Zoltán H; Blandizzi, Corrado; Fornai, Matteo

    2016-01-01

    Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular mechanisms underlying the pathophysiology of chronic inflammatory diseases. AMPK is expressed in several immune cell types including macrophages, lymphocytes, neutrophils and dendritic cells, and governs a broad array of cell functions, which include cytokine production, chemotaxis, cytotoxicity, apoptosis and proliferation. Based on its wide variety of immunoregulatory actions, the AMPK system has been targeted to reveal its impact on the course of immune-related diseases, such as atherosclerosis, psoriasis, joint inflammation and inflammatory bowel diseases. The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.

  19. Theoretical investigation of the reaction mechanism for the phosphate diester hydrolysis using an asymmetric dinuclear metal complex as a biomimetic model of the purple acid phosphatase enzyme.

    Science.gov (United States)

    Ferreira, Dalva E C; De Almeida, Wagner B; Neves, Ademir; Rocha, Willian R

    2008-12-14

    In this work we have applied quantum mechanical calculations, at the density functional theory level, to investigate the phosphate diester hydrolysis promoted by a cationic heterodinuclear Fe(III)...Zn(II) complex that mimics the structural and functional properties of the purple acid phosphatase (PAP) enzymes. The hydrolysis of the dimethyl phosphate diester was investigated in the gas phase and in solution by means of the continuum PCM model, using the B3LYP hybrid exchange-correlation functional. Our computed results showed that the hydrolysis of the dimethyl phosphate ester takes place in two steps. The first step corresponds to a slow P-O bond formation through nucleophilic attack of the coordinated (Fe(III))-OH group. The second step consists of a proton transfer process followed by the release of a methanol molecule. The first step is rate determining with activation free energy of 12.3 kcal mol(-1), which is about 3 times lower than the activation free energy for the uncatalyzed reaction. We also show that the heterodinuclear site plays an important role favoring an associative mechanism for the phosphate diester hydrolysis, favoring the formation of a high energy intermediate phosphorane, and orienting the phosphate group to the nucleophilic attack.

  20. Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.

    Science.gov (United States)

    Miyazaki, Takatsugu; Ichikawa, Megumi; Yokoi, Gaku; Kitaoka, Motomitsu; Mori, Haruhide; Kitano, Yoshikazu; Nishikawa, Atsushi; Tonozuka, Takashi

    2013-09-01

    Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α-glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with β-D-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O-α-D-glucopyranosyl-α-D-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK. © 2013 FEBS.

  1. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  2. A study of overproduction and enhanced secretion of enzymes. Quarterly report

    Energy Technology Data Exchange (ETDEWEB)

    Dashek, W.V.

    1993-09-01

    Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are two major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.

  3. FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota

    Directory of Open Access Journals (Sweden)

    Sophie Comtet-Marre

    2018-02-01

    Full Text Available Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6 were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.

  4. FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota.

    Science.gov (United States)

    Comtet-Marre, Sophie; Chaucheyras-Durand, Frédérique; Bouzid, Ourdia; Mosoni, Pascale; Bayat, Ali R; Peyret, Pierre; Forano, Evelyne

    2018-01-01

    Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme) families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6) were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi) represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.

  5. Mitigation of Membrane Biofouling in MBR Using a Cellulolytic Bacterium, Undibacterium sp. DM-1, Isolated from Activated Sludge.

    Science.gov (United States)

    Nahm, Chang Hyun; Lee, Seonki; Lee, Sang Hyun; Lee, Kibaek; Lee, Jaewoo; Kwon, Hyeokpil; Choo, Kwang-Ho; Lee, Jung-Kee; Jang, Jae Young; Lee, Chung-Hak; Park, Pyung-Kyu

    2017-03-28

    Biofilm formation on the membrane surface results in the loss of permeability in membrane bioreactors (MBRs) for wastewater treatment. Studies have revealed that cellulose is not only produced by a number of bacterial species but also plays a key role during formation of their biofilm. Hence, in this study, cellulase was introduced to a MBR as a cellulose-induced biofilm control strategy. For practical application of cellulase to MBR, a cellulolytic ( i.e ., cellulase-producing) bacterium, Undibacterium sp. DM-1, was isolated from a lab-scale MBR for wastewater treatment. Prior to its application to MBR, it was confirmed that the cell-free supernatant of DM-1 was capable of inhibiting biofilm formation and of detaching the mature biofilm of activated sludge and cellulose-producing bacteria. This suggested that cellulase could be an effective anti-biofouling agent for MBRs used in wastewater treatment. Undibacterium sp. DM-1-entrapping beads ( i.e ., cellulolytic-beads) were applied to a continuous MBR to mitigate membrane biofouling 2.2-fold, compared with an MBR with vacant-beads as a control. Subsequent analysis of the cellulose content in the biofilm formed on the membrane surface revealed that this mitigation was associated with an approximately 30% reduction in cellulose by cellulolytic-beads in MBR.

  6. One-step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic bacillus subtilis: opportunities and challenges

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiao-Zhou [Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); Zhang, Yi-Heng P. [Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); Institute for Critical Technology and Applied Science, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); BioEnergy Science Center of Department of Energy, Oak Ridge, TN (United States)

    2010-10-15

    One-step consolidated bioprocessing that integrates cellulase production, cellulose hydrolysis, and product fermentation into a single step for decreasing costly cellulase use, increasing volumetric productivity, and reducing capital investment is widely accepted for low-cost production of biofuels or other value-added biochemicals. Considering the narrow margins between biomass and low-value biocommodities, good physiological performance of industrial microbes is crucial for economically viable production. Bacillus subtilis, the best-characterized Gram-positive microorganism, is a major industrial microorganism with numerous valuable features such as hexose and pentose utilization, low-nutrient needs, fast growth rate, high protein secretion capacity, industrial safety, etc. As compared with other potential consolidated bioprocessing microorganisms such as Clostridium spp., Escherichia coli, and the yeast Saccharomyces cerevisiae, recombinant cellulolytic B. subtilis strains would be a potential platform for biocommodity production from nonfood biomass. Here, we review the advances in recombinant cellulolytic B. subtilis development and metabolic engineering for biocommodity production, and discuss the opportunities and challenges of cellulolytic B. subtilis for biocommodity production. (Copyright copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  7. Pyrosequencing reveals high-temperature cellulolytic microbial consortia in Great Boiling Spring after in situ lignocellulose enrichment.

    Directory of Open Access Journals (Sweden)

    Joseph P Peacock

    Full Text Available To characterize high-temperature cellulolytic microbial communities, two lignocellulosic substrates, ammonia fiber-explosion-treated corn stover and aspen shavings, were incubated at average temperatures of 77 and 85°C in the sediment and water column of Great Boiling Spring, Nevada. Comparison of 109,941 quality-filtered 16S rRNA gene pyrosequences (pyrotags from eight enrichments to 37,057 quality-filtered pyrotags from corresponding natural samples revealed distinct enriched communities dominated by phylotypes related to cellulolytic and hemicellulolytic Thermotoga and Dictyoglomus, cellulolytic and sugar-fermenting Desulfurococcales, and sugar-fermenting and hydrogenotrophic Archaeoglobales. Minor enriched populations included close relatives of hydrogenotrophic Thermodesulfobacteria, the candidate bacterial phylum OP9, and candidate archaeal groups C2 and DHVE3. Enrichment temperature was the major factor influencing community composition, with a negative correlation between temperature and richness, followed by lignocellulosic substrate composition. This study establishes the importance of these groups in the natural degradation of lignocellulose at high temperatures and suggests that a substantial portion of the diversity of thermophiles contributing to consortial cellulolysis may be contained within lineages that have representatives in pure culture.

  8. A novel copper complex induces ROS generation in doxorubicin resistant Ehrlich ascitis carcinoma cells and increases activity of antioxidant enzymes in vital organs in vivo

    International Nuclear Information System (INIS)

    Mookerjee, Ananda; Roy, Syamal; Choudhuri, Soumitra K; Basu, Jayati Mookerjee; Majumder, Surajit; Chatterjee, Shilpak; Panda, Gouri S; Dutta, Pranabananda; Pal, Smarajit; Mukherjee, Pratima; Efferth, Thomas

    2006-01-01

    In search of a suitable GSH-depleting agent, a novel copper complex viz., copper N-(2-hydroxyacetophenone) glycinate (CuNG) has been synthesized, which was initially found to be a potential resistance modifying agent and later found to be an immunomodulator in mice model in different doses. The objective of the present work was to decipher the effect of CuNG on reactive oxygen species (ROS) generation and antioxidant enzymes in normal and doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing Swiss albino mice. The effect of CuNG has been studied on ROS generation, multidrug resistance-associated protein1 (MRP1) expression and on activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). CuNG increased ROS generation and reduced MRP1 expression in EAC/Dox cells while only temporarily depleted glutathione (GSH) within 2 h in heart, kidney, liver and lung of EAC/Dox bearing mice, which were restored within 24 h. The level of liver Cu was observed to be inversely proportional to the level of GSH. Moreover, CuNG modulated SOD, CAT and GPx in different organs and thereby reduced oxidative stress. Thus nontoxic dose of CuNG may be utilized to reduce MRP1 expression and thus sensitize EAC/Dox cells to standard chemotherapy. Moreover, CuNG modulated SOD, CAT and and GPx activities to reduce oxidative stress in some vital organs of EAC/Dox bearing mice. CuNG treatment also helped to recover liver and renal function in EAC/Dox bearing mice. Based on our studies, we conclude that CuNG may be a promising candidate to sensitize drug resistant cancers in the clinic

  9. Enzyme recycling in lignocellulosic biorefineries

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Pinelo, Manuel

    2017-01-01

    platform. Cellulases are the most important enzymes required in this process, but the complex nature of lignocellulose requires several other enzymes (hemicellulases and auxiliary enzymes) for efficient hydrolysis. Enzyme recycling increases the catalytic productivity of the enzymes by reusing them...... for several batches of hydrolysis, and thereby reduces the overall cost associated with the hydrolysis. Research on this subject has been ongoing for many years and several promising technologies and methods have been developed and demonstrated. But only in a very few cases have these technologies been...... upscaled and tested in industrial settings, mainly because of many difficulties with recycling of enzymes from the complex lignocellulose hydrolyzate at industrially relevant conditions, i.e., high solids loadings. The challenges are associated with the large number of different enzymes required...

  10. Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources.

    Science.gov (United States)

    Kaushal, Prem Singh; Talawar, Ramappa K; Krishna, P D V; Varshney, Umesh; Vijayan, M

    2008-05-01

    Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs.

  11. Vertical zonation and seed germination indices of chromium resistant cellulolytic and nitrogen fixing bacteria from a chronically metal exposed land area

    International Nuclear Information System (INIS)

    Aslam, S.; Qazi, J.I.

    2014-01-01

    Twenty eight cellulolytic and 25 nitrogen fixing bacteria were isolated from 20, 40 and 60 cm depths of the chromium contaminated land area. The cellulolytic as well as nitrogen fixing microbial communities in soil profiles were dominated by genus Bacillus. More diverse nitrogen fixing bacterial isolates belonging to different genera Paenibacillus, Corynebacterium and Pseudomonas were observed as compared to cellulolytic bacterial community. Majority of the cellulolytic bacteria were found inhabitants of 20 cm soil layer while 40 cm depth was the preferred zone for the nitrogen fixing bacteria. Screening of the bacterial isolates for chromium resistance showed that isolates designated as ASK15 and ASK16 were able to resist up to 1800 mg/l of chromium while the nitrogen fixing isolates which offered a maximum resistant level up to 1650 mg/l of chromium were ASNt10 and ASNS13. Nitrogen fixing isolates enhanced seed germination by 33% and expressed efficient nitrogenase activity up to 0.80 (C/sub 2/H/sub 2/ nmol/ml/hr). Growth promoting assay proved ASNt10 a potential isolate which produced 90 meu g/ml of indoleacetic acid (IAA). Though cellulolytic isolates did not affect seed germination, a significant influence on root length similar to that of ASNt10 and ASNS13 with nearly 5-fold increase in comparison with uninoculated control was observed. The isolates ASK15, ASK16 were identified as Bacillus cereus while ASNt10 and ASNS13 as Paenibacillus barcinonensis and Bacillus megaterium, respectively. (author)

  12. Dimeric Fe (II, III) complex of quinoneoxime as functional model of PAP enzyme: Mössbauer, magneto-structural and DNA cleavage studies

    Science.gov (United States)

    Salunke-Gawali, Sunita; Ahmed, Khursheed; Varret, François; Linares, Jorge; Zaware, Santosh; Date, Sadgopal; Rane, Sandhya

    2008-07-01

    Purple acid phosphatase, ( PAP), is known to contain dinuclear Fe2 + 2, + 3 site with characteristic Fe + 3 ← Tyr ligand to metal charge transfer in coordination. Phthiocoloxime (3-methyl-2-hydroxy-1,4-naphthoquinone-1-oxime) ligand L, mimics (His/Tyr) ligation with controlled and unique charge transfers resulting in valence tautomeric coordination with mixed valent diiron site in model compound Fe-1: [μ-OH-Fe2 + 2, + 3 ( o-NQCH3ox) ( o-NSQCH3ox)2 (CAT) H2O]. Fe-2: [Fe + 3( o-NQCH3ox) ( p-NQCH3ox)2]2 a molecularly associated dimer of phthiocoloxime synthesized for comparison of charge transfer. 57Fe Mössbauer studies was used to quantitize unusual valences due to ligand in dimeric Fe-1 and Fe-2 complexes which are supported by EPR and SQUID studies. 57Fe Mössbauer spectra for Fe-1 at 300 K indicates the presence of two quadrupole split asymmetric doublets due to the differences in local coordination geometries of [Fe + 3]A and [Fe + 2]B sites. The hyperfine interaction parameters are δ A = 0.152, (Δ E Q)A = 0.598 mm/s with overlapping doublet at δ B = 0.410 and (Δ E Q)B = 0.468 mm/s. Due to molecular association tendency of ligand, dimer Fe-2 possesses 100% Fe + 3(h.s.) hexacoordinated configuration with isomer shift δ = 0.408 mm/s. Slightly distorted octahedral symmetry created by NQCH3ox ligand surrounding Fe + 3(h.s.) state generates small field gradient indicated by quadrupole split Δ E Q = 0.213 mm/s. Decrease of isomer shifts together with variation of quadrupole splits with temperature in Fe-1 dimer compared to Fe-2 is result of charge transfers in [Fe2 + 2, + 3 SQ] complexes. EPR spectrum of Fe-1 shows two strong signals at g 1 = 4.17 and g 2 = 2.01 indicative of S = 3/2 spin state with an intermediate spin of Fe + 3(h.s.) configuration. SQUID data of χ _m^{corr} .T were best fitted by using HDVV spin pair model S = 2, 3/2 resulting in antiferromagnetic exchange ( J = -13.5 cm - 1 with an agreement factor of R = 1.89 × 10 - 5). The lower J

  13. Effect of two fungicides, benlate and phenyl mercury acetate, on a population of cellulolytic fungi in soil and in pure culture

    Energy Technology Data Exchange (ETDEWEB)

    Smith, R.N.; Long, P.A.

    1980-01-01

    Cellulolytic fungi were successfully isolated from an arable loam type soil using the polythene rod technique and the screened substrate technique. Phenyl mercury acetate (PMA) applied to the soil at 100 ppm severely reduced the incidence of many cellulolytic fungi normally present, with a concomitant increase in the incidence of Penicillium spp and Trichoderma lignorum. The application of benlate at 100 ppm had little effect other than to increase the incidence of Doratomyces mircosporus, Dreschlera sp. and Papulospora sp. Both benlate and PMA were much more toxic when examined in-vitro in cellulose agar than when added to soil. Nevertheless, the incidence of cellulolytic isolates from soil reflected the relative sensitivity of the isolates to the respective fungicides in-vitro. The activity of benlate in-vitro decreased with an increase in glucose concentration in the media, but the activity of PMA was independent of glucose concentration.

  14. Cellulolytic Protist Numbers Rise and Fall Dramatically in Termite Queens and Kings during Colony Foundation

    Science.gov (United States)

    Shimada, Keisuke; Lo, Nathan; Kitade, Osamu; Wakui, Akane

    2013-01-01

    Among the best-known examples of mutualistic symbioses is that between lower termites and the cellulolytic flagellate protists in their hindguts. Although the symbiosis in worker termites has attracted much attention, there have been only a few studies of protists in other castes. We have performed the first examination of protist population dynamics in queens and kings during termite colony foundation. Protist numbers, as well as measurements of hindgut and reproductive tissue sizes, were undertaken at five time points over 400 days in incipient colonies of Reticulitermes speratus, as well as in other castes of mature colonies of this species. We found that protist numbers increased dramatically in both queens and kings during the first 50 days of colony foundation but began to decrease by day 100, eventually disappearing by day 400. Hindgut width followed a pattern similar to that of protist numbers, while ovary and testis widths increased significantly only at day 400. Kings were found to contain higher numbers of protists than queens in incipient colonies, which may be linked to higher levels of nutrient transfer from kings to queens than vice versa, as is known in some other termite species. Protists were found to be abundant in soldiers from mature colonies but absent in neotenics. This probably reflects feeding of soldiers by workers via proctodeal trophallaxis and of reproductives via stomodeal trophallaxis. The results reveal the dynamic nature of protist numbers during colony foundation and highlight the trade-offs that exist between reproduction and parental care during this critical phase of the termite life cycle. PMID:23376945

  15. Ethanol from wood. Cellulase enzyme production

    Energy Technology Data Exchange (ETDEWEB)

    Szengyel, Zsolt

    2000-03-01

    Conversion of biomass to liquid fuels, such as ethanol, has been investigated during the past decades. First due to the oil crisis of the 1970s and lately because of concerns about greenhouse effect, ethanol has been found to be a suitable substitute for gasoline in transportation. Although ethanol is produced in large quantities from corn starch, the conversion of lignocellulosic biomass to ethanol is rather problematic. However, cellulosic raw materials are important as they are available in large quantities from agriculture and forestry. One of the most extensively investigated processes is the enzymatic process, in which fungal cellulolytic enzymes are used to convert the cellulose content of the biomass to glucose, which is then fermented to ethanol. In order to make the raw material accessible to biological attack, it has to be pretreated first. The most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment. In this thesis the utilization of steam pretreated willow (hardwood) and spruce (softwood) was examined for enzyme production using a filamentous fungus T. reesei RUT C30. Various carbon sources originating from the steam pretreated materials have been investigated. The replacement of the solid carbon source with a liquid carbon source, as well as the effect of pH, was studied. The effect of toxic compounds generated during pretreatment was also examined. Comparative study of softwood and hardwood showed that steam pretreated hardwood is a better carbon source than softwood. The hydrolytic potential of enzyme solutions produced on wood derived carbon sources was better compared to commercial cellulases. Also enzyme solutions produced on steam pretreated spruce showed less sensitivity towards toxic compounds formed during steam pretreatment.

  16. Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

    Science.gov (United States)

    Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

    2008-09-01

    We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

  17. Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

    Directory of Open Access Journals (Sweden)

    Dashtban Mehdi

    2012-05-01

    Full Text Available Abstract Background Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH. Results We successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg higher β-glucosidase activity compared to the parent strain (2.2 IU/mg after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. Conclusions The recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation

  18. [The efficiency of the enzyme immunoassay test system opisthorchiasis-CIC-EIA-best to detect circulating immune complexes containing opisthorchis antigens in the serum of patients with opisthorchiasis].

    Science.gov (United States)

    Starkova, T V; Poletaeva, O G; Kovrova, E A; Krasovskaia, N N; Tkachenko, T N; Masiago, A V; Ofitserov, V I; Tereshchenko, A Iu

    2011-01-01

    The efficacy of a kit of Opisthorchiasis-CIC-EIA-Best reagents was evaluated using 270 sera from patients in the study and control groups. The kit showed a sufficient sensitivity (not less than 87.2%) and a high specificity (not less than 97.9%). The use of the above kit of the reagents for enzyme immunoassay in practical healthcare enables one to increase detection rates among the infested subjects on comprehensive examination of those with suspected opisthorchiasis.

  19. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

    Science.gov (United States)

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-05

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

    Science.gov (United States)

    Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

    2016-02-01

    Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

  1. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  2. Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

    Energy Technology Data Exchange (ETDEWEB)

    Cossar, D; Canevascini, G

    1986-07-01

    The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellubiose dehydrogenase remain to be elucidated.

  3. VTVH-MCD and DFT studies of thiolate bonding to [FeNO]7/[FeO2]8 complexes of isopenicillin N synthase: substrate determination of oxidase versus oxygenase activity in nonheme Fe enzymes.

    Science.gov (United States)

    Brown, Christina D; Neidig, Michael L; Neibergall, Matthew B; Lipscomb, John D; Solomon, Edward I

    2007-06-13

    Isopenicillin N synthase (IPNS) is a unique mononuclear nonheme Fe enzyme that catalyzes the four-electron oxidative double ring closure of its substrate ACV. A combination of spectroscopic techniques including EPR, absorbance, circular dichroism (CD), magnetic CD, and variable-temperature, variable-field MCD (VTVH-MCD) were used to evaluate the geometric and electronic structure of the [FeNO]7 complex of IPNS coordinated with the ACV thiolate ligand. Density Function Theory (DFT) calculations correlated to the spectroscopic data were used to generate an experimentally calibrated bonding description of the Fe-IPNS-ACV-NO complex. New spectroscopic features introduced by the binding of the ACV thiolate at 13 100 and 19 800 cm-1 are assigned as the NO pi*(ip) --> Fe dx2-y2 and S pi--> Fe dx2-y2 charge transfer (CT) transitions, respectively. Configuration interaction mixes S CT character into the NO pi*(ip) --> Fe dx2-y2 CT transition, which is observed experimentally from the VTVH-MCD data from this transition. Calculations on the hypothetical {FeO2}8 complex of Fe-IPNS-ACV reveal that the configuration interaction present in the [FeNO]7 complex results in an unoccupied frontier molecular orbital (FMO) with correct orientation and distal O character for H-atom abstraction from the ACV substrate. The energetics of NO/O2 binding to Fe-IPNS-ACV were evaluated and demonstrate that charge donation from the ACV thiolate ligand renders the formation of the FeIII-superoxide complex energetically favorable, driving the reaction at the Fe center. This single center reaction allows IPNS to avoid the O2 bridged binding generally invoked in other nonheme Fe enzymes that leads to oxygen insertion (i.e., oxygenase function) and determines the oxidase activity of IPNS.

  4. The action of Saraca asoca Roxb. de Wilde bark on the PGH2 synthetase enzyme complex of the sheep vesicular gland.

    Science.gov (United States)

    Middelkoop, T B; Labadie, R P

    1985-01-01

    Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH2 synthetase. They were assayed spectrophotometrically with adrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.

  5. Rational optimization of drug-target residence time: Insights from inhibitor binding to the S. aureus FabI enzyme-product complex

    Science.gov (United States)

    Chang, Andrew; Schiebel, Johannes; Yu, Weixuan; Bommineni, Gopal R.; Pan, Pan; Baxter, Michael V.; Khanna, Avinash; Sotriffer, Christoph A.; Kisker, Caroline; Tonge, Peter J.

    2013-01-01

    Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme. PMID:23697754

  6. Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

    Science.gov (United States)

    Ruane, Karen M; Lloyd, Adrian J; Fülöp, Vilmos; Dowson, Christopher G; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I

    2013-11-15

    Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

  7. Genome-Centric Analysis of a Thermophilic and Cellulolytic Bacterial Consortium Derived from Composting

    Science.gov (United States)

    Lemos, Leandro N.; Pereira, Roberta V.; Quaggio, Ronaldo B.; Martins, Layla F.; Moura, Livia M. S.; da Silva, Amanda R.; Antunes, Luciana P.; da Silva, Aline M.; Setubal, João C.

    2017-01-01

    Microbial consortia selected from complex lignocellulolytic microbial communities are promising alternatives to deconstruct plant waste, since synergistic action of different enzymes is required for full degradation of plant biomass in biorefining applications. Culture enrichment also facilitates the study of interactions among consortium members, and can be a good source of novel microbial species. Here, we used a sample from a plant waste composting operation in the São Paulo Zoo (Brazil) as inoculum to obtain a thermophilic aerobic consortium enriched through multiple passages at 60°C in carboxymethylcellulose as sole carbon source. The microbial community composition of this consortium was investigated by shotgun metagenomics and genome-centric analysis. Six near-complete (over 90%) genomes were reconstructed. Similarity and phylogenetic analyses show that four of these six genomes are novel, with the following hypothesized identifications: a new Thermobacillus species; the first Bacillus thermozeamaize genome (for which currently only 16S sequences are available) or else the first representative of a new family in the Bacillales order; the first representative of a new genus in the Paenibacillaceae family; and the first representative of a new deep-branching family in the Clostridia class. The reconstructed genomes from known species were identified as Geobacillus thermoglucosidasius and Caldibacillus debilis. The metabolic potential of these recovered genomes based on COG and CAZy analyses show that these genomes encode several glycoside hydrolases (GHs) as well as other genes related to lignocellulose breakdown. The new Thermobacillus species stands out for being the richest in diversity and abundance of GHs, possessing the greatest potential for biomass degradation among the six recovered genomes. We also investigated the presence and activity of the organisms corresponding to these genomes in the composting operation from which the consortium was built

  8. Advances in enzyme bioelectrochemistry

    Directory of Open Access Journals (Sweden)

    ANDRESSA R. PEREIRA

    Full Text Available ABSTRACT Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

  9. Discriminated release of phenolic substances from red wine grape skins (Vitis vinifera L.) by multicomponent enzymes treatment

    DEFF Research Database (Denmark)

    Arnous, Anis; Meyer, Anne S.

    2010-01-01

    Detailed insight into the effects of enzymatic treatments on grape phenolics is of significant importance for grape processing for wine making. This study examined the release of phenols during enzymatic (pectinolytic and cellulolytic) degradation of the cell wall polysaccharides in skins of Merlot...... the enzymatic treatment; phenolic acids, including hydroxybenzoic acids and hydroxycinnamic acids, were released as a function of monosaccharides liberation, i.e. as a function of the enzyme catalyzed cell wall degradation of the skins, and with some of the phenolic acids perhaps released from the lignin...

  10. Photosynthetic fuel for heterologous enzymes

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

    2017-01-01

    of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

  11. Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: genetic markers and characterization of cellulolytic potential.

    Science.gov (United States)

    Koeck, Daniela E; Zverlov, Vladimir V; Liebl, Wolfgang; Schwarz, Wolfgang H

    2014-07-01

    Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach - the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) - was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters. Copyright © 2014 Elsevier GmbH. All rights reserved.

  12. Dietary Corn Bran Fermented by Bacillus subtilis MA139 Decreased Gut Cellulolytic Bacteria and Microbiota Diversity in Finishing Pigs

    Directory of Open Access Journals (Sweden)

    Ping Liu

    2017-12-01

    Full Text Available Solid-state fermentation of feedstuffs by Bacillus subtilis MA139 can reduce insoluble dietary fiber content in vitro and improve growth performance in pigs. This study was conducted to investigate the effects of dietary corn bran (CB fermented by B. subtilis on growth performance and gut microbiota composition in finishing pigs. A total of 60 finishing pigs were allocated to 3 dietary treatments consisting of a control (CON diet, a 10% CB diet, and a 10% fermented CB (FCB diet in a 21 d feeding trial. Growth performance and nutrient digestibility were evaluated. Fecal samples were determined for bacterial community diversity by 16S rRNA gene amplicon sequencing. The dietary CB and FCB did not affect growth performance of finishing pigs. The digestibility of organic matter was decreased in both CB and FCB treatments compared with CON group (P < 0.05. The α-diversity for bacterial community analysis of Chao 1 in FCB treatment was lower than CON treatment (P < 0.05. The Fibrobacteres phylum belongs to cellulolytic bacteria was isolated, and their relative abundance in CB group showed no difference between CON and FCB treatments. The abundance of Lachnospiraceae_NK4A136_group in CB treatment was higher than CON and FCB groups (P < 0.05, whereas the population of norank_f_Prevotellaceae was higher in FCB group compared to CON and CB groups (P < 0.05. In conclusion, dietary FCB decreased the abundance of bacterial communities, particularly the population of bacteria related to cellulolytic degradation.

  13. The Leishmania donovani complex: Genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH and FH), new targets for multilocus sequence typing

    Czech Academy of Sciences Publication Activity Database

    Zemanová, Eva; Jirků, Milan; Mauricio, I. L.; Horák, Aleš; Miles, M. A.; Lukeš, Julius

    2007-01-01

    Roč. 37, č. 2 (2007), s. 149-160 ISSN 0020-7519 R&D Projects: GA MŠk 2B06129 Grant - others:EU(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Source of funding: R - rámcový projekt EK Keywords : Leishmania donovani complex * zymodeme * multilocus sequence typing * Leishmania * phylogenetic network Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.392, year: 2007

  14. Quantitative Analysis of Complex Drug-Drug Interactions Between Repaglinide and Cyclosporin A/Gemfibrozil Using Physiologically Based Pharmacokinetic Models With In Vitro Transporter/Enzyme Inhibition Data.

    Science.gov (United States)

    Kim, Soo-Jin; Toshimoto, Kota; Yao, Yoshiaki; Yoshikado, Takashi; Sugiyama, Yuichi

    2017-09-01

    Quantitative analysis of transporter- and enzyme-mediated complex drug-drug interactions (DDIs) is challenging. Repaglinide (RPG) is transported into the liver by OATP1B1 and then is metabolized by CYP2C8 and CYP3A4. The purpose of this study was to describe the complex DDIs of RPG quantitatively based on unified physiologically based pharmacokinetic (PBPK) models using in vitro K i values for OATP1B1, CYP3A4, and CYP2C8. Cyclosporin A (CsA) or gemfibrozil (GEM) increased the blood concentrations of RPG. The time profiles of RPG and the inhibitors were analyzed by PBPK models, considering the inhibition of OATP1B1 and CYP3A4 by CsA or OATP1B1 inhibition by GEM and its glucuronide and the mechanism-based inhibition of CYP2C8 by GEM glucuronide. RPG-CsA interaction was closely predicted using a reported in vitro K i,OATP1B1 value in the presence of CsA preincubation. RPG-GEM interaction was underestimated compared with observed data, but the simulation was improved with the increase of f m,CYP2C8 . These results based on in vitro K i values for transport and metabolism suggest the possibility of a bottom-up approach with in vitro inhibition data for the prediction of complex DDIs using unified PBPK models and in vitro f m value of a substrate for multiple enzymes should be considered carefully for the prediction. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  15. The Cellulolytic Activity And Volatile Fatty Acid Product Of Rumen Bacteria Of Buffalo And Cattle On Rice Straw, Elephant Grass, and Sesbania Leaves Substrates

    Directory of Open Access Journals (Sweden)

    Caribu Hadi Prayitno

    1999-01-01

    Full Text Available Experiment on The Cellulolytic Activity and Volatile Fatty Acid Product of Rumen Bacteria of Buffalo and Cattle on Rice Straw, Elephant Grass, and Sesbania Leaves Substrates had been conducted at Feedstuff Laboratory of Animal Science Soedirman University. The basic design  that was used in this experiment was Completely Randomized Design (CRD with factorial pattern of 6 x 3, three replications. The bacteria isolate as the factors were cellulolytic rumen bacteria isolate of buffalo (A1, A2, and A3 and cattle (A4, A5 and A6 while the substrates (second factor  were NDF rice straw (S1, elephant grass (S2, and sesbania leaves (S3 Cell walls. The result of this experiment showed that the interaction between bacteria isolate and substrate  type were significant on pH, NDF digestibility, cellulase activity, pH was  6.28 until 6.43.  The NDF digestibility range was 12.27 until 55.61 percent. The lowers of cellulase activity was 5.11 IU/ml and the higher was 24.47 IU/ml. The range of acetic acid yield was 63.37 to 307.467 mg/100 ml. Range of  propionic production was 15.17 to 352.20 mg/ 100 ml. The production of butiric acid was 8.77 to 40.87 mg/ 100 ml. The cellulase activity  of cellulolytic rumen bacteria of buffalo was higher than cattle, and also their effect on NDF digestibility of rice straw, elephant grass, and sesbania leaves cell walls. The A3 of cellulolytic rumen bacteria isolate of  buffalo changed cell walls substrat to volatile fatty  acid was more effective than cattle, especially on cell elephant grass. Propionic and butiric  acid that was produced by cellulolytic rumen bacteria isolate of buffalo more higher than cattle (Animal Production 1 (1 : 1-9 (1999 Key Words: Cellulolytic, VFA, Rumen Bacteria, Buffalo, Cattle.

  16. Spatial heterogeneity of cellulolytic activity and fungal communities within individual decomposing Quercus petraea leaves

    Czech Academy of Sciences Publication Activity Database

    Navrátilová, Diana; Větrovský, Tomáš; Baldrian, Petr

    27 Part A, JUNE (2017), s. 125-133 ISSN 1754-5048 R&D Projects: GA ČR GA13-06763S; GA MŠk(CZ) LD15086 Institutional support: RVO:61388971 Keywords : Cellulose decomposition * Cellobiohydrolase * Enzyme activity Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.219, year: 2016

  17. Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.

    Science.gov (United States)

    Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin

    2016-06-08

    The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

  18. Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNAIle2 and ATP

    International Nuclear Information System (INIS)

    Osawa, Takuo; Inanaga, Hideko; Kimura, Satoshi; Terasaka, Naohiro; Suzuki, Tsutomu; Numata, Tomoyuki

    2011-01-01

    A. fulgidus TiaS was cocrystallized with tRNA Ile2 and ATP and X-ray diffraction data were collected to 2.9 Å resolution using a synchrotron-radiation source. The cytidine at the first anticodon position of archaeal tRNA Ile2 , which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm 2 C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA Ile -agm 2 C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA Ile2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA Ile2 and ATP by the vapour-diffusion method. The crystals of the TiaS–tRNA Ile2 –ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3 2 21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS–tRNA Ile2 –ATP complex, with a Matthews coefficient of 2.8 Å 3 Da −1 and a solvent content of 61%

  19. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with /sup 125/I-labelled Clq binding and Raji-cell RIA tests

    Energy Technology Data Exchange (ETDEWEB)

    Casali, P; Bossus, A; Carpentier, N A; Lambert, P H [Hopital Cantonal Geneve (Switzerland)

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample was incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes were detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test did not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG was 3 ..mu..g/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 ..mu..g/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the /sup 125/I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, /sup 125/I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application.

  20. Complexo enzimático para suínos: digestão, metabolismo, desempenho e impacto ambiental Enzyme complex for swine: nutrient digestion, metabolism, performance and environmental impact

    Directory of Open Access Journals (Sweden)

    Urbano dos Santos Ruiz

    2008-03-01

    energy, protein and amino acids levels. In the Exp 2, growth performance, production and composition of feces in total solids, volatile solids, nitrogen, macro and micro minerals, in relation to growth performance, were measured in barrows from 50 to 151 days of age and 18.34 ± 1.35 kg initial body weight. Three experimental diets were used, one formulated to meet or exceed the nutritional requirements of swine according to NRC (1998 and the others with reduced energy, protein and amino acids levels, with or without the enzyme complex. It was used a complete randomized block design. Dietary enzyme supplementation in feeds based mainly on corn and soybean meal did not improve nutrient digestibilities and growth performance neither reduced nutrient excretion in feces. More research is necessary to test new nutritional matrix and different levels of the enzyme complex.

  1. Effect of gamma irradiation and environmental factors on the production of extracellular cellulase enzyme by trichoderma Spp. using banana waste under solid state bio processing

    International Nuclear Information System (INIS)

    El-Shafey, H.M.; Matar, Z.A.I.; Ghanem, S.M.A.

    2007-01-01

    Fungal strains were isolated from degraded banana waste including leaves, pseudo stems and skins. Many isolated strains showed cellulolytic activities using the plate screening medium. The hyper cellulolytic isolates were selected on the basis of the diameter of the hydrolysis zone surrounding the colonies and identified to the genus level. The identified strains were found to belong to one of the genera Trichoderma, Aspergillus, Pleurotus or Penicillium. The strain with the larger diameter of the hydrolysis zone was found to belong to the genus Trichoderma. It was further identified to be Trichoderma harzianum, which was selected to be studied. Banana waste including leaves and pseudo stems were inoculated by the selected fungus and the production of the carboxymethyl cellulase (CMCase) and filter paper cellulase (FPCase) was followed during changes of the growth conditions under solid state fermentation. It was found that the two enzymes shared the same incubation temperature (25 degree C) and incubation period (18 days) for the maximum enzyme production. The gamma radiation dose of 1.5 KGy increased the production of CMCase produced on leaves by 4.0% and on pseudo stems by 5.6% and the production of FPCase produced on leaves by 2.4% and on pseudo stems by 2.3%. The results also suggest that FPCase and CMCase enzymes produced on leaves were higher than those produced from pseudo stems and the level of CMCase enzyme produced was higher than that of FPCase

  2. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  3. lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

    Directory of Open Access Journals (Sweden)

    Nagy Olga

    2012-03-01

    Full Text Available Abstract Background Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, lemming (lmg mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila melanogaster. Results The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite

  4. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  5. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  6. Rethinking fundamentals of enzyme action.

    Science.gov (United States)

    Northrop, D B

    1999-01-01

    Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

  7. Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity.

    Science.gov (United States)

    Cunha, Eva S; Hatem, Christine L; Barrick, Doug

    2016-08-01

    Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Caldicellulosiruptor obsidiansis sp. nov., an anaerobic, extremely thermophilic, cellulolytic bacterium isolated from Obsidian Pool, Yellowstone National Park.

    Science.gov (United States)

    Hamilton-Brehm, Scott D; Mosher, Jennifer J; Vishnivetskaya, Tatiana; Podar, Mircea; Carroll, Sue; Allman, Steve; Phelps, Tommy J; Keller, Martin; Elkins, James G

    2010-02-01

    A novel, obligately anaerobic, extremely thermophilic, cellulolytic bacterium, designated OB47(T), was isolated from Obsidian Pool, Yellowstone National Park, WY. The isolate was a nonmotile, non-spore-forming, Gram-positive rod approximately 2 microm long by 0.2 microm wide and grew at temperatures between 55 and 85 degrees C, with the optimum at 78 degrees C. The pH range for growth was 6.0 to 8.0, with values of near 7.0 being optimal. Growth on cellobiose produced the fastest specific growth rate at 0.75 h(-1). The organism also displayed fermentative growth on glucose, maltose, arabinose, fructose, starch, lactose, mannose, sucrose, galactose, xylose, arabinogalactan, Avicel, xylan, filter paper, processed cardboard, pectin, dilute acid-pretreated switchgrass, and Populus. OB47(T) was unable to grow on mannitol, fucose, lignin, Gelrite, acetate, glycerol, ribose, sorbitol, carboxymethylcellulose, and casein. Yeast extract stimulated growth, and thiosulfate, sulfate, nitrate, and sulfur were not reduced. Fermentation end products were mainly acetate, H2, and CO2, although lactate and ethanol were produced in 5-liter batch fermentations. The G+C content of the DNA was 35 mol%, and sequence analysis of the small subunit rRNA gene placed OB47(T) within the genus Caldicellulosiruptor. Based on its phylogenetic and phenotypic properties, the isolate is proposed to be designated Caldicellulosiruptor obsidiansis sp. nov. and OB47 is the type strain (ATCC BAA-2073).

  9. Caldicellulosiruptor obsidiansis sp. nov., an Anaerobic, Extremely Thermophilic, Cellulolytic Bacterium Isolated from Obsidian Pool, Yellowstone National Park▿

    Science.gov (United States)

    Hamilton-Brehm, Scott D.; Mosher, Jennifer J.; Vishnivetskaya, Tatiana; Podar, Mircea; Carroll, Sue; Allman, Steve; Phelps, Tommy J.; Keller, Martin; Elkins, James G.

    2010-01-01

    A novel, obligately anaerobic, extremely thermophilic, cellulolytic bacterium, designated OB47T, was isolated from Obsidian Pool, Yellowstone National Park, WY. The isolate was a nonmotile, non-spore-forming, Gram-positive rod approximately 2 μm long by 0.2 μm wide and grew at temperatures between 55 and 85°C, with the optimum at 78°C. The pH range for growth was 6.0 to 8.0, with values of near 7.0 being optimal. Growth on cellobiose produced the fastest specific growth rate at 0.75 h−1. The organism also displayed fermentative growth on glucose, maltose, arabinose, fructose, starch, lactose, mannose, sucrose, galactose, xylose, arabinogalactan, Avicel, xylan, filter paper, processed cardboard, pectin, dilute acid-pretreated switchgrass, and Populus. OB47T was unable to grow on mannitol, fucose, lignin, Gelrite, acetate, glycerol, ribose, sorbitol, carboxymethylcellulose, and casein. Yeast extract stimulated growth, and thiosulfate, sulfate, nitrate, and sulfur were not reduced. Fermentation end products were mainly acetate, H2, and CO2, although lactate and ethanol were produced in 5-liter batch fermentations. The G+C content of the DNA was 35 mol%, and sequence analysis of the small subunit rRNA gene placed OB47T within the genus Caldicellulosiruptor. Based on its phylogenetic and phenotypic properties, the isolate is proposed to be designated Caldicellulosiruptor obsidiansis sp. nov. and OB47 is the type strain (ATCC BAA-2073). PMID:20023107

  10. Dominant ectosymbiotic bacteria of cellulolytic protists in the termite gut also have the potential to digest lignocellulose.

    Science.gov (United States)

    Yuki, Masahiro; Kuwahara, Hirokazu; Shintani, Masaki; Izawa, Kazuki; Sato, Tomoyuki; Starns, David; Hongoh, Yuichi; Ohkuma, Moriya

    2015-12-01

    Wood-feeding lower termites harbour symbiotic gut protists that support the termite nutritionally by degrading recalcitrant lignocellulose. These protists themselves host specific endo- and ectosymbiotic bacteria, functions of which remain largely unknown. Here, we present draft genomes of a dominant, uncultured ectosymbiont belonging to the order Bacteroidales, 'Candidatus Symbiothrix dinenymphae', which colonizes the cell surface of the cellulolytic gut protists Dinenympha spp. We analysed four single-cell genomes of Ca. S. dinenymphae, the highest genome completeness was estimated to be 81.6-82.3% with a predicted genome size of 4.28-4.31 Mb. The genome retains genes encoding large parts of the amino acid, cofactor and nucleotide biosynthetic pathways. In addition, the genome contains genes encoding various glycoside hydrolases such as endoglucanases and hemicellulases. The genome indicates that Ca. S. dinenymphae ferments lignocellulose-derived monosaccharides to acetate, a major carbon and energy source of the host termite. We suggest that the ectosymbiont digests lignocellulose and provides nutrients to the host termites, and hypothesize that the hydrolytic activity might also function as a pretreatment for the host protist to effectively decompose the crystalline cellulose components. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.

    Science.gov (United States)

    Denman, Stuart E; McSweeney, Christopher S

    2006-12-01

    Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.

  12. Patterns of functional enzyme activity in fungus farming ambrosia beetles

    Directory of Open Access Journals (Sweden)

    De Fine Licht Henrik H

    2012-06-01

    Full Text Available Abstract Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily

  13. Patterns of functional enzyme activity in fungus farming ambrosia beetles.

    Science.gov (United States)

    De Fine Licht, Henrik H; Biedermann, Peter H W

    2012-06-06

    In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray

  14. Efeito da suplementação enzimática no desempenho e qualidade dos ovos de poedeiras comerciais = Effects of the enzyme complex supplementation on the performance and egg quality of laying hens

    Directory of Open Access Journals (Sweden)

    Alice Eiko Murakami

    2007-04-01

    Full Text Available O objetivo deste trabalho foi determinar o efeito da utilização de um complexo multienzimático sobre o desempenho e qualidade dos ovos de poedeiras comerciais. Foram utilizadas 336 poedeiras comerciais, HyLine W36, com 29 semanas de idade distribuídas em um delineamento inteiramente casualizado com 7 tratamentos e 6 repetições de 8 aves cada. Os tratamentos consistiram de uma dieta basal suplementada com o complexo enzimático nos níveis de 400 ou 500 ppm com valorização do nível de proteína, energia metabolizável e aminoácidos do farelo de soja em 5/5/5, 7/7/7 e 7/7/9%, respectivamente. As variáveisestudadas foram: consumo de ração (g ave-1 dia-1, conversão alimentar (kg dz-1 de ovos, peso médio dos ovos (g, produção de ovos (%, gravidade específica (g mL-1, Unidade Haugh, coloração da gema, percentuais de sólidos totais, de gema, clara e casca (%,espessura da casca (mm e composição bromatológica das excretas. A adição do complexo multienzimático apresentou resultados produtivos e de qualidade dos ovos semelhantes à dieta controle.The present study aimed to evaluate the effects of a commercial enzyme complex on the performance and egg quality of commerciallaying hens. A total of 336 29-week old Hyline W36 layers were allocated to a completely randomized experimental design with seven treatments and six replications of eight birds each. The treatments consisted of the basal diet supplemented with inclusion of (500 or 400ppm, overestimating the percentages of protein, ME and amino acids of the soybean meal by 5:5:5, 7:7:7 and 7:7:9, respectively. The studied variables were: feed intake (g bird-1 day-1, feed:egg (kg feed-1 egg dozen-1, egg weight (g, egg laying percentage (hen day-1 specific gravity (g ml-1, Haugh Unit, color of the egg yolk, total solids percentage (%, egg yolk percentage, albumen and eggshell (%, eggshell thickness (mm and chemical compositionof hen manure. The inclusion of enzyme complex in the

  15. Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea Hare Aplysia kurodai. Efficient glucose release from sea lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa ß-glucosidase.

    Directory of Open Access Journals (Sweden)

    Akihiko Tsuji

    Full Text Available Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds. In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa. Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase and 2 ß-glucosidases (110K and 210K were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K

  16. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

    Science.gov (United States)

    Adav, Sunil S; Li, An A; Manavalan, Arulmani; Punt, Peter; Sze, Siu Kwan

    2010-08-06

    The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

  17. Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

    Science.gov (United States)

    Leatham, Gary F.

    1985-01-01

    Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting. PMID:16346918

  18. Modeling the Syn-Disposition of Nitrogen Donors in Non-Heme Diiron Enzymes. Synthesis, Characterization, and Hydrogen Peroxide Reactivity of Diiron(III) Complexes with the Syn N-Donor Ligand H2BPG2DEV

    Science.gov (United States)

    Friedle, Simone; Kodanko, Jeremy J.; Morys, Anna J.; Hayashi, Takahiro; Moënne-Loccoz, Pierre; Lippard, Stephen J.

    2009-01-01

    In order to model the syn disposition of histidine residues in carboxylate-bridged non-heme diiron enzymes, we prepared a new dinucleating ligand, H2BPG2DEV, that provides this geometric feature. The ligand incorporates biologically relevant carboxylate functionalities, which have not been explored as extensively as nitrogen-only analogs. Three novel oxo-bridged diiron(III) complexes [Fe2(μ-O)(H2O)2-(BPG2DEV)](ClO4)2 (6), [Fe2(μ-O)(μ-O CAriPrO)(BPG2DEV)](ClO4) (7), and [Fe2(μ-O)(μ-CO3)(BPG2DEV)] (8) were prepared. Single crystal X-ray structural characterization confirms that two pyridines are bound syn with respect to the Fe–Fe vector in these compounds. The carbonato-bridged complex 8 forms quantitatively from 6 in a rapid reaction with gaseous CO2 in organic solvents. A common maroon-colored intermediate (λmax = 490 nm; ε = 1500 M−1 cm−1) forms in reactions of 6, 7, or 8 with H2O2 and NEt3 in CH3CN/H2O solutions. Mass spectrometric analyses of this species, formed using 18O-labeled H2O2, indicate the presence of a peroxide ligand bound to the oxo-bridged diiron(III) center. The Mössbauer spectrum at 90 K of the EPR-silent intermediate exhibits a quadrupole doublet with δ. = 0.58 mm/s and ΔEQ = 0.58 mm/s. The isomer shift is typical for a peroxodiiron(III) species, but the quadrupole splitting parameter is unusually small compared to related complexes. These Mössbauer parameters are comparable to those observed for a peroxo intermediate formed in the reaction of reduced toluene/o-xylene monooxygenase hydroxylase (ToMOH) with dioxygen. Resonance Raman studies reveal an unusually low-energy O–O stretching mode in the peroxo intermediate that is consistent with a short diiron distance. Although peroxodiiron(III) intermediates generated from 6, 7, and 8 are poor O-atom transfer catalysts, they display highly efficient catalase activity, with turnover numbers up to 10,000. In contrast to hydrogen peroxide reactions of diiron(III) complexes that lack

  19. Genome Sequence and Analysis of the Soil Cellulolytic ActinomyceteThermobifida fusca

    Energy Technology Data Exchange (ETDEWEB)

    Lykidis, Athanasios; Mavromatis, Konstantinos; Ivanova, Natalia; Anderson, Iain; Land, Miriam; DiBartolo, Genevieve; Martinez, Michele; Lapidus, Alla; Lucas, Susan; Copeland, Alex; Richardson, Paul; Wilson,David B.; Kyrpides, Nikos

    2007-02-01

    Thermobifida fusca is a moderately thermophilic soilbacterium that belongs to Actinobacteria. 3 It is a major degrader ofplant cell walls and has been used as a model organism for the study of 4secreted, thermostable cellulases. The complete genome sequence showedthat T. fusca has a 5 single circular chromosome of 3642249 bp predictedto encode 3117 proteins and 65 RNA6 species with a coding densityof 85percent. Genome analysis revealed the existence of 29 putative 7glycoside hydrolases in addition to the previously identified cellulasesand xylanases. The 8 glycosyl hydrolases include enzymes predicted toexhibit mainly dextran/starch and xylan 9 degrading functions. T. fuscapossesses two protein secretion systems: the sec general secretion 10system and the twin-arginine translocation system. Several of thesecreted cellulases have 11 sequence signatures indicating theirsecretion may be mediated by the twin-arginine12 translocation system. T.fusca has extensive transport systems for import of carbohydrates 13coupled to transcriptional regulators controlling the expression of thetransporters and14 glycosylhydrolases. In addition to providing anoverview of the physiology of a soil 15 actinomycete, this study presentsinsights on the transcriptional regulation and secretion of16 cellulaseswhich may facilitate the industrial exploitation of thesesystems.

  20. Diversity of Cellulolytic Microbes and the Biodegradation of Municipal Solid Waste by a Potential Strain

    Science.gov (United States)

    Gautam, S. P.; Bundela, P. S.; Pandey, A. K.; Jamaluddin; Awasthi, M. K.; Sarsaiya, S.

    2012-01-01

    Municipal solid waste contains high amounts of cellulose, which is an ideal organic waste for the growth of most of microorganism as well as composting by potential microbes. In the present study, Congo red test was performed for screening of microorganism, and, after selecting a potential strains, it was further used for biodegradation of organic municipal solid waste. Forty nine out of the 250 different microbes tested (165 belong to fungi and 85 to bacteria) produced cellulase enzyme and among these Trichoderma viride was found to be a potential strain in the secondary screening. During the biodegradation of organic waste, after 60 days, the average weight losses were 20.10% in the plates and 33.35% in the piles. There was an increase in pH until 20 days. pH however, stabilized after 30 days in the piles. Temperature also stabilized as the composting process progressed in the piles. The high temperature continued until 30 days of decomposition, after which the temperature dropped to 40°C and below during the maturation. Good quality compost was obtained in 60 days. PMID:22518141

  1. Diversity of Cellulolytic Microbes and the Biodegradation of Municipal Solid Waste by a Potential Strain

    Directory of Open Access Journals (Sweden)

    S. P. Gautam

    2012-01-01

    Full Text Available Municipal solid waste contains high amounts of cellulose, which is an ideal organic waste for the growth of most of microorganism as well as composting by potential microbes. In the present study, Congo red test was performed for screening of microorganism, and, after selecting a potential strains, it was further used for biodegradation of organic municipal solid waste. Forty nine out of the 250 different microbes tested (165 belong to fungi and 85 to bacteria produced cellulase enzyme and among these Trichoderma viride was found to be a potential strain in the secondary screening. During the biodegradation of organic waste, after 60 days, the average weight losses were 20.10% in the plates and 33.35% in the piles. There was an increase in pH until 20 days. pH however, stabilized after 30 days in the piles. Temperature also stabilized as the composting process progressed in the piles. The high temperature continued until 30 days of decomposition, after which the temperature dropped to 40°C and below during the maturation. Good quality compost was obtained in 60 days.

  2. Engineering yeast consortia for surface-display of complex cellulosome structures

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wilfred [University of Delaware

    2014-03-31

    As our society marches toward a more technologically advanced future, energy and environmental sustainability are some of the most challenging problems we face today. Biomass is one of the most abundant renewable-feedstock for sustainable production of biofuels. However, the main technological obstacle to more widespread uses of this resource is the lack of low-cost technologies to overcome the recalcitrant nature of the cellulosic structure, especially the hydrolysis step on highly ordered celluloses. In this proposal, we successfully engineered several efficient and inexpensive whole-cell biocatalysts in an effort to produce economically compatible and sustainable biofuels, namely cellulosic ethanol. Our approach was to display of a highly efficient cellulolytic enzyme complex, named cellulosome, on the surface of a historical ethanol producer Saccharomyces cerevisiae for the simultaneous and synergistic saccharification and fermentation of cellulose to ethanol. We first demonstrated the feasibility of assembling a mini-cellulosome by incubating E. coli lysates expressing three different cellulases. Resting cells displaying mini-cellulosomes produced 4-fold more ethanol from phosphoric acid-swollen cellulose (PASC) than cultures with only added enzymes. The flexibility to assemble the mini-cellulosome structure was further demonstrated using a synthetic yeast consortium through intracellular complementation. Direct ethanol production from PASC was demonstrated with resting cell cultures. To create a microorganism suitable for a more cost-effective process, called consolidated bioprocessing (CBP), a synthetic consortium capable of displaying mini-cellulosomes on the cell surface via intercellular complementation was created. To further improve the efficiency, a new adaptive strategy of employing anchoring and adaptor scaffoldins to amplify the number of enzymatic subunits was developed, resulting in the creation of an artificial tetravalent cellulosome on the

  3. Enzymes in Human Milk.

    Science.gov (United States)

    Dallas, David C; German, J Bruce

    2017-01-01

    Milk proteins are a complex and diverse source of biological activities. Beyond their function, intact milk proteins also act as carriers of encrypted functional sequences that, when released as peptides, exert biological functions, including antimicrobial and immunomodulatory activity, which could contribute to the infant's competitive success. Research has now revealed that the release of these functional peptides begins within the mammary gland itself. A complex array of proteases produced in mother's milk has been shown to be active in the milk, releasing these peptides. Moreover, our recent research demonstrates that these milk proteases continue to digest milk proteins within the infant's stomach, possibly even to a larger extent than the infant's own proteases. As the neonate has relatively low digestive capacity, the activity of milk proteases in the infant may provide important assistance to digesting milk proteins. The coordinated release of these encrypted sequences is accomplished by selective proteolytic action provided by an array of native milk proteases and infant-produced enzymes. The task for scientists is now to discover the selective advantages of this protein-protease-based peptide release system. © 2017 Nestec Ltd., Vevey/S. Karger AG, Basel.

  4. Effect of feeding palm oil by-products based diets on total bacteria, cellulolytic bacteria and methanogenic archaea in the rumen of goats.

    Science.gov (United States)

    Abubakr, Abdelrahim; Alimon, Abdul Razak; Yaakub, Halimatun; Abdullah, Norhani; Ivan, Michael

    2014-01-01

    Rumen microorganisms are responsible for digestion and utilization of dietary feeds by host ruminants. Unconventional feed resources could be used as alternatives in tropical areas where feed resources are insufficient in terms of quality and quantity. The objective of the present experiment was to evaluate the effect of diets based on palm oil (PO), decanter cake (DC) or palm kernel cake (PKC) on rumen total bacteria, selected cellulolytic bacteria, and methanogenic archaea. Four diets: control diet (CD), decanter cake diet (DCD), palm kernel cake diet (PKCD) and CD plus 5% PO diet (CPOD) were fed to rumen cannulated goats and rumen samples were collected at the start of the experimental diets (day 0) and on days 4, 6, 8, 12, 18, 24 and 30 post dietary treatments. Feeding DCD and PKCD resulted in significantly higher (Pgoats fed PKCD and CPOD and the trend showed a severe reduction on days 4 and 6 post experimental diets. In conclusion, results indicated that feeding DCD and PKC increased the populations of cellulolytic bacteria and decreased the density of methanogenic archaea in the rumen of goats.

  5. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  6. Enzymic hydrolysis of cellulosic wastes to glucose

    Energy Technology Data Exchange (ETDEWEB)

    Spano, L A; Medeiros, J; Mandels, M

    1976-01-01

    An enzymic process for the conversion of cellulose to glucose is based on the use of a specific enzyme derived from mutant strains of the fungus trichoderma viride which is capable of reacting with the crystalline fraction of the cellulose molecule. The production and mode of action of the cellulase complex produced during the growth of trichoderma viride is discussed as well as the application of such enzymes for the conversion of cellulosic wastes to crude glucose syrup for use in production of chemical feedstocks, single-cell proteins, fuels, solvents, etc.

  7. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A

    1981-04-04

    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  8. Profiling the orphan enzymes

    Science.gov (United States)

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  9. The ultrasound technology for modifying enzyme activity

    Directory of Open Access Journals (Sweden)

    Meliza Lindsay Rojas

    2016-01-01

    Full Text Available Enzymes are protein complexes compounds widely studied and used due to their ability to catalyze reactions. The food processing mainly a ims the inactivation of enzymes due to various undesirable effects. However, there are many processes that can be optimized by its catalytic activity. In this context, different technologies have been applied both to inactivate or to improve the enzymes ef ficiency. The Ultrasound technology emerges as an alternative mainly applied to achieve the enzyme inactivation. On the contrary, very few investigations show the ability of this technology under certain conditions to achieve the opposite effect (i.e. increase the catalytic activity of enzymes. The objective of this study was to correlate the ultrasonic energy delivered to the sample (J/mL with the residual enzymatic activity and explain the possible mechanisms which results in the enzymatic activation/in activation complex behavior. The activity of POD in coconut water was evaluated as a model. The enzymatic activity initially increased, followed by reduction with a trend to enzyme inactivation. This complex behavior is directly related to the applied ultr asonic energy and their direct mechanical effects on the product, as well as the effect in the enzymatic infinite intermediate states and its structural conformation changes. The obtained results are useful for both academic and industrial perspectives.

  10. The ultrasound technology for modifying enzyme activity

    Directory of Open Access Journals (Sweden)

    Meliza Lindsay

    2016-06-01

    Full Text Available Enzymes are protein complexes compounds widely studied and used due to their ability to catalyze reactions. The food processing mainly aims the inactivation of enzymes due to various undesirable effects. However, there are many processes that can be optimized by its catalytic activity. In this context, different technologies have been applied both to inactivate or to improve the enzymes efficiency. The Ultrasound technology emerges as an alternative mainly applied to achieve the enzyme inactivation. On the contrary, very few investigations show the ability of this technology under certain conditions to achieve the opposite effect (i.e. increase the catalytic activity of enzymes. The objective of this study was to correlate the ultrasonic energy delivered to the sample (J/mL with the residual enzymatic activity and explain the possible mechanisms which results in the enzymatic activation/inactivation complex behavior. The activity of POD in coconut water was evaluated as a model. The enzymatic activity initially increased, followed by reduction with a trend to enzyme inactivation. This complex behavior is directly related to the applied ultrasonic energy and their direct mechanical effects on the product, as well as the effect in the enzymatic infinite intermediate states and its structural conformation changes. The obtained results are useful for both academic and industrial perspectives.

  11. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  12. Does cypermethrin affect enzyme activity, respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

    Institute of Scientific and Technical Information of China (English)

    Ronnie Von Santos Veloso; Eliseu José G.Pereira; Raul Narciso C.Guedes; Maria Goreti A.Oliveira

    2013-01-01

    Insecticides cause a range of sub-lethal effects on targeted insects,which are frequently detrimental to them.However,targeted insects are able to cope with insecticides within sub-lethal ranges,which vary with their susceptibility.Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera:Curculionidae) to sub-lethal exposure to the pyrethoid insecticide cypermethrin.We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure.Lower respiration rate and lower activity were also expected in insecticide-resistant insects since these traits are also likely to favor survivorship under insecticide exposure.Curiously though,cypermethrin did not affect activity of digestive and energy metabolism enzymes,and even reduced the activity of some enzymes (particularly for cellulase and cysteine-proteinase activity in this case).There was strain variation in response,which may be (partially) related to insecticide resistance in some strains.Sub-lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects,which is likely to impair their fitness.However,such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced).Walking activity varies with strain and may minimize insecticide exposure,which should be a concern,particularly if associated with (physiological) insecticide resistance.

  13. An approach to mitigating soil CO2 emission by biochemically inhibiting cellulolytic microbial populations through mediation via the medicinal herb Isatis indigotica

    Science.gov (United States)

    Wu, Hong-Sheng; Chen, Su-Yun; Li, Ji; Liu, Dong-Yang; Zhou, Ji; Xu, Ya; Shang, Xiao-Xia; Wei, Dong-yang; Yu, Lu-ji; Fang, Xiao-hang; Li, Shun-yi; Wang, Ke-ke

    2017-06-01

    Greenhouse gases (GHGs, particularly carbon dioxide (CO2)) emissions from soil under wheat production are a significant source of agricultural carbon emissions that have not been mitigated effectively. A field experiment and a static incubation study in a lab were conducted to stimulate wheat growth and investigate its potential to reduce CO2 emissions from soil through intercropping with a traditional Chinese medicinal herb called Isatis indigotica. This work was conducted by adding I. indigotica root exudates based on the quantitative real-time PCR (qPCR) analysis of the DNA copy number of the rhizosphere or bulk soil microbial populations. This addition was performed in relation to the CO2 formation by cellulolytic microorganisms (Penicillium oxalicum, fungi and Ruminococcus albus) to elucidate the microbial ecological basis for the molecular mechanism that decreases CO2 emissions from wheat fields using I. indigotica. The results showed that the panicle weight and full grains per panicle measured through intercropping with I. indigotica (NPKWR) increased by 39% and 28.6%, respectively, compared to that of the CK (NPKW). Intercropping with I. indigotica significantly decreased the CO2 emissions from soil under wheat cultivation. Compared with CK, the total CO2 emission flux during the wheat growth period in the I. indigotica (NPKWR) intercropping treatment decreased by 29.26%. The intensity of CO2 emissions per kg of harvested wheat grain declined from 7.53 kg CO2/kg grain in the NPKW (CK) treatment to 5.55 kg CO2/kg grain in the NPKWR treatment. The qPCR analysis showed that the DNA copy number of the microbial populations of cellulolytic microorganisms (P. oxalicum, fungi and R. albus) in the field rhizosphere around I. indigotica or in the bulk soil under laboratory incubation was significantly lower than that of CK. This finding indicated that root exudates from I. indigotica inhibited the activity and number of cellulolytic microbial populations, which led

  14. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  15. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  16. Targeted enzyme prodrug therapies.

    Science.gov (United States)

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  17. Enzymes in Fermented Fish.

    Science.gov (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  18. Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

    Science.gov (United States)

    Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

    2016-08-01

    Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme

  19. Discovery of enzymes for toluene synthesis from anoxic microbial communities

    DEFF Research Database (Denmark)

    Beller, Harry R.; Rodrigues, Andria V.; Zargar, Kamrun

    2018-01-01

    Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes...... phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from...... a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic...

  20. Kallotenue papyrolyticum gen. nov., sp. nov., a cellulolytic and filamentous thermophile that represents a novel lineage (Kallotenuales ord. nov., Kallotenuaceae fam. nov.) within the class Chloroflexia

    Energy Technology Data Exchange (ETDEWEB)

    Cole, Jesse; Gieler, Brandon; Heisler, Devon; Palisoc, Maryknoll; Williams, Amanda; Dohnalkova, Alice; Ming, Hong; Yu, Tian T.; Dodsworth, Jeremy A.; Li, Wen J.; Hedlund, Brian P.

    2013-08-15

    Several closely-related, thermophilic, and cellulolytic bacterial strains, designated JKG1T, JKG2, JKG3, JKG4, and JKG5, were isolated from a cellulolytic enrichment (corn stover) incubated in the water column of Great Boiling Spring, NV. Strain JKG1T had cells of a diameter of 0.7 - 0.9 μm and length of ~2.0 μm that formed non-branched multicellular filaments reaching >300 μm. Spores were not formed and dense liquid cultures were red. The temperature range for growth was 45-65 °C, with an optimum of 55 °C. The pH range for growth was 5.6-9.0, with an optimum of 7.5. JKG1T grew as an aerobic heterotroph, utilizing glucose, sucrose, xylose, arabinose, cellobiose, carboxymethylcellulose, filter paper, microcrystalline cellulose, xylan, starch, casamino acids, tryptone, peptone, yeast extract, acetate, citrate, lactate, pyruvate, and glycerol as sole carbon sources, and was not observed to photosynthesize. The cells stained Gram-negative. Phylogenetic analysis using 16S rRNA gene sequences placed the new isolates in the class Chloroflexia, but distant from other cultivated members, with the highest sequence identity of 82.5% to Roseiflexus castenholzii. The major quinone was menaquinone-9; no ubiquinones were detected. The major cellular fatty acids (>5%) were C18:0, anteiso-C17:0, iso-C18:0, and iso-C17:0. C16:0, iso-C16:0, and C17:0. The peptidoglycan amino acids were alanine, ornithine, glutamic acid, serine, and asparagine. Whole-cell sugars included mannose, rhamnose, glucose, galactose, ribose, arabinose, and xylose. Morphological, phylogenetic, and chemotaxonomic results suggest that JKG1T is representative of a new lineage within the class Chloroflexia, which we propose to designate Kallotenue papyrolyticum gen. nov., sp. nov., Kallotenuaceae fam. nov., Kallotenuales ord. nov.

  1. Expression of lignocellulolytic enzymes in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mellitzer Andrea

    2012-05-01

    Full Text Available Abstract Background Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic proteins due to several advantages. Results In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. Conclusion In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.

  2. Regulation of the cellulolytic system in Trichoderma reesei by sophorose: induction of cellulase and repression of beta-glucosidase.

    OpenAIRE

    Sternberg, D; Mandels, G R

    1980-01-01

    Sophorose has two regulatory roles in the production of cellulase enzymes in Trichoderma reesei: beta-glucosidase repression and cellulase induction. Sophorose also is hydrolyzed by the mycelial-associated beta-glucosidase. Repression of beta-glucosidase reduces sophorose hydrolysis and thus may increase cellulase induction.

  3. Enzymic lactose hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J J; Brand, J C

    1980-01-01

    Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

  4. Indicators: Sediment Enzymes

    Science.gov (United States)

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  5. Enzyme Vs. Extremozyme -32 ...

    Indian Academy of Sciences (India)

    Enzymes are biocatalytic protein molecules that enhance the rates of ... to physical forces (hydrogen bonds, hydrophobic 1, electrostatic and Van der ... conformation. In 1995 ... surface against 14.7% in Klenow poll (some of the hydrophobic.

  6. Valores energéticos do farelo de arroz integral suplementado com complexos enzimáticos para frangos de corte Energy values of whole rice bran supplemented with enzymes complexes for broilers

    Directory of Open Access Journals (Sweden)

    Renato Alberto Giacometti

    2003-06-01

    Full Text Available Este experimento foi realizado com o objetivo de estudar o efeito de enzimas carboidrases exógenas com atividade xilanase sobre a energia metabolizável aparente (EMA e aparente corrigida (EMAn do farelo de arroz integral (FAI. As enzimas carboidrases com atividade xilanase utilizadas foram as da marca comercial Rovabio TM Excel AP, Allzyme Rice e Ronozyme WX, denominadas, respectivamente, neste trabalho, de enzimas A, B, e C. Foram utilizadas 240 aves de corte da linhagem Coob, com 21 dias de idade, alojadas em gaiolas metálicas com bandejas coletoras de excretas instaladas em uma sala de metabolismo com ambiente controlado. O delineamento foi inteiramente casualizado, constituído por 4 dietas-referência e 4 dietas-teste e 6 repetições, totalizando 48 parcelas com 5 aves cada uma. As dietas-referência e as dietas-teste estudadas foram as seguintes: Dieta-referência à base de milho, farelo de soja, vitaminas e minerais (DR; DR + enzima A; DR + enzima B; DR + enzima C; 70% de DR + 30% de FAI; 70% de DR + 30% de FAI + enzima A; 70% de DR + 30% de FAI + enzima B e 70% de DR + 30% de FAI + enzima C. Utilizou-se o método de coleta total de excretas (Sibbald & Slinger, 1963 durante 5 dias em cada unidade experimental, precedido de 5 dias de adaptação das aves às dietas. As determinações de EMA e EMAn foram realizadas conforme metodologia de Matterson et al. (1965. A EMAn do FAI foi de 2897 kcal/kg de MS. Com o uso das enzimas carboidrases, o melhor valor de EMAn do FAI foi obtido quando utilizou-se a enzima C, sendo de 3083 kcal/kg de MS, com um aumento percentual de 6,4%. A dieta-referência que recebeu a adição da enzima C teve sua EMAn aumentada, o que não ocorreu com a utilização das outras carboidrases.An experiment was conducted to study the effect of exogen carbohydrase enzymes with xylanase activity on the apparent metabolizable energy (AME and apparent corrected metabolizable energy (AMEn of whole rice bran (WRB. The

  7. Overproduction of ligninolytic enzymes

    Science.gov (United States)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  8. Development of eco-friendly process for the production of bioethanol from banana peel using inhouse developed cocktail of thermo-alkali-stable depolymerizing enzymes.

    Science.gov (United States)

    Prakash, Heena; Chauhan, Prakram Singh; General, Thiyam; Sharma, A K

    2018-03-29

    Conversion of agro-industrial wastes to energy is an innovative approach for waste valorization and management which also mitigates environmental pollution. In this view, present study investigated the feasibility of producing bioethanol from banana peels using cocktail of depolymerizing enzyme/s. We isolated Geobacillus stearothermophilus HPA19 from natural resource which produces cocktail of thermo-alkali-stable xylano-pectino-cellulolytic enzyme/s using wheat bran within 24 h. The optimal temperature and pH for xylanase, filter paper cellulase and pectinase were 80, 70 and 80 °C, and 9.0, 8.0 and 9.0, respectively. Cocktail enzymes showed stability at high temperature (80 °C) and pH (10.0). Ni 2+ and Zn 2+ promoted the relative activity of xylanase and FPase, whereas Na + , Ca 2+ and K + promoted pectinase activity. Cocktail was assessed in saccharification of banana peel. Reducing sugar obtained (37.06 mg ml -1 ) after one variable at a time (OVAT) method is greatly influenced by enzyme dose. Further, response surface methodology was used to optimize saccharification leading to twofold increase in reducing sugar. Maximum ethanol production (21.1 gl -1 ) was achieved through fermentation giving the efficiency of 76.5% within 30 h. Hence utilization of waste biomass for production of value-added products through biotechnological intervention not only helps to combat environmental pollution but also contributes significantly to the economy.

  9. Measurement of enzyme activity.

    Science.gov (United States)

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  10. Furin is a chemokine-modifying enzyme

    DEFF Research Database (Denmark)

    Hensbergen, Paul J; Verzijl, Dennis; Balog, Crina I A

    2004-01-01

    Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the tota...

  11. 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.

  12. Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

    Science.gov (United States)

    Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

    2016-01-01

    This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    Science.gov (United States)

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  14. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  15. Decolorization of complex dyes and textile effluent by extracellular enzymes of Cyathus bulleri cultivated on agro-residues/domestic wastes and proposed pathway of degradation of Kiton blue A and reactive orange 16.

    Science.gov (United States)

    Vats, Arpita; Mishra, Saroj

    2017-04-01

    In this study, the white-rot fungus Cyathus bulleri was cultivated on low-cost agro-residues, namely wheat bran (WB), wheat straw (WS), and domestic waste orange peel (OP) for production of ligninolytic enzymes. Of the three substrates, WB and OP served as good materials for the production of laccase with no requirement of additional carbon or nitrogen source. Specific laccase activity of 94.4 U mg -1 extracellular protein and 21.01 U mg -1 protein was obtained on WB and OP, respectively. Maximum decolorization rate of 13.6 μmol h -1  U -1 laccase for reactive black 5 and 22.68 μmol h -1  U -1 laccase for reactive orange 16 (RO) was obtained with the WB culture filtrate, and 11.7 μmol h -1  U -1 laccase for reactive violet 5 was observed with OP culture filtrate. Importantly, Kiton blue A (KB), reported not to be amenable to enzymatic degradation, was degraded by culture filtrate borne activities. Products of degradation of KB and RO were identified by mass spectrometry, and a pathway of degradation proposed. WB-grown culture filtrate decolorized and detoxified real and simulated textile effluents by about 40%. The study highlights the use of inexpensive materials for the production of enzymes effective on dyes and effluents.

  16. Depolymerization of chitosan by enzymes from the digestive tract of ...

    African Journals Online (AJOL)

    A complex of enzymes was isolated in a preparation derived from the digestive tract of sea cucumber, Stichopus japonicus. Hydrolysis of chitosan using this enzyme preparation decreased its molecular weight (Mw), increased its water solubility and produced water-soluble chitosan (WSC). The conditions for hydrolysis were ...

  17. Matrix Metalloproteinase Enzyme Family

    Directory of Open Access Journals (Sweden)

    Ozlem Goruroglu Ozturk

    2013-04-01

    Full Text Available Matrix metalloproteinases play an important role in many biological processes such as embriogenesis, tissue remodeling, wound healing, and angiogenesis, and in some pathological conditions such as atherosclerosis, arthritis and cancer. Currently, 24 genes have been identified in humans that encode different groups of matrix metalloproteinase enzymes. This review discuss the members of the matrix metalloproteinase family and their substrate specificity, structure, function and the regulation of their enzyme activity by tissue inhibitors. [Archives Medical Review Journal 2013; 22(2.000: 209-220

  18. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  19. Translational control of an intestinal microvillar enzyme

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Sjöström, H

    1986-01-01

    The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is only...... about 3% of the adult rate. The small amount synthesized occurs in a high-mannose-glycosylated, membrane-bound, form that is processed to the mature, complex-glycosylated, form at a markedly slower rate than that of the adult enzyme. Extracts of total RNA from adult and foetal intestine contained...

  20. The surface science of enzymes

    DEFF Research Database (Denmark)

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    2002-01-01

    One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function......? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe...... catalysis by enzymes, and in particular the analogies between enzyme catalyzed reactions and surface catalyzed reactions. We do this by discussing two concrete examples of reactions catalyzed both in nature (by enzymes) and in industrial reactors (by inorganic materials), and show that although analogies...

  1. Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

    Science.gov (United States)

    Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2018-02-01

    In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Compounds Released from Biomass Deconstruction: Understanding Their Effect on Cellulose Enzyme Hydrolysis and Their Biological Activity

    Science.gov (United States)

    Djioleu, Angele Mezindjou

    The effect of compounds produced during biomass pretreatment on cellulolytic enzyme was investigated. Liquid prehydrolyzates were prepared by pretreating switchgrass using 24 combinations of temperature, time, and sulfuric acid concentration based on a full factorial design. Temperature was varied from 140°C to 180°C; time ranged from 10 to 40 min; and the sulfuric acid concentrations were 0.5% or 1% (v/v). Identified products in the prehydrolyzates included xylose, glucose, hydroxymethylfurfural (HMF), furfural, acetic acid, formic acid, and phenolic compounds at concentration ranging from 0 to 21.4 g/L. Pretreatment conditions significantly affected the concentrations of compounds detected in prehydrolyzates. When assayed in the presence of switchgrass prehydrolyzates against model substrates, activities of cellulase, betaglucosidase, and exoglucanase, were significantly reduced by at least 16%, 31.8%, and 57.8%, respectively, as compared to the control. A strong positive correlation between inhibition of betaglucosidase and concentration of glucose, acetic acid, and furans in prehydrolyzate was established. Exoglucanase inhibition correlated with the presence of phenolic compounds and acetic acid. The prehydrolyzate, prepared at 160°C, 30 min, and 1% acid, was fractionated by centrifugal partition chromatography (CPC) into six fractions; the inhibition effect of these fractions on betaglucosidase and exoglucanase was determined. The initial hydrolysis rate of cellobiose by betaglucosidase was significantly reduced by the CPC sugar-rich fraction; however, exoglucanase was deactivated by the CPC phenolic-rich fraction. Finally, biological activities of water-extracted compounds from sweetgum bark and their effect on cellulase was investigated. It was determined that 12% of solid content of the bark extract could be accounted by phenolic compounds with gallic acid identified as the most concentrated phytochemical. Sweetgum bark extract inhibited Staphylococcus

  3. Magnetically responsive enzyme powders

    Czech Academy of Sciences Publication Activity Database

    Pospišková, K.; Šafařík, Ivo

    2015-01-01

    Roč. 380, APR 2015 (2015), s. 197-200 ISSN 0304-8853 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : enzyme powders * cross-linking * magnetic modification * magnetic separation * magnetic iron oxides particles * microwave-assisted synthesis Subject RIV: CE - Biochemistry Impact factor: 2.357, year: 2015

  4. Enzyme with rhamnogalacturonase activity.

    NARCIS (Netherlands)

    Kofod, L.V.; Andersen, L.N.; Dalboge, H.; Kauppinen, M.S.; Christgau, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A.G.J.; Schols, H.A.

    1998-01-01

    An enzyme exhibiting rhamnogalacturonase activity, capable of cleaving a rhamnogalacturonan backbone in such a manner that galacturonic acids are left as the non-reducing ends, and which exhibits activity on hairy regions from a soy bean material and/or on saponified hairy regions from a sugar beet

  5. Implantable enzyme amperometric biosensors.

    Science.gov (United States)

    Kotanen, Christian N; Moussy, Francis Gabriel; Carrara, Sandro; Guiseppi-Elie, Anthony

    2012-05-15

    The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Cold-Adapted Enzymes

    Science.gov (United States)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  7. Embedded enzymes catalyse capture

    Science.gov (United States)

    Kentish, Sandra

    2018-05-01

    Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

  8. Photoperiodism and Enzyme Activity

    Science.gov (United States)

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  9. ISFET based enzyme sensors

    NARCIS (Netherlands)

    van der Schoot, Bart H.; Bergveld, Piet

    1987-01-01

    This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the

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

    Energy Technology Data Exchange (ETDEWEB)

    Khadempour, Lily [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Zoology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA; Burnum-Johnson, Kristin E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Baker, Erin S. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Nicora, Carrie D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Webb-Robertson, Bobbie-Jo M. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; White, Richard A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Huang, Eric L. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Currie, Cameron R. [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA

    2016-10-26

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

  11. On the Temperature Dependence of Enzyme-Catalyzed Rates.

    Science.gov (United States)

    Arcus, Vickery L; Prentice, Erica J; Hobbs, Joanne K; Mulholland, Adrian J; Van der Kamp, Marc W; Pudney, Christopher R; Parker, Emily J; Schipper, Louis A

    2016-03-29

    One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems.

  12. The Enzyme Function Initiative†

    Science.gov (United States)

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

    2011-01-01

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

  13. The Enzyme Function Initiative.

    Science.gov (United States)

    Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

    2011-11-22

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.

  14. Bioremediation of Industrial Waste Through Enzyme Producing Marine Microorganisms.

    Science.gov (United States)

    Sivaperumal, P; Kamala, K; Rajaram, R

    Bioremediation process using microorganisms is a kind of nature-friendly and cost-effective clean green technology. Recently, biodegradation of industrial wastes using enzymes from marine microorganisms has been reported worldwide. The prospectus research activity in remediation area would contribute toward the development of advanced bioprocess technology. To minimize industrial wastes, marine enzymes could constitute a novel alternative in terms of waste treatment. Nowadays, the evidence on the mechanisms of bioremediation-related enzymes from marine microorganisms has been extensively studied. This review also will provide information about enzymes from various marine microorganisms and their complexity in the biodegradation of comprehensive range of industrial wastes. © 2017 Elsevier Inc. All rights reserved.

  15. Enzymes for Enhanced Oil Recovery (EOR)

    Energy Technology Data Exchange (ETDEWEB)

    Nasiri, Hamidreza

    2011-04-15

    Primary oil recovery by reservoir pressure depletion and secondary oil recovery by waterflooding usually result in poor displacement efficiency. As a consequence there is always some trapped oil remaining in oil reservoirs. Oil entrapment is a result of complex interactions between viscous, gravity and capillary forces. Improving recovery from hydrocarbon fields typically involves altering the relative importance of the viscous and capillary forces. The potential of many EOR methods depends on their influence on fluid/rock interactions related to wettability and fluid/fluid interactions reflected in IFT. If the method has the potential to change the interactions favorably, it may be considered for further investigation, i.e. core flooding experiment, pilot and reservoir implementation. Enzyme-proteins can be introduced as an enhanced oil recovery method to improve waterflood performance by affecting interactions at the oil-water-rock interfaces. An important part of this thesis was to investigate how selected enzymes may influence wettability and capillary forces in a crude oil-brine-rock system, and thus possibly contribute to enhanced oil recovery. To investigate further by which mechanisms selected enzyme-proteins may contribute to enhance oil recovery, groups of enzymes with different properties and catalytic functions, known to be interfacially active, were chosen to cover a wide range of possible effects. These groups include (1) Greenzyme (GZ) which is a commercial EOR enzyme and consists of enzymes and stabilizers (surfactants), (2) The Zonase group consists of two types of pure enzyme, Zonase1 and Zonase2 which are protease enzymes and whose catalytic functions are to hydrolyze (breakdown) peptide bonds, (3) The Novozyme (NZ) group consists of three types of pure enzyme, NZ2, NZ3 and NZ6 which are esterase enzymes and whose catalytic functions are to hydrolyze ester bonds, and (4) Alpha-Lactalbumin ( -La) which is an important whey protein. The effect of

  16. Early evolution of efficient enzymes and genome organization

    Directory of Open Access Journals (Sweden)

    Szilágyi András

    2012-10-01

    Full Text Available Abstract Background Cellular life with complex metabolism probably evolved during the reign of RNA, when it served as both information carrier and enzyme. Jensen proposed that enzymes of primordial cells possessed broad specificities: they were generalist. When and under what conditions could primordial metabolism run by generalist enzymes evolve to contemporary-type metabolism run by specific enzymes? Results Here we show by numerical simulation of an enzyme-catalyzed reaction chain that specialist enzymes spread after the invention of the chromosome because protocells harbouring unlinked genes maintain largely non-specific enzymes to reduce their assortment load. When genes are linked on chromosomes, high enzyme specificity evolves because it increases biomass production, also by reducing taxation by side reactions. Conclusion The constitution of the genetic system has a profound influence on the limits of metabolic efficiency. The major evolutionary transition to chromosomes is thus proven to be a prerequisite for a complex metabolism. Furthermore, the appearance of specific enzymes opens the door for the evolution of their regulation. Reviewers This article was reviewed by Sándor Pongor, Gáspár Jékely, and Rob Knight.

  17. The effect of varying organosolv pretreatment chemicals on the physicochemical properties and cellulolytic hydrolysis of mountain pine beetle-killed lodgepole pine.

    Science.gov (United States)

    Del Rio, Luis F; Chandra, Richard P; Saddler, Jack N

    2010-05-01

    Mountain pine beetle-killed lodgepole pine (Pinus contorta) chips were pretreated using the organosolv process, and their ease of subsequent enzymatic hydrolysis was assessed. The effect of varying pretreatment chemicals and solvents on the substrate's physicochemical characteristics was also investigated. The chemicals employed were MgCl2, H2SO4, SO2, and NaOH, and the solvents were ethanol and butanol. It was apparent that the different pretreatments resulted in variations in both the chemical composition of the solid and liquid fractions as well in the extent of cellulolytic hydrolysis (ranging from 21% to 82% hydrolysis after 12 h). Pretreatment under acidic conditions resulted in substrates that were readily hydrolyzed despite the apparent contradiction that pretreatment under alkaline conditions resulted in increased delignification (approximately 7% and 10% residual lignin for alkaline conditions versus 17% to 19% for acidic conditions). Acidic pretreatments also resulted in lower cellulose degree of polymerization, shorter fiber lengths, and increased substrate porosity. The substrates generated when butanol/water mixtures were used as the pretreatment solvent were also hydrolyzed more readily than those generated with ethanol/water. This was likely due to the limited miscibility of the solvents resulting in an increased concentration of pretreatment chemicals in the aqueous layer and thus a higher pretreatment severity.

  18. Identification and characterization of an anaerobic ethanol-producing cellulolytic bacterial consortium from Great Basin hot springs with agricultural residues and energy crops.

    Science.gov (United States)

    Zhao, Chao; Deng, Yunjin; Wang, Xingna; Li, Qiuzhe; Huang, Yifan; Liu, Bin

    2014-09-01

    In order to obtain the cellulolytic bacterial consortia, sediments from Great Basin hot springs (Nevada, USA) were sampled and enriched with cellulosic biomass as the sole carbon source. The bacterial composition of the resulting anaerobic ethanol-producing celluloytic bacterial consortium, named SV79, was analyzed. With methods of the full-length 16S rRNA librarybased analysis and denaturing gradient gel electrophoresis, 21 bacteria belonging to eight genera were detected from this consortium. Clones with closest relation to the genera Acetivibrio, Clostridium, Cellulosilyticum, Ruminococcus, and Sporomusa were predominant. The cellulase activities and ethanol productions of consortium SV79 using different agricultural residues (sugarcane bagasse and spent mushroom substrate) and energy crops (Spartina anglica, Miscanthus floridulus, and Pennisetum sinese Roxb) were studied. During cultivation, consortium SV79 produced the maximum filter paper activity (FPase, 9.41 U/ml), carboxymethylcellulase activity (CMCase, 6.35 U/ml), and xylanase activity (4.28 U/ml) with sugarcane bagasse, spent mushroom substrate, and S. anglica, respectively. The ethanol production using M. floridulus as substrate was up to 2.63 mM ethanol/g using gas chromatography analysis. It has high potential to be a new candidate for producing ethanol with cellulosic biomass under anoxic conditions in natural environments.

  19. Synthesis and Characterization of Magnetic Carriers Based on Immobilized Enzyme

    Science.gov (United States)

    Li, F. H.; Tang, N.; Wang, Y. Q.; Zhang, L.; Du, W.; Xiang, J.; Cheng, P. G.

    2018-05-01

    Several new types of carriers and technologies have been implemented to improve traditional enzyme immobilization in industrial biotechnology. The magnetic immobilized enzyme is a kind of new method of enzyme immobilization developed in recent years. An external magnetic field can be used to control the motion mode and direction of immobilized enzyme, and to improve the catalytic efficiency of immobilized enzyme. In this paper, Fe3O4-CaCO3-PDA complex and CaCO3/Fe3O4 composite modified by PEI were prepared. The results show that the morphology of Fe3O4-CaCO3-PDA complex formation is irregular, while the morphology of CaCO3/Fe3O4 composite modified by PEI is regular and has a porous structure.

  20. Concentration profiles near an activated enzyme.

    Science.gov (United States)

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  1. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    Science.gov (United States)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  2. Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes.

    Science.gov (United States)

    Merz, Michael; Eisele, Thomas; Berends, Pieter; Appel, Daniel; Rabe, Swen; Blank, Imre; Stressler, Timo; Fischer, Lutz

    2015-06-17

    Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zambare, Vasudeo; Zambare, Archana; Christopher, Lew P. [Center for Bioprocessing Research & Development, South Dakota School of Mines and Technology, Rapid City 57701, SD (United States); Muthukumarappan, Kasiviswanath [Center for Bioprocessing Research & Development, South Dakota State University, Brookings 57007, SD (United States)

    2011-07-01

    A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 60C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 60C and 70C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli

  4. NRSA enzyme decomposition model data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme activities measured at more than 2000 US streams and rivers. These enzyme data were then used to predict organic matter decomposition and microbial...

  5. Cellulase enzyme and biomass utilization

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... human population grows and economic development. However, the current .... conditions and the production cost of the related enzyme system. Therefore ... Given the importance of this enzyme to these so many industries,.

  6. Effects of isobutyrate supplementation on ruminal microflora, rumen enzyme activities and methane emissions in Simmental steers.

    Science.gov (United States)

    Wang, C; Liu, Q; Zhang, Y L; Pei, C X; Zhang, S L; Wang, Y X; Yang, W Z; Bai, Y S; Shi, Z G; Liu, X N

    2015-02-01

    The objective of this study was to evaluate the effects of isobutyrate supplementation on rumen microflora, enzyme activities and methane emissions in Simmental steers consuming a corn stover-based diet. Eight ruminally cannulated Simmental steers were used in a replicated 4 × 4 Latin square experiment. The treatments were control (without isobutyrate), low isobutyrate (LIB), moderate isobutyrate (MIB) and high isobutyrate (HIB) with 8.4, 16.8 and 25.2 g isobutyrate per steer per day respectively. Isobutyrate was hand-mixed into the concentrate portion. Diet consisted of 60% corn stover and 40% concentrate [dry matter (DM) basis]. Dry matter intake (averaged 9 kg/day) was restricted to a maximum of 90% of ad libitum intake. Population of total bacteria, cellulolytic bacteria and anaerobic fungi were linearly increased, whereas that of protozoa and total methanogens was linearly reduced with increasing isobutyrate supplementation. Real-time PCR quantification of population of Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and Fibrobacter succinogenes was linearly increased with increasing isobutyrate supplementation. Activities of carboxymethyl cellulase, xylanase and β-glucosidase were linearly increased, whereas that of protease was linearly reduced. Methane production was linearly decreased with increasing isobutyrate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover were linearly increased, whereas that of crude protein in diet was linearly decreased with increasing isobutyrate supplementation. The present results indicate that isobutyrate supplemented improved microflora, rumen enzyme activities and methane emissions in steers. It was suggested that the isobutyrate stimulated the digestive micro-organisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum isobutyrate dose was approximately 16.8 g isobutyrate per steer per day. Journal of Animal

  7. 31P and 1H NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: Relaxation measurements with Mn(II) and Co(II)

    International Nuclear Information System (INIS)

    Jarori, G.K.; Ray, B.D.; Rao, B.D.N.

    1989-01-01

    The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31 P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the δ protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31 P relaxation rates in E-MnADP and E-MnATP yields activation energies (ΔE) in the range 6-10 kcal/mol. Thus, the 31 P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E-CoADP and E-CoATP exhibit frequency dependence and ΔE values in the range 1-2 kcal/mol; i.e., these rates depend upon 31 P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 angstrom, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1 H spin-lattice relaxation rate of the δ protons of arginine in the E-MnADP-Arg complex was also measured at three frequencies. From the frequency dependence of the relaxation rate an effective τ C of 0.6 ns has also been calculated, which is most likely to be the electron spin relaxation rate (τ S1 ) for Mn(II) in this complex. The distance estimated on the basis of the reciprocal sixth root of the average relaxation rate of the δ protons was 10.9 ± 0.3 angstrom

  8. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger.

    Science.gov (United States)

    Reilly, Morgann C; Kim, Joonhoon; Lynn, Jed; Simmons, Blake A; Gladden, John M; Magnuson, Jon K; Baker, Scott E

    2018-02-01

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

  9. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Reilly, Morgann C. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kim, Joonhoon [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lynn, Jed [Joint BioEnergy Institute, Emeryville, CA (United States); Wright-Patterson Air Force Base, Dayton, OH (United States); Simmons, Blake A. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gladden, John M. [Joint BioEnergy Institute, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States); Magnuson, Jon K. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Baker, Scott E. [Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2018-01-06

    Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. This strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

  10. Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

    Science.gov (United States)

    Riekki, R; Palomäki, T; Virtaharju, O; Kokko, H; Romantschuk, M; Saarilahti, H T

    2000-07-01

    A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

  11. The FPase properties and morphology changes of a cellulolytic bacterium, Sporocytophaga sp. JL-01, on decomposing filter paper cellulose.

    Science.gov (United States)

    Wang, Xiuran; Peng, Zhongqi; Sun, Xiaoling; Liu, Dongbo; Chen, Shan; Li, Fan; Xia, Hongmei; Lu, Tiancheng

    2012-01-01

    Sporocytophaga sp. JL-01 is a sliding cellulose degrading bacterium that can decompose filter paper (FP), carboxymethyl cellulose (CMC) and cellulose CF11. In this paper, the morphological characteristics of S. sp. JL-01 growing in FP liquid medium was studied by Scanning Electron Microscope (SEM), and one of the FPase components of this bacterium was analyzed. The results showed that the cell shapes were variable during the process of filter paper cellulose decomposition and the rod shape might be connected with filter paper decomposing. After incubating for 120 h, the filter paper was decomposed significantly, and it was degraded absolutely within 144 h. An FPase1 was purified from the supernatant and its characteristics were analyzed. The molecular weight of the FPase1 was 55 kDa. The optimum pH was pH 7.2 and optimum temperature was 50°C under experiment conditions. Zn(2+) and Co(2+) enhanced the enzyme activity, but Fe(3+) inhibited it.

  12. INDUCTION OF AUTOANTIBODIES TO HUMAN ENZYMES FOLLOWING VIRAL-INFECTION - A BIOLOGICALLY RELEVANT HYPOTHESIS

    NARCIS (Netherlands)

    WEIJERS, RNM; LAWSON, C; LEUNISSEN, J

    Macro enzymes, i. e. complexes of normal (iso-)enzymes with an immunoglobulin, may be due to immunological cross-reactions evoked by specific viral antigenic determinants that are homologous to regions in the target enzymes. A search of the National Biomedical Research Foundation protein databank

  13. Size determination of an equilibrium enzymic system by radiation inactivation

    International Nuclear Information System (INIS)

    Simon, P.; Swillens, S.; Dumont, J.E.

    1982-01-01

    Radiation inactivation of complex enzymic systems is currently used to determine the enzyme size and the molecular organization of the components in the system. An equilibrium model was simulated describing the regulation of enzyme activity by association of the enzyme with a regulatory unit. It is assumed that, after irradiation, the system equilibrates before the enzyme activity is assayed. The theoretical results show that the target-size analysis of these numerical data leads to a bad estimate of the enzyme size. Moreover, some implicit assumptions such as the transfer of radiation energy between non-covalently bound molecules should be verified before interpretation of target-size analysis. It is demonstrated that the apparent target size depends on the parameters of the system, namely the size and the concentration of the components, the equilibrium constant, the relative activities of free enzyme and enzymic complex, the existence of energy transfer, and the distribution of the components between free and bound forms during the irradiation. (author)

  14. Utilization of detrital complexes by the oyster, Crassostrea virginica (Gmelin)

    International Nuclear Information System (INIS)

    Crosby, M.P.

    1987-01-01

    The contribution of bacteria and nonliving particulate organic matter of detrital complexes to the nutrition of the American oyster, Crassostrea virginica, was investigated in the laboratory under normal feeding conditions. Results indicate the oysters were capable of assimilating crude fiber extracted from 14 C-Spartina alterniflora with an efficiency of approximately 3% and that enteric bacteria did not enhance this process. Less than 1% of an oyster's energetic demands could be met by direct utilization of this substrate, in the Choptank River subestuary of the Chesapeake Bay. The potential contribution of refractory organics to oysters in large salt marshes having crude fiber concentration greater than in the Choptank system, are discussed. The ability of the oyster to utilize 14 C and 15 N from cellulolytic marine bacteria, isolated from a S. alterniflora dominated salt marsh, was also studied

  15. Mesoscopic dynamics of diffusion-influenced enzyme kinetics.

    Science.gov (United States)

    Chen, Jiang-Xing; Kapral, Raymond

    2011-01-28

    A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t(-1/2) and t(-3/2) power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

  16. Mesoscopic dynamics of diffusion-influenced enzyme kinetics

    Science.gov (United States)

    Chen, Jiang-Xing; Kapral, Raymond

    2011-01-01

    A particle-based mesoscopic model for enzyme kinetics is constructed and used to investigate the influence of diffusion on the reactive dynamics. Enzymes and enzyme-substrate complexes are modeled as finite-size soft spherical particles, while substrate, product, and solvent molecules are point particles. The system is evolved using a hybrid molecular dynamics-multiparticle collision dynamics scheme. Both the nonreactive and reactive dynamics are constructed to satisfy mass, momentum, and energy conservation laws, and reversible reaction steps satisfy detailed balance. Hydrodynamic interactions among the enzymes and complexes are automatically accounted for in the dynamics. Diffusion manifests itself in various ways, notably in power-law behavior in the evolution of the species concentrations. In accord with earlier investigations, regimes where the product production rate exhibits either monotonic or nonmonotonic behavior as a function of time are found. In addition, the species concentrations display both t^{-1/2} and t^{-3/2} power-law behavior, depending on the dynamical regime under investigation. For high enzyme volume fractions, cooperative effects influence the enzyme kinetics. The time dependent rate coefficient determined from the mass action rate law is computed and shown to depend on the enzyme concentration. Lifetime distributions of substrate molecules newly released in complex dissociation events are determined and shown to have either a power-law form for rebinding to the same enzyme from which they were released or an exponential form for rebinding to different enzymes. The model can be used and extended to explore a variety of issues related concentration effects and diffusion on enzyme kinetics.

  17. Isolation and Screening of Thermo-Stable Cellulase Enzyme Fungal Producer at Different Temperature

    International Nuclear Information System (INIS)

    Noor Ashiqin Jamroo; Noor Azrimi Umor; Kamsani

    2015-01-01

    Thermo stable cellulase from fungi has high potential for industrial application. In this study, wild -type of fungal were isolate from different sources such as hot spring water, sea water, soft wood, rice straw and cow dung. The isolates were characterized by cultural and morphological observation. Based on morphological characteristics, the genera of all fungal cultures were identified namely Aspergillus fumigatus. The screening for thermo stable cellulase were done using 2 % carboxymethyl cellulose and congo red as an indicator at temperature 30, 37, 45 and 50 degree Celsius respectively. Out of 26 fungal isolates, only eight isolates were selected for further screening and showed the abilities to secrete cellulases by forming distinct halo zones on selective agar plate. The maximum halo zone ranging from 32 mm to 35 mm were obtained after 72 hour incubation at 50 degree Celsius by H2, SW1 and C1 isolates. As contrary other isolates showed halo zone range from 22 mm to 29 mm at same temperature. All the isolates showed the abilities to secrete cellulase enzyme at other temperature but lower when compared to 50 degree Celsius referred to the halo zone obtained. The SW1 isolates showed highest cellulolytic index which was 2.93 measured at 37 degree Celsius and 2.67 at 50 degree Celsius respectively. (author)

  18. Sekuensing 16S DNA Bakteri Selulolitik Asal Limbah Cairan Rumen Sapi Peranakan Ongole (SEQUENCING OF 16S DNA OF CELLULOLYTIC BACTERIA FROM BOVINE RUMEN FLUID WASTE ONGOLE CROSSBREED

    Directory of Open Access Journals (Sweden)

    Widya Paramita Lokapirnasari

    2017-04-01

    Full Text Available This study aimed to identified cellulolytic inoculant code WPL 214 isolated from bovine rumen fluid waste of Ongole Cross Breed of Surabaya Slaughter house. A single colony of isolates celulolytic grown on 5 mL of liquid media Luria Bertani (LB consist of 1 % NaCl , 1% tripton , 0.5 % yeast extract, containing1 % carboxymethyl cellulose (CMC at temperature 37°C, using a shaker of incubator during 16-18 hours. That isolate determined by 16S DNA gen analysis using High Fidelity Platinum Taq DNA Polymerase with primer forward PB36 5’-AGR GTT TGA TCM TGG CTC AG-3’ and primer reverse PB38 5’-GMT ACCTTG TTA CGA CTT-3’ for PCR. Nucleotide sequence of 16S DNA fragment was determined through the sequencing method. The result was then compared with GenBank database to recognize the type of the sample bacteria. DNA isolation and 16S DNA coding genes amplification were carried out using Kit High Fidelity Platinum Taq DNA Polymerase. Afterward, BLAST was applied to identify the phylogenetic tree. The bacteria was capable of indicating the existence of clear zone in a media CMC by congo red staining. The existence of the clear zone associated with the activity of microbes to degrade cellulose. The conclusión of this research based on the results was the sequencing nucleotides genome 16S DNA showed that cellulolytic inoculant was identified as Enterobacter cloacae WPL 214. ABSTRAK Penelitian ini bertujuan untuk mengidentifikasi lebih lanjut isolat selulolitik kode WPL 214 yang telah diisolasi dari cairan rumen sapi peranakan ongole dari limbah Rumah Potong Hewan Surabaya. Koloni tunggal dari isolat selulolitik ditumbuhkan pada 5 mL media cair Luria Bertani (LB dengan komposisisi 1% NaCl, 1% tripton, 0,5% yeast ekstrak, yang mengandung 1% substrat carboxymethyl cellulose (CMC pada suhu 37°C, dengan pengocokan menggunakan shaker incubator selama ±16-18 jam. Penelitian ini terdiri dari dua tahap, tahap pertama dilakukan isolasi DNA, tahap kedua

  19. Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

    Directory of Open Access Journals (Sweden)

    Hui Wei

    Full Text Available Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory, 3 β-D-glucosidases (2 of them secretory and 243 other glycoside hydrolase (GH proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP strain by introducing a CBH (e.g. CBHI into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

  20. Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

    Science.gov (United States)

    Wei, Hui; Wang, Wei; Yarbrough, John M; Baker, John O; Laurens, Lieve; Van Wychen, Stefanie; Chen, Xiaowen; Taylor, Larry E; Xu, Qi; Himmel, Michael E; Zhang, Min

    2013-01-01

    Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

  1. Cellulolytic potential of probiotic Bacillus Subtilis AMS6 isolated from traditional fermented soybean (Churpi): An in-vitro study with regards to application as an animal feed additive.

    Science.gov (United States)

    Manhar, Ajay K; Bashir, Yasir; Saikia, Devabrata; Nath, Dhrubajyoti; Gupta, Kuldeep; Konwar, Bolin K; Kumar, Rahul; Namsa, Nima D; Mandal, Manabendra

    2016-01-01

    The aim of the present study is to evaluate the probiotic attributes of Bacillus subtilis AMS6 isolated from fermented soybean (Churpi). This isolate exhibited tolerance to low pH (pH 2.0) and bile salt (0.3%), capability to autoaggregate and coaggregate. AMS6 also showed highest antibacterial activity against the pathogenic indicator strain Salmonella enterica typhimurium (MTCC 1252) and susceptibility towards different antibiotics tested. The isolate was effective in inhibiting the adherence of food borne pathogens to Caco-2 epithelial cell lines, and was also found to be non-hemolytic which further strengthen the candidature of the isolate as a potential probiotic. Further studies revealed B. subtilis AMS6 showed cellulolytic activity (0.54±0.05 filter paper units mL(-1)) at 37°C. The isolate was found to hydrolyze carboxymethyl cellulose, filter paper and maize (Zea mays) straw. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96h of incubation. A full length cellulase gene of AMS6 was amplified using degenerate primers consisting of 1499 nucleotides. The ORF encoded for a protein of 499 amino acids residues with a predicted molecular mass of 55.04kDa. The amino acids sequence consisted of a glycosyl hydrolase family 5 domain at N-terminal; Glycosyl hydrolase catalytic core and a CBM-3 cellulose binding domain at its C terminal. The study suggests potential probiotic B. subtilis AMS6 as a promising candidate envisaging its application as an animal feed additive for enhanced fiber digestion and gut health of animal. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase.

    Science.gov (United States)

    Delabona, Priscila da Silva; Cota, Júnio; Hoffmam, Zaira Bruna; Paixão, Douglas Antonio Alvaredo; Farinas, Cristiane Sanchez; Cairo, João Paulo Lourenço Franco; Lima, Deise Juliana; Squina, Fábio Marcio; Ruller, Roberto; Pradella, José Geraldo da Cruz

    2013-03-01

    Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Determination of the activity signature of key carbohydrate metabolism enzymes in phenolic-rich grapevine tissues

    DEFF Research Database (Denmark)

    Covington, Elizabeth Dunn; Roitsch, Thomas Georg; Dermastia, Marina

    2016-01-01

    Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity...... assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf...

  4. Novel Enzymes for Targeted Hydrolysis of Algal Cell Walls

    DEFF Research Database (Denmark)

    Schultz-Johansen, Mikkel

    Seaweeds, also known as macroalgae, constitute a rich source of valuable biomolecules which have a potential industrial application in food and pharma products. The use of enzymes can optimize the extraction and separation of these molecules from the seaweed biomass, but most commercial enzymes...... 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...... degradation. In addition, three carrageenases were characterised; one as a GH16 κ-carrageenase whereas the other two belong to a new GH16 subfamily of enzymes that degrade furcellaran (κ/β-carrageenan). From metagenome sequence data three putative GH107 fucanases were identified and characterized...

  5. EVOLUTIONARY TRANSITIONS IN ENZYME ACTIVITY OF ANT FUNGUS GARDENS

    DEFF Research Database (Denmark)

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G

    2010-01-01

    an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across...... the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens...... are targeted primarily towards partial degradation of plant cell walls, reflecting a plesiomorphic state of non-domesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major...

  6. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  7. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr

    2014-09-01

    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  8. DGAT enzymes and triacylglycerol biosynthesis

    Science.gov (United States)

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

  9. Enzyme stabilization for pesticide degradation

    Energy Technology Data Exchange (ETDEWEB)

    Rivers, D.B.; Frazer, F.R. III; Mason, D.W.; Tice, T.R.

    1988-01-01

    Enzymes offer inherent advantages and limitations as active components of formulations used to decontaminate soil and equipment contaminated with toxic materials such as pesticides. Because of the catalytic nature of enzymes, each molecule of enzyme has the potential to destroy countless molecules of a contaminating toxic compound. This degradation takes place under mild environmental conditions of pH, temperature, pressure, and solvent. The basic limitation of enzymes is their degree of stability during storage and application conditions. Stabilizing methods such as the use of additives, covalent crosslinking, covalent attachment, gel entrapment, and microencapsulation have been directed developing an enzyme preparation that is stable under extremes of pH, temperature, and exposure to organic solvents. Initial studies were conducted using the model enzymes subtilisin and horseradish peroxidase.

  10. Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme

    NARCIS (Netherlands)

    van Noorden, Cornelis J. F.

    2002-01-01

    Immunohistochemical localization of enzymes is compared directly with localization of enzyme activity with (catalytic) enzyme histochemical methods. The two approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is

  11. Enzyme Mimics: Advances and Applications.

    Science.gov (United States)

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Phage lytic enzymes: a history.

    Science.gov (United States)

    Trudil, David

    2015-02-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  13. [The rise of enzyme engineering in China].

    Science.gov (United States)

    Li, Gaoxiang

    2015-06-01

    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China.

  14. Enzyme structure, enzyme function and allozyme diversity in ...

    African Journals Online (AJOL)

    In estimates of population genetic diversity based on allozyme heterozygosity, some enzymes are regularly more variable than others. Evolutionary theory suggests that functionally less important molecules, or parts of molecules, evolve more rapidly than more important ones; the latter enzymes should then theoretically be ...

  15. Studies on a photoreactivating enzyme from Drosophila melanogaster cultured cells

    International Nuclear Information System (INIS)

    Beck, L.A.

    1982-01-01

    A photoreactivating enzyme was purified from Schneider's Line No. 2 Drosophila melanogaster cultured cells. DEAE cellulose chromatography with high potassium phosphate buffer conditions was used to separate nucleic acids from the protein component of the crude cell extract. The protein pass-through fraction from DEAE cellulose was chromatographed on phosphocellulose followed by hydroxylapatite, using linear potassium phosphate gradients to elute the enzyme. Gel filtration chromatography on Sephacryl S-200 resulted in a 4500-fold purification of the enzyme with a final recovery of 4%. The enzyme has an apparent gel filtration molecular weight of 32,900 (+/- 1350 daltons) and an isoelectric pH of 4.9. Optimum ionic strength for activity is 0.17 at pH 6.5 in potassium phosphate buffer. The action spectrum for photoreactivation in Drosophila has an optimum at 365 nm with a response to wavelengths in the range of 313 to 465 nm. Drosophila photoreactivating enzyme contains an essential RNA that is necessary for activity in vitro. The ability of the enzyme to photoreactivate dimers in vitro is abolished by treatment of the enzyme with ribonucleases, or by disruption of the enzyme-RNA complex by electrophoresis or adsorption to DEAE cellulose. The essential RNA is heterogeneous in size but contains a 10-12 base region that may interact with the active site of the enzyme, and thus is protected from degradation by contaminating RNase activities during purification. The RNA is thought to stabilize the photoreactivating enzyme by maintaining the enzyme in the proper configuration for binding to dimer-containing DNA. It is not known whether this RNA is essential for in vivo photoreactivation

  16. Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

    Directory of Open Access Journals (Sweden)

    José Maria Rodrigues da Luz

    2012-12-01

    Full Text Available The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse. The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase and hydrolytic enzymes (cellulases, xylanases and tanases. Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6. These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

  17. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    Science.gov (United States)

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

  18. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  19. Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

    DEFF Research Database (Denmark)

    Biran, Suzan; Bach, Poul; Simonsen, Ole

    Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care....... However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered detergent formulations, these issues can be partly overcome by physically isolating the enzymes in separate...... particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis...

  20. Lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier rather than by inducing nonproductive adsorption of enzymes.

    Science.gov (United States)

    Djajadi, Demi T; Jensen, Mads M; Oliveira, Marlene; Jensen, Anders; Thygesen, Lisbeth G; Pinelo, Manuel; Glasius, Marianne; Jørgensen, Henning; Meyer, Anne S

    2018-01-01

    Lignin is known to hinder efficient enzymatic conversion of lignocellulose in biorefining processes. In particular, nonproductive adsorption of cellulases onto lignin is considered a key mechanism to explain how lignin retards enzymatic cellulose conversion in extended reactions. Lignin-rich residues (LRRs) were prepared via extensive enzymatic cellulose degradation of corn stover ( Zea mays subsp. mays L.), Miscanthus  ×  giganteus stalks (MS) and wheat straw ( Triticum aestivum L.) (WS) samples that each had been hydrothermally pretreated at three severity factors (log R 0 ) of 3.65, 3.83 and 3.97. The LRRs had different residual carbohydrate levels-the highest in MS; the lowest in WS. The residual carbohydrate was not traceable at the surface of the LRRs particles by ATR-FTIR analysis. The chemical properties of the lignin in the LRRs varied across the three types of biomass, but monolignols composition was not affected by the severity factor. When pure cellulose was added to a mixture of LRRs and a commercial cellulolytic enzyme preparation, the rate and extent of glucose release were unaffected by the presence of LRRs regardless of biomass type and severity factor, despite adsorption of the enzymes to the LRRs. Since the surface of the LRRs particles were covered by lignin, the data suggest that the retardation of enzymatic cellulose degradation during extended reaction on lignocellulosic substrates is due to physical blockage of the access of enzymes to the cellulose caused by the gradual accumulation of lignin at the surface of the biomass particles rather than by nonproductive enzyme adsorption. The study suggests that lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier blocking the access of enzymes to cellulose rather than by inducing retardation through nonproductive adsorption of enzymes.

  1. Effects of replacing dietary starch with neutral detergent-soluble fibre on ruminal fermentation, microbial synthesis and populations of ruminal cellulolytic bacteria using the rumen simulation technique (RUSITEC).

    Science.gov (United States)

    Zhao, X H; Liu, C J; Liu, Y; Li, C Y; Yao, J H

    2013-12-01

    A rumen simulation technique (RUSITEC) apparatus with eight 800 ml fermenters was used to investigate the effects of replacing dietary starch with neutral detergent-soluble fibre (NDSF) by inclusion of sugar beet pulp in diets on ruminal fermentation, microbial synthesis and populations of ruminal cellulolytic bacteria. Experimental diets contained 12.7, 16.4, 20.1 or 23.8% NDSF substituted for starch on a dry matter basis. The experiment was conducted over two independent 15-day incubation periods with the last 8 days used for data collection. There was a tendency that 16.4% NDSF in the diet increased the apparent disappearance of organic matter (OM) and neutral detergent fibre (NDF). Increasing dietary NDSF level increased carboxymethylcellulase and xylanase activity in the solid fraction and apparent disappearance of acid detergent fibre (ADF) but reduced the 16S rDNA copy numbers of Ruminococcus albus in both liquid and solid fractions and R. flavefaciens in the solid fraction. The apparent disappearance of dietary nitrogen (N) was reduced by 29.6% with increased dietary NDSF. Substituting NDSF for starch appeared to increase the ratios of acetate/propionate and methane/volatile fatty acids (VFA) (mol/mol). Replacing dietary starch with NDSF reduced the daily production of ammonia-N and increased the growth of the solid-associated microbial pellets (SAM). Total microbial N flow and efficiency of microbial synthesis (EMS), expressed as g microbial N/kg OM fermented, tended to increase with increased dietary NDSF, but the numerical increase did not continue as dietary NDSF exceeded 20.1% of diet DM. Results suggested that substituting NDSF for starch up to 16.4% of diet DM increased digestion of nutrients (except for N) and microbial synthesis, and further increases (from 16.4% to 23.8%) in dietary NDSF did not repress microbial synthesis but did significantly reduce digestion of dietary N. © 2012 Blackwell Verlag GmbH.

  2. Digestive enzymes of some earthworms.

    Science.gov (United States)

    Mishra, P C; Dash, M C

    1980-10-15

    4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

  3. Photoreactivating enzyme from Escherichia coli

    International Nuclear Information System (INIS)

    Snapka, R.M.; Fuselier, C.O.

    1977-01-01

    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm. (author)

  4. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-01-01

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  5. Photoreactivating enzyme from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Snapka, R M; Fuselier, C O [California Univ., Irvine (USA)

    1977-05-01

    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm.

  6. Efeito da suplementação enzimática no desempenho e qualidade dos ovos de poedeiras comerciais - DOI: 10.4025/actascianimsci.v29i2.221 Effects of the enzyme complex supplementation on the performance and egg quality of laying hens - DOI: 10.4025/actascianimsci.v29i2.221

    Directory of Open Access Journals (Sweden)

    Márcia Izumi Sakamoto

    2007-11-01

    Full Text Available O objetivo deste trabalho foi determinar o efeito da utilização de um complexo multienzimático sobre o desempenho e qualidade dos ovos de poedeiras comerciais. Foram utilizadas 336 poedeiras comerciais, HyLine W36, com 29 semanas de idade distribuídas em um delineamento inteiramente casualizado com 7 tratamentos e 6 repetições de 8 aves cada. Os tratamentos consistiram de uma dieta basal suplementada com o complexo enzimático nos níveis de 400 ou 500 ppm com valorização do nível de proteína, energia metabolizável e aminoácidos do farelo de soja em 5/5/5, 7/7/7 e 7/7/9%, respectivamente. As variáveis estudadas foram: consumo de ração (g ave-1 dia-1, conversão alimentar (kg dz-1 de ovos, peso médio dos ovos (g, produção de ovos (%, gravidade específica (g mL-1, Unidade Haugh, coloração da gema, percentuais de sólidos totais, de gema, clara e casca (%, espessura da casca (mm e composição bromatológica das excretas. A adição do complexo multienzimático apresentou resultados produtivos e de qualidade dos ovos semelhantes à dieta controle.The present study aimed to evaluate the effects of a commercial enzyme complex on the performance and egg quality of commercial laying hens. A total of 336 29-week old Hyline W36 layers were allocated to a completely randomized experimental design with seven treatments and six replications of eight birds each. The treatments consisted of the basal diet supplemented with inclusion of (500 or 400 ppm, overestimating the percentages of protein, ME and amino acids of the soybean meal by 5:5:5, 7:7:7 and 7:7:9, respectively. The studied variables were: feed intake (g bird-1 day- 1, feed:egg (kg feed-1 egg dozen-1, egg weight (g, egg laying percentage (hen day-1 specific gravity (g ml-1, Haugh Unit, color of the egg yolk, total solids percentage (%, egg yolk percentage, albumen and eggshell (%, eggshell thickness (mm and chemical composition of hen manure. The inclusion of enzyme complex in

  7. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

    Science.gov (United States)

    Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

    2017-08-30

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

  8. Hfq stimulates the activity of the CCA-adding enzyme

    Directory of Open Access Journals (Sweden)

    Betat Heike

    2007-10-01

    Full Text Available Abstract Background The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A polymerase I (PAP. As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme. Therefore, it was assumed that Hfq might not only influence the poly(A polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme. Results Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially accelerated in the presence of Hfq. Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition. Conclusion The increase of the CCA-addition in the presence of Hfq suggests that this protein acts as a stimulating factor not only for PAP, but also for the CCA-adding enzyme. In both cases, Hfq interacts with RNA substrates, while a direct binding to the corresponding enzymes was not demonstrated up to now (although experimental data indicate a possible interaction of PAP and Hfq. So far, the basic principle of these stimulatory effects is not clear yet. In case of the CCA-adding enzyme, however, the presented data indicate that the complex between Hfq and tRNA substrate might enhance the product release from the enzyme.

  9. Base metal complexes as homogeneous catalysts and enzyme mimics.

    Science.gov (United States)

    Hu, Xile

    2011-01-01

    This article is a short overview of some recent research activity in the Laboratory of Inorganic Synthesis and Catalysis (LSCI) at EPFL-ISIC. It summarizes the work on Ni-catalyzed cross-coupling reactions of non-activated alkyl halides. It then describes and discusses the work on the bio-mimetic chemistry of [Fe]-hydrogenase.

  10. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    Science.gov (United States)

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

    2016-07-01

    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  11. Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin.

    Science.gov (United States)

    A L Rocha, Vanessa; N Maeda, Roberto; Pereira, Nei; F Kern, Marcelo; Elias, Luisa; Simister, Rachael; Steele-King, Clare; Gómez, Leonardo D; McQueen-Mason, Simon J

    2016-03-01

    This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS-MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono-oxygenases (AA9), carbohydrate-binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two-fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327-336, 2016. © 2016 American Institute of Chemical Engineers.

  12. BAKERY ENZYMES IN CEREAL TECHNOLOGIES

    Directory of Open Access Journals (Sweden)

    Václav Koman

    2012-10-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE Bread is the most common and traditional food in the world. For years, enzymes such as malt and fungal alpha-amylase have been used in bread making. Due to the changes in the baking industry and the ever-increasing demand for more natural products, enzymes have gained real importance in bread-making. If an enzyme is added, it is often destroyed by the heat during the baking process. For generations, enzymes have been used for the improvement of texture and appearance, enhancement of nutritional values and generation of appealing flavours and aromas. Enzymes used in bakery industry constitute nearly one third of the market. The bakery products have undergone radical improvements in quality over the past years in terms of flavour, texture and shelf-life. The the biggest contributor for these improvementsis the usage of enzymes. Present work seeks to systematically describe bakery enzymes, their classification, benefits, usage and chemical reactions in the bread making process.doi:10.5219/193

  13. Rhodium complexes as therapeutic agents.

    Science.gov (United States)

    Ma, Dik-Lung; Wang, Modi; Mao, Zhifeng; Yang, Chao; Ng, Chan-Tat; Leung, Chung-Hang

    2016-02-21

    The landscape of inorganic medicinal chemistry has been dominated by the investigation of platinum, and to a lesser extent ruthenium, complexes over the past few decades. Recently, complexes based on other metal centers such as rhodium have attracted attention due to their tunable chemical and biological properties as well as distinct mechanisms of action. This perspective highlights recent examples of rhodium complexes that show diverse biological activities against various targets, including enzymes and protein-protein interactions.

  14. [Automated analyzer of enzyme immunoassay].

    Science.gov (United States)

    Osawa, S

    1995-09-01

    Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

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

    Science.gov (United States)

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

  16. Notable fibrolytic enzyme production by Aspergillus spp. isolates from the gastrointestinal tract of beef cattle fed in lignified pastures.

    Directory of Open Access Journals (Sweden)

    Flávia Oliveira Abrão

    Full Text Available Fungi have the ability to degrade vegetal cell wall carbohydrates, and their presence in the digestive tract of ruminants can minimize the effects of lignified forage on ruminal fermentation. Here, we evaluated enzyme production by Aspergillus spp. isolates from the digestive tracts of cattle grazed in tropical pastures during the dry season. Filamentous fungi were isolated from rumen and feces by culture in cellulose-based medium. Ninety fungal strains were isolated and identified by rDNA sequence analysis, microculture, or both. Aspergillus terreus was the most frequently isolated species, followed by Aspergillus fumigatus. The isolates were characterized with respect to their cellulolytic, xylanolytic, and lignolytic activity through qualitative evaluation in culture medium containing a specific corresponding carbon source. Carboxymethyl cellulase (CMCase activity was quantified by the reducing sugar method. In the avicel and xilan degradation test, the enzyme activity (EA at 48 h was significantly higher other periods (P < 0.05. Intra- and inter-specific differences in EA were verified, and high levels of phenoloxidases, which are crucial for lignin degradation, were observed in 28.9% of the isolates. Aspergillus terreus showed significantly higher EA for avicelase (3.96 ±1.77 and xylanase (3.13 ±.091 than the other Aspergillus species at 48 h of incubation. Isolates AT13 and AF69 showed the highest CMCase specific activity (54.84 and 33.03 U mg-1 protein, respectively. Selected Aspergillus spp. isolates produced remarkable levels of enzymes involved in vegetal cell wall degradation, suggesting their potential as antimicrobial additives or probiotics in ruminant diets.

  17. A metal-based inhibitor of NEDD8-activating enzyme.

    Directory of Open Access Journals (Sweden)

    Hai-Jing Zhong

    Full Text Available A cyclometallated rhodium(III complex [Rh(ppy(2(dppz](+ (1 (where ppy=2-phenylpyridine and dppz=dipyrido[3,2-a:2',3'-c]phenazine dipyridophenazine has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE. The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme.

  18. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    are affected (in a positive or negative way) by the presence of the other enzymes and compounds in the media. In this thesis the concept of multi-enzyme in-pot term is adopted for processes that are carried out by the combination of enzymes in a single reactor and implemented at pilot or industrial scale...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective....... In this way the model parameters that drives the main dynamic behavior can be identified and thus a better understanding of this type of processes. In order to develop, test and verify the methodology, three case studies were selected, specifically the bi-enzyme process for the production of lactobionic acid...

  19. PIXE analysis of Zn enzymes

    International Nuclear Information System (INIS)

    Solis, C.; Oliver, A.; Andrade, E.; Ruvalcaba-Sil, J.L.; Romero, I.; Celis, H.

    1999-01-01

    Zinc is a necessary component in the action and structural stability of many enzymes. Some of them are well characterized, but in others, Zn stoichiometry and its association is not known. PIXE has been proven to be a suitable technique for analyzing metallic proteins embedded in electrophoresis gels. In this study, PIXE has been used to investigate the Zn content of enzymes that are known to carry Zn atoms. These include the carbonic anhydrase, an enzyme well characterized by other methods and the cytoplasmic pyrophosphatase of Rhodospirillum rubrum that is known to require Zn to be stable but not how many metal ions are involved or how they are bound to the enzyme. Native proteins have been purified by polyacrylamide gel electrophoresis and direct identification and quantification of Zn in the gel bands was performed with an external proton beam of 3.7 MeV energy

  20. GRE Enzymes for Vector Analysis

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  1. Watching Individual Enzymes at Work

    Science.gov (United States)

    Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

    Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

  2. DGAT enzymes and triacylglycerol biosynthesis

    OpenAIRE

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, ...

  3. Enzymes: principles and biotechnological applications

    Science.gov (United States)

    Robinson, Peter K.

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  4. Investigation of some characteristics of enzymes that ensure the process of membrane digestion in paddlefish and Russian sturgeon

    Directory of Open Access Journals (Sweden)

    A. N. Nevalennyy

    2010-01-01

    Full Text Available Complex research of characteristics of some enzymes which are carrying out membrane hydrolysis of food at a spoonbilled cat and Russian sturgeon is carried out. High thermostability enzymes the squirrel of all investigated enzymes is marked.

  5. de novo computational enzyme design.

    Science.gov (United States)

    Zanghellini, Alexandre

    2014-10-01

    Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Svensson, Birte

    2016-01-01

    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...

  7. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  8. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    Science.gov (United States)

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  9. Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.

    Science.gov (United States)

    Su, Xiaoyun; Han, Yejun; Dodd, Dylan; Moon, Young Hwan; Yoshida, Shosuke; Mackie, Roderick I; Cann, Isaac K O

    2013-03-01

    Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k(cat) values of ∼8,000 and ∼4,500 s(-1), respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.

  10. Heme-containing enzymes and inhibitors for tryptophan metabolism.

    Science.gov (United States)

    Yan, Daojing; Lin, Ying-Wu; Tan, Xiangshi

    2017-09-20

    Iron-containing enzymes such as heme enzymes play crucial roles in biological systems. Three distinct heme-containing dioxygenase enzymes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the initial and rate-limiting step of l-tryptophan catabolism through the kynurenine pathway in mammals. Overexpression of these enzymes causes depletion of tryptophan and the accumulation of metabolic products, which contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. In the past few decades, IDO1 has garnered the most attention as a therapeutic target with great potential in cancer immunotherapy. Many potential inhibitors of IDO1 have been designed, synthesized and evaluated, among which indoximod (d-1-MT), INCB024360, GDC-0919 (formerly NLG-919), and an IDO1 peptide-based vaccine have advanced to the clinical trial stage. However, recently, the roles of TDO and IDO2 have been elucidated in immune suppression. In this review, the current drug discovery landscape for targeting TDO, IDO1 and IDO2 is highlighted, with particular attention to the recent use of drugs in clinical trials. Moreover, the crystal structures of these enzymes, in complex with inhibitors, and the mechanisms of Trp catabolism in the first step, are summarized to provide information for facilitating the discovery of new enzyme inhibitors.

  11. Subcellular localization of pituitary enzymes

    Science.gov (United States)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  12. Enzymes in CO2 Capture

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gladis, Arne; Thomsen, Kaj

    The enzyme Carbonic Anhydrase (CA) can accelerate the absorption rate of CO2 into aqueous solutions by several-fold. It exist in almost all living organisms and catalyses different important processes like CO2 transport, respiration and the acid-base balances. A new technology in the field...... of carbon capture is the application of enzymes for acceleration of typically slow ternary amines or inorganic carbonates. There is a hidden potential to revive currently infeasible amines which have an interesting low energy consumption for regeneration but too slow kinetics for viable CO2 capture. The aim...... of this work is to discuss the measurements of kinetic properties for CA promoted CO2 capture solvent systems. The development of a rate-based model for enzymes will be discussed showing the principles of implementation and the results on using a well-known ternary amine for CO2 capture. Conclusions...

  13. Substrate mediated enzyme prodrug therapy

    DEFF Research Database (Denmark)

    Fejerskov, Betina; Jarlstad Olesen, Morten T; Zelikin, Alexander N

    2017-01-01

    Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug administra......Substrate mediated enzyme prodrug therapy (SMEPT) is a biomedical platform developed to perform a localized synthesis of drugs mediated by implantable biomaterials. This approach combines the benefits and at the same time offers to overcome the drawbacks for traditional pill-based drug...

  14. Shotgun Approach to Increasing Enzymatic Saccharification Yields of Ammonia Fiber Expansion Pretreated Cellulosic Biomass

    International Nuclear Information System (INIS)

    Chundawat, Shishir P. S.; Uppugundla, Nirmal; Gao, Dahai; Curran, Paul G.; Balan, Venkatesh; Dale, Bruce E.

    2017-01-01

    Most cellulolytic enzyme blends, either procured from a commercial vendor or isolated from a single cellulolytic microbial secretome, do not efficiently hydrolyze ammonia-pretreated (e.g., ammonia fiber expansion, AFEX) lignocellulosic agricultural crop residues like corn stover to fermentable sugars. Typically reported commercial enzyme loading (30–100 mg protein/g glucan) necessary to achieve >90% total hydrolysis yield (to monosaccharides) for AFEX-treated biomass, within a short saccharification time frame (24–48 h), is economically unviable. Unlike acid-based pretreatments, AFEX retains most of the hemicelluloses in the biomass and therefore requires a more complex suite of enzymes for efficient hydrolysis of cellulose and hemicellulose at industrially relevant high solids loadings. One strategy to reduce enzyme dosage while improving cocktail effectiveness for AFEX-treated biomass has been to use individually purified enzymes to determine optimal enzyme combinations to maximize hydrolysis yields. However, this approach is limited by the selection of heterologous enzymes available or the labor required for isolating low-abundance enzymes directly from the microbial secretomes. Here, we show that directly blending crude cellulolytic and hemicellulolytic enzymes-rich microbial secretomes can maximize specific activity on AFEX-treated biomass without having to isolate individual enzymes. Fourteen commercially available cellulolytic and hemicellulolytic enzymes were procured from leading enzyme companies (Novozymes ® , Genencor ® , and Biocatalysts ® ) and were mixed together to generate several hundred unique cocktail combinations. The mixtures were assayed for activity on AFEX-treated corn stover (AFEX-CS) using a previously established high-throughput methodology. The optimal enzyme blend combinations identified from these screening assays were enriched in various low-abundance hemicellulases and accessory enzymes typically absent in most commercial

  15. One-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production using the whole solid-state fermentation medium of mixed filamentous fungi.

    Science.gov (United States)

    Maehara, Larissa; Pereira, Sandra C; Silva, Adilson J; Farinas, Cristiane S

    2018-02-01

    The efficient use of renewable lignocellulosic feedstocks to obtain biofuels and other bioproducts is a key requirement for a sustainable biobased economy. This requires novel and effective strategies to reduce the cost contribution of the cellulolytic enzymatic cocktails needed to convert the carbohydrates into simple sugars, in order to make large-scale commercial processes economically competitive. Here, we propose the use of the whole solid-state fermentation (SSF) medium of mixed filamentous fungi as an integrated one-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production. Ten different individual and mixed cultivations of commonly used industrial filamentous fungi (Aspergillus niger, Aspergillus oryzae, Trichoderma harzianum, and Trichoderma reesei) were performed under SSF and the whole media (without the extraction step) were used in the hydrolysis of pretreated sugarcane bagasse. The cocultivation of T. reesei with A. oryzae increased the amount of glucose released by around 50%, compared with individual cultivations. The release of glucose and reducing sugars achieved using the whole SSF medium was around 3-fold higher than obtained with the enzyme extract. The addition of soybean protein (0.5% w/w) during the hydrolysis reaction further significantly improved the saccharification performance by blocking the lignin and avoiding unproductive adsorption of enzymes. The results of the alcoholic fermentation validated the overall integrated process, with a volumetric ethanol productivity of 4.77 g/L.h, representing 83.5% of the theoretical yield. These findings demonstrate the feasibility of the proposed one-pot integrated strategy using the whole SSF medium of mixed filamentous fungi for on-site enzymes production, biomass hydrolysis, and ethanol production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

  16. Nuclear magnetic resonance characterization of apple juice containing enzyme preparations

    International Nuclear Information System (INIS)

    Prestes, Rosilene A.; Almeida, Denise Milleo; Barison, Andersson; Pinheiro, Luis Antonio; Wosiacki, Gilvan

    2012-01-01

    In this work, 1 H nuclear magnetic resonance ( 1 H NMR) was employed to evaluate changes in apple juice in response to the addition of Panzym Yieldmash and Ultrazym AFP-L enzymatic complexes and compare it with premium apple juice. The juice was processed at different temperatures and concentrations of enzymatic complexes. The differences in the results were attributed mainly to the enzyme concentrations, since temperature did not cause any variation. A quantitative analysis indicated that the concentration of fructose increased while the concentrations of sucrose and glucose decreased in response to increasing concentrations of the enzymatic complexes. (author)

  17. Potential enzyme toxicity of oxytetracycline to catalase

    International Nuclear Information System (INIS)

    Chi Zhenxing; Liu Rutao; Zhang Hao

    2010-01-01

    Oxytetracycline (OTC) is a kind of widely used veterinary drugs. The residue of OTC in the environment is potentially harmful. In the present work, the non-covalent toxic interaction of OTC with catalase was investigated by the fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy at physiological pH 7.4. OTC can interact with catalase to form a complex mainly by van der Waals' interactions and hydrogen bonds with one binding site. The association constants K were determined to be K 293K = 7.09 x 10 4 L mol -1 and K 311K = 3.31 x 10 4 L mol -1 . The thermodynamic parameters (ΔH o , ΔG o and ΔS o ) of the interaction were calculated. Based on the Foerster theory of non-radiative energy transfer, the distance between bound OTC and the tryptophan residues of catalase was determined to be 6.48 nm. The binding of OTC can result in change of the micro-environment of the tryptophan residues and the secondary structure of catalase. The activity of catalase was also inhibited for the bound OTC. This work establishes a new strategy to probe the enzyme toxicity of veterinary drug residues and is helpful for clarifying the molecular toxic mechanism of OTC in vivo. The established strategy can be used to investigate the potential enzyme toxicity of other small organic pollutants and drugs.

  18. Complexity explained

    CERN Document Server

    Erdi, Peter

    2008-01-01

    This book explains why complex systems research is important in understanding the structure, function and dynamics of complex natural and social phenomena. Readers will learn the basic concepts and methods of complex system research.

  19. From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

    Science.gov (United States)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-10

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

  20. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

    Science.gov (United States)

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-01

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

  1. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    Science.gov (United States)

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  2. Thermodynamics of Enzyme-Catalyzed Reactions Database

    Science.gov (United States)

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  3. Curious Cases of the Enzymes.

    Science.gov (United States)

    Ulusu, Nuriye Nuray

    2015-07-01

    Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts… Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This paper is dedicated to these early moments of this remarkable science that touched our lives in the past and will make life a lot more efficient for humanity in the future. There was almost always a delicate, fundamentally essential relationship between mankind and the enzymes. Challenged by a very alien and hostile Nature full of predators, prehistoric men soon discovered the medicinal properties of the plants, through trial and error. In fact, they accidently discovered the enzyme inhibitors and thus, in crude terms, kindled a sparkling area of research. These plant-derivatives that acted as enzyme inhibitors helped prehistoric men in their pursuit of survival and protection from predators; in hunting and fishing… Later in history, while the underlying purposes of survival and increasing the quality of life stayed intact, the ways and means of enzymology experienced a massive transformation, as the 'trial and error' methodology of the ancients is now replaced with rational scientific theories.

  4. Enzymes with activity toward Xyloglucan

    NARCIS (Netherlands)

    Vincken, J.P.

    2003-01-01

    Xyloglucans are plant cell wall polysaccharides, which belong to the hemicellulose class. Here the structural variations of xyloglucans will be reviewed. Subsequently, the anchoring of xyloglucan in the plant cell wall will be discussed. Enzymes involved in degradation or modification of xyloglucan

  5. Complex chemistry

    International Nuclear Information System (INIS)

    Kim, Bong Gon; Kim, Jae Sang; Kim, Jin Eun; Lee, Boo Yeon

    2006-06-01

    This book introduces complex chemistry with ten chapters, which include development of complex chemistry on history coordination theory and Warner's coordination theory and new development of complex chemistry, nomenclature on complex with conception and define, chemical formula on coordination compound, symbol of stereochemistry, stereo structure and isomerism, electron structure and bond theory on complex, structure of complex like NMR and XAFS, balance and reaction on solution, an organo-metallic chemistry, biology inorganic chemistry, material chemistry of complex, design of complex and calculation chemistry.

  6. Production and characterization of β-glucosidase from Gongronella ...

    African Journals Online (AJOL)

    Among the enzymes of the cellulolytic complex, β-glucosidases are noteworthy due to the possibility of their application in different industrial processes, such as production of biofuels, winemaking, and development of functional foods. This study aimed to evaluate the production and characterization of β-glucosidase from ...

  7. Production and characterization of β-glucosidase from Gongronella ...

    African Journals Online (AJOL)

    sunny t

    2016-04-20

    Apr 20, 2016 ... Among the enzymes of the cellulolytic complex, β-glucosidases are noteworthy due to the possibility of their application in different industrial processes, such as production of biofuels, winemaking, and development of functional foods. This study aimed to evaluate the production and characterization of β-.

  8. Dietary modulation of thymic enzymes.

    Science.gov (United States)

    Susana, Feliu María; Paula, Perris; Slobodianik, Nora

    2014-01-01

    Malnutrition is a complex syndrome caused by an inadequate intake of energy, protein, minerals and vitamins which affects the immune system. Nutritional imbalances, present in children with energy-protein malnutrition and infections, make defining the specific effects of each of them on the thymus difficult. For this reason, it is necessary to design an experimental model in animals that could define a single variable. As the thymus atrophy described in humans is similar to that observed in murines, a rat experimental model makes the extrapolation to man possible. Some authors suggest that the activity of Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP)--involved in purine metabolism--have an influence on T lymphocyte development and the immune system, due to intracellular accumulation of toxic levels of deoxynucleotides. Studies in our group, performed in an experimental model on Wistar growing rats, have demonstrated that protein deficiency or imbalance in the profile of essential amino acids in the diet, produce loss of thymus weight, reduction in the number of thymocytes, a diminished proportion of T cells presenting the W3/13 antigenic determinant and DNA content with concomitant increase in cell size, and the proportion of immature T cells and activity of ADA and PNP, without modifying the activity of 5´Nucleotidase in the thymus. It is important to point out that there were neither differences in energy intake between experimental groups and their controls, nor clinical symptoms of deficiency of other nutrients. The increase in these thymic enzyme activities was an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which would be toxic for T lymphocytes. On the other hand, the administration of a recovery diet, with a high amount of high quality protein, was able to reverse the mentioned effects. The quick reply of Adenosine Deaminase to nutritional disorders and the following nutritional recovery, points

  9. Visualization of Enzyme Activities in Earthworm Biopores by In Situ Soil Zymography.

    Science.gov (United States)

    Razavi, Bahar S; Hoang, Duyen; Kuzyakov, Yakov

    2017-01-01

    Earthworms produce biopores with strongly increased microbial and enzyme activities and consequently they form microbial hotspots in soil. In extremely dynamic microhabitats and hotspots such as earthworm biopores, the in situ enzyme activities are a footprint of process rates and complex biotic interactions. The effect of earthworms on enzyme activities inside biopores, relative to earthworm-free soil, can be visualized by in situ soil zymography. Here, we describe the details of the approach and discuss its advantages and limitations. Direct zymography provides high spatial resolution for quantitative images of enzyme activities in biopores.

  10. Continuous glycerolysis in an immobilized enzyme packed reactor for industrial monoacylglycerol production

    DEFF Research Database (Denmark)

    . In spite of optimal reaction conditions a complex heterogeneous reactant mixture with a glycerol in oil emulsion occurs. Hence, the movement of material from phase to phase as well as through the catalyst pores becomes important since it can influence the performance of the immobilized enzyme reactor...... and sunflower oil dissolved in a binary tert-butanol:tert-pentanol medium. Practical design-related issues such as required reaction time, enzyme capacity, expansion of the enzyme during wetting, and the effect of different column length-to-diameter ratios, fluid velocities and particle sizes of the enzymes...

  11. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

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

    DEFF Research Database (Denmark)

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

    Algal cell wall polysaccharides, and their derived oligosaccharides, display a range of health beneficial bioactive properties. Enzymes capable of degrading algal polysaccharides into oligosaccharides may be used to produce biomolecules with new functionalities for the food and pharma industry....... Some marine bacteria are specialized in degrading algal biomass and secrete enzymes that can decompose the complex algal cell wall polysaccharides. In order to identify such bacteria and enzymatic activities, we have used a combination of traditional cultivation and isolation methods, bioinformatics...... 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...

  13. Wiring of heme enzymes by methylene-blue labeled dendrimers

    DEFF Research Database (Denmark)

    Álvarez-Martos, Isabel; Shahdost-fard, Faezeh; Ferapontova, Elena

    2017-01-01

    Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme- and moli......Redox-modified branched 3D dendrimeric nanostructures may be considered as perspective wires for electrical connection between redox enzymes and electrodes. Here, we studied electron transfer (ET) reactions and bioelectrocatalysis of heme-containing horseradish peroxidase (HRP) and heme......- and molibdopterin-containing sulfite oxidase (SOx), wired to gold by the methylene blue (MB)-labeled polyamidoamine (PAMAM) dendrimers. The enzymes’ electrochemical transformation and bioelectrocatalytic function could be followed at both unlabeled and MB-labeled dendrimer-modified electrodes with the formal redox......, optimization of bioelectrocatalysis of complex intermembrane and, possibly, membrane enzymes....

  14. Heavy enzymes--experimental and computational insights in enzyme dynamics.

    Science.gov (United States)

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki

    2014-08-01

    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Actinomycete enzymes and activities involved in straw saccharification

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, A J; Ball, A S [Liverpool Univ. (UK). Dept. of Genetics and Microbiology

    1990-01-01

    This research programme has been directed towards the analysis of actinomycete enzyme systems involved in the degradation of plant biomass (lignocellulose.) The programme was innovative in that a novel source of enzymes was systematically screened and wheat straw saccharifying activity was the test criterion. Over 200 actinomycete strains representing a broad taxonomic range were screened. A range of specific enzyme activities were involved and included cellulase, xylanase, arabinofuranosidase, acetylesterase, {beta}-xylosidase and {beta}-glucosidase. Since hemicellulose (arabinoxylan) was the primary source of sugar, xylanases were characterized. The xylan-degrading systems of actinomycetes were complex and nonuniform, with up to six separate endoxylanases identified in active strains. Except for microbispora bispora, actinomycetes were found to be a poor source of cellulase activity. Evidence for activity against the lignin fraction of straw was produced for a range of actinomycete strains. While modification reactions were common, cleavage of inter-monomer bonds, and utilization of complex polyphenolic compounds were restricted to two strains: Thermomonospora mesophila and Streptomyces badius. Crude enzyme preparations from actinomycetes can be used to generate sugar, particularly pentoses, directly from cereal straw. The potential for improvements in yield rests with the formulation to cooperative enzyme combinations from different strains. The stability properties of enzymes from thermophilic strains and the general neutral to alkali pH optima offer advantages in certain process situations. Actinomycetes are a particularly rich source of xylanases for commercial application and can rapidly solubilise a lignocarbohydrate fraction of straw which may have both product and pretreatment potential. 31 refs., 4 figs., 5 tabs.

  16. Enzyme technology: Key to selective biorefining

    DEFF Research Database (Denmark)

    Meyer, Anne S.

    2014-01-01

    to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

  17. Pectin-modifying enzymes and pectin-derived materials: applications and impacts.

    Science.gov (United States)

    Bonnin, Estelle; Garnier, Catherine; Ralet, Marie-Christine

    2014-01-01

    Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.

  18. Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism.

    Science.gov (United States)

    Shi, Yuguang; Cheng, Dong

    2009-07-01

    Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications.

  19. Fungal Enzymes and Yeasts for Conversion of Plant Biomass to Bioenergy and High-Value Products

    DEFF Research Database (Denmark)

    Lange, Lene

    2017-01-01

    Fungi and fungal enzymes play important roles in the new bioeconomy. Enzymes from filamentous fungi can unlock the potential of recalcitrant lignocellulose structures of plant cell walls as a new resource, and fungi such as yeast can produce bioethanol from the sugars released after enzyme treatm...... contributed to mycology and environmental research? Future perspectives and approaches are listed, highlighting the importance of fungi in development of the bioeconomy.......Fungi and fungal enzymes play important roles in the new bioeconomy. Enzymes from filamentous fungi can unlock the potential of recalcitrant lignocellulose structures of plant cell walls as a new resource, and fungi such as yeast can produce bioethanol from the sugars released after enzyme...... treatment. Such processes reflect inherent characteristics of the fungal way of life, namely, that fungi as heterotrophic organisms must break down complex carbon structures of organic materials to satisfy their need for carbon and nitrogen for growth and reproduction. This chapter describes major steps...

  20. ExplorEnz: the primary source of the IUBMB enzyme list

    Science.gov (United States)

    McDonald, Andrew G.; Boyce, Sinéad; Tipton, Keith F.

    2009-01-01

    ExplorEnz is the MySQL database that is used for the curation and dissemination of the International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature. A simple web-based query interface is provided, along with an advanced search engine for more complex Boolean queries. The WWW front-end is accessible at http://www.enzyme-database.org, from where downloads of the database as SQL and XML are also available. An associated form-based curatorial application has been developed to facilitate the curation of enzyme data as well as the internal and public review processes that occur before an enzyme entry is made official. Suggestions for new enzyme entries, or modifications to existing ones, can be made using the forms provided at http://www.enzyme-database.org/forms.php. PMID:18776214

  1. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

    Science.gov (United States)

    Guo, Qing; He, Yufan; Lu, H. Peter

    2015-01-01

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions. PMID:26512103

  2. Fungal enzyme production in seeds of transgenic canola plants for conversion of cellulosic materials to ethanol

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, K.J.; Beauchemin, K.A. [Agriculture and Agri-Food Canada, Lethbridge, AB (Canada); Moloney, M.M. [Calgary Univ., AB (Canada). Dept. of Biological Sciences

    1997-07-01

    The fuel alcohol industry makes use of industrial enzymes to effectively degrade fibrous plant cell walls. Carbohydrates in cellulosic materials are in the form of complex sugars that can be hydrolyzed to simple sugars by fungal fibrolytic enzymes such as cellulases and xylanases. This study was conducted to find a cost effective way to produce fibrolytic enzymes using gene fusion technology in which a xylanase gene and a cellulase gene from two fungal species are introduced into canola to be a carrier for the production of these enzymes. The two genes had been analyzed for maximal enzymatic activity to minimize side effects. Results of the study demonstrated the stability and potential of transgenic oil-bodies as an immobilized enzyme matrix, and showed that it is possible to express fibrolytic enzymes in canola.

  3. ExplorEnz: a MySQL database of the IUBMB enzyme nomenclature.

    Science.gov (United States)

    McDonald, Andrew G; Boyce, Sinéad; Moss, Gerard P; Dixon, Henry B F; Tipton, Keith F

    2007-07-27

    We describe the database ExplorEnz, which is the primary repository for EC numbers and enzyme data that are being curated on behalf of the IUBMB. The enzyme nomenclature is incorporated into many other resources, including the ExPASy-ENZYME, BRENDA and KEGG bioinformatics databases. The data, which are stored in a MySQL database, preserve the formatting of chemical and enzyme names. A simple, easy to use, web-based query interface is provided, along with an advanced search engine for more complex queries. The database is publicly available at http://www.enzyme-database.org. The data are available for download as SQL and XML files via FTP. ExplorEnz has powerful and flexible search capabilities and provides the scientific community with the most up-to-date version of the IUBMB Enzyme List.

  4. Flavonoids as modulators of metabolic enzymes and drug transporters.

    Science.gov (United States)

    Miron, Anca; Aprotosoaie, Ana Clara; Trifan, Adriana; Xiao, Jianbo

    2017-06-01

    Flavonoids, natural compounds found in plants and in plant-derived foods and beverages, have been extensively studied with regard to their capacity to modulate metabolic enzymes and drug transporters. In vitro, flavonoids predominantly inhibit the major phase I drug-metabolizing enzyme CYP450 3A4 and the enzymes responsible for the bioactivation of procarcinogens (CYP1 enzymes) and upregulate the enzymes involved in carcinogen detoxification (UDP-glucuronosyltransferases, glutathione S-transferases (GSTs)). Flavonoids have been reported to inhibit ATP-binding cassette (ABC) transporters (multidrug resistance (MDR)-associated proteins, breast cancer-resistance protein) that contribute to the development of MDR. P-glycoprotein, an ABC transporter that limits drug bioavailability and also induces MDR, was differently modulated by flavonoids. Flavonoids and their phase II metabolites (sulfates, glucuronides) inhibit organic anion transporters involved in the tubular uptake of nephrotoxic compounds. In vivo studies have partially confirmed in vitro findings, suggesting that the mechanisms underlying the modulatory effects of flavonoids are complex and difficult to predict in vivo. Data summarized in this review strongly support the view that flavonoids are promising candidates for the enhancement of oral drug bioavailability, chemoprevention, and reversal of MDR. © 2017 New York Academy of Sciences.

  5. Spatial distribution of enzyme driven reactions at micro-scales

    Science.gov (United States)

    Kandeler, Ellen; Boeddinghaus, Runa; Nassal, Dinah; Preusser, Sebastian; Marhan, Sven; Poll, Christian

    2017-04-01

    Studies of microbial biogeography can often provide key insights into the physiologies, environmental tolerances, and ecological strategies of soil microorganisms that dominate in natural environments. In comparison with aquatic systems, soils are particularly heterogeneous. Soil heterogeneity results from the interaction of a hierarchical series of interrelated variables that fluctuate at many different spatial and temporal scales. Whereas spatial dependence of chemical and physical soil properties is well known at scales ranging from decimetres to several hundred metres, the spatial structure of soil enzymes is less clear. Previous work has primarily focused on spatial heterogeneity at a single analytical scale using the distribution of individual cells, specific types of organisms or collective parameters such as bacterial abundance or total microbial biomass. There are fewer studies that have considered variations in community function and soil enzyme activities. This presentation will give an overview about recent studies focusing on spatial pattern of different soil enzymes in the terrestrial environment. Whereas zymography allows the visualization of enzyme pattern in the close vicinity of roots, micro-sampling strategies followed by MUF analyses clarify micro-scale pattern of enzymes associated to specific microhabitats (micro-aggregates, organo-mineral complexes, subsoil compartments).

  6. Evolutionary transitions in enzyme activity of ant fungus gardens.

    Science.gov (United States)

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

    2010-07-01

    Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

  7. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    Science.gov (United States)

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  8. Study of DNA reconstruction enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Sekiguchi, M [Kyushu Univ., Fukuoka (Japan). Faculty of Science

    1976-12-01

    Description was made of the characteristics and mechanism of 3 reconstructive enzymes which received from M. luteus or E. coli or T4, and of which natures were clarified as reconstructive enzymes of DNA irradiated with ultraviolet rays. As characteristics, the site of breaking, reaction, molecular weight, electric charge in the neutrality and a specific adhesion to DNA irradiated with ultraviolet rays were mentioned. As to mutant of ultraviolet ray sensitivity, hereditary control mechanism of removal and reconstruction by endo-nuclease activation was described, and suggestion was referred to removal and reconstruction of cells of xedoderma pigmentosum which is a hereditary disease of human. Description was also made as to the mechanism of exonuclease activation which separates dimer selectively from irradiated DNA.

  9. Metrological aspects of enzyme production

    International Nuclear Information System (INIS)

    Kerber, T M; Pereira-Meirelles, F V; Dellamora-Ortiz, G M

    2010-01-01

    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies

  10. Consumer attitudes to enzymes in food production

    DEFF Research Database (Denmark)

    Søndergaard, Helle Alsted; Grunert, Klaus G.; Scholderer, Joachim

    2005-01-01

    The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in three...... European countries. Results show that consumers are most positive towards non-GM enzyme production methods. The enzyme production method is by far the most important factor for the formation of buying intentions compared to price and benefits. Results also show that environmental concern and attitudes...... to technological progress are the socio-political attitudes that have the highest predictive value regarding attitudes to enzyme production methods....

  11. Research progress of nanoparticles as enzyme mimetics

    Science.gov (United States)

    Hu, XiaoNa; Liu, JianBo; Hou, Shuai; Wen, Tao; Liu, WenQi; Zhang, Ke; He, WeiWei; Ji, YingLu; Ren, HongXuan; Wang, Qi; Wu, XiaoChun

    2011-10-01

    Natural enzymes as biological catalysts possess remarkable advantages, especially their highly efficient and selective catalysis under mild conditions. However, most natural enzymes are proteins, thus exhibiting an inherent low durability to harsh reaction conditions. Artificial enzyme mimetics have been pursued extensively to avoid this drawback. Quite recently, some inorganic nanoparticles (NPs) have been found to exhibit unique enzyme mimetics. In addition, their much higher stability overcomes the inherent disadvantage of natural enzymes. Furthermore, easy mass-production and low cost endow them more benefits. As a new member of artificial enzyme mimetics, they have received intense attention. In this review article, major progress in this field is summarized and future perspectives are highlighted.

  12. Allosteric regulation of epigenetic modifying enzymes.

    Science.gov (United States)

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Enzyme-assisted extraction of stabilized chlorophyll from spinach.

    Science.gov (United States)

    Özkan, Gülay; Ersus Bilek, Seda

    2015-06-01

    Zinc complex formation with chlorophyll derivatives in spinach pulp was studied by adding 300ppm Zn(2+) for production of stable food colorant, followed by the heating at 110°C for 15min. Zinc complex formation increased at pH values of 7.0 or greater. Pectinex Ultra SP-L was selected for enzyme-assisted release of zinc-chlorophyll derivatives from spinach pulp. Effect of enzyme concentration (1-9%), treatment temperature (30-60°C), and time (30-210min) on total chlorophyll content (TCC) were optimized using response surface methodology. A quadratic regression model (R(2)=0.9486) was obtained from the experimental design. Optimum treatment conditions were 8% enzyme concentration, 45°C, and 30min, which yielded a 50.747mgTCC/100g spinach pulp. Enzymatic treatment was followed by solvent extraction with ethanol at a solvent-to-sample ratio of 2.5:1 at 60°C for 45min for the highest TCC recovery. Pretreatment with enzyme and extraction in ethanol resulted in 39% increase in Zn-chlorophyll derivative yield. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

    Science.gov (United States)

    Ghoshroy, Sohini; Robertson, Deborah L

    2015-01-01

    Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex his