WorldWideScience

Sample records for nonmonotonic solute-induced thermostability

  1. Specification of Nonmonotonic Reasoning.

    NARCIS (Netherlands)

    Engelfriet, J.; Treur, J.

    2000-01-01

    Two levels of description of nonmonotonic reasoning are distinguished. For these levels semantical formalizations are given. The first Level is defined semantically by the notion of belief state frame, the second Level by the notion of reasoning frame. We introduce two specification languages to

  2. Specification of Nonmonotonic Reasoning

    NARCIS (Netherlands)

    Engelfriet, J.; Treur, J.

    1996-01-01

    Two levels of description of nonmonotonic reasoning are distinguished. For these levels semantical formalizations are given. The first level is defined semantically by the notion of belief state frame, the second level by the notion of reasoning frame. We introduce two specification languages to

  3. Regularization of Nonmonotone Variational Inequalities

    International Nuclear Information System (INIS)

    Konnov, Igor V.; Ali, M.S.S.; Mazurkevich, E.O.

    2006-01-01

    In this paper we extend the Tikhonov-Browder regularization scheme from monotone to rather a general class of nonmonotone multivalued variational inequalities. We show that their convergence conditions hold for some classes of perfectly and nonperfectly competitive economic equilibrium problems

  4. AAAI Workshop on Nonmonotonic Reasoning

    OpenAIRE

    Etherington, David

    1985-01-01

    On October 17-19 1984 a workshop on non-monotonic reasoning was held at Mohonk Mountain House, outside New Paltz, New York. The workshop was organized by Raymond Reiter and Bonnie Webber, and was sponsored by the Association for the Advancement of Artificial Intelligence.

  5. Thermostating highly confined fluids.

    Science.gov (United States)

    Bernardi, Stefano; Todd, B D; Searles, Debra J

    2010-06-28

    In this work we show how different use of thermostating devices and modeling of walls influence the mechanical and dynamical properties of confined nanofluids. We consider a two dimensional fluid undergoing Couette flow using nonequilibrium molecular dynamics simulations. Because the system is highly inhomogeneous, the density shows strong fluctuations across the channel. We compare the dynamics produced by applying a thermostating device directly to the fluid with that obtained when the wall is thermostated, considering also the effects of using rigid walls. This comparison involves an analysis of the chaoticity of the fluid and evaluation of mechanical properties across the channel. We look at two thermostating devices with either rigid or vibrating atomic walls and compare them with a system only thermostated by conduction through vibrating atomic walls. Sensitive changes are observed in the xy component of the pressure tensor, streaming velocity, and density across the pore and the Lyapunov localization of the fluid. We also find that the fluid slip can be significantly reduced by rigid walls. Our results suggest caution in interpreting the results of systems in which fluid atoms are thermostated and/or wall atoms are constrained to be rigid, such as, for example, water inside carbon nanotubes.

  6. Aspects and modular reasoning in nonmonotonic logic

    DEFF Research Database (Denmark)

    Ostermann, Klaus

    2008-01-01

    Nonmonotonic logic is a branch of logic that has been developed to model situations with incomplete information. We argue that there is a connection between AOP and nonmonotonic logic which deserves further study. As a concrete technical contribution and "appetizer", we outline an AO semantics de...... defined in default logic (a form of nonmonotonic logic), propose a definition of modular reasoning, and show that the default logic version of the language semantics admits modular reasoning whereas a conventional language semantics based on weaving does not....

  7. Nonmonotonicity of the Frictional Bimaterial Effect

    Science.gov (United States)

    Aldam, Michael; Xu, Shiqing; Brener, Efim A.; Ben-Zion, Yehuda; Bouchbinder, Eran

    2017-10-01

    Sliding along frictional interfaces separating dissimilar elastic materials is qualitatively different from sliding along interfaces separating identical materials due to the existence of an elastodynamic coupling between interfacial slip and normal stress perturbations in the former case. This bimaterial coupling has important implications for the dynamics of frictional interfaces, including their stability and rupture propagation along them. We show that while this bimaterial coupling is a monotonically increasing function of the bimaterial contrast, when it is coupled to interfacial shear stress perturbations through a friction law, various physical quantities exhibit a nonmonotonic dependence on the bimaterial contrast. In particular, we show that for a regularized Coulomb friction, the maximal growth rate of unstable interfacial perturbations of homogeneous sliding is a nonmonotonic function of the bimaterial contrast and provides analytic insight into the origin of this nonmonotonicity. We further show that for velocity-strengthening rate-and-state friction, the maximal growth rate of unstable interfacial perturbations of homogeneous sliding is also a nonmonotonic function of the bimaterial contrast. Results from simulations of dynamic rupture along a bimaterial interface with slip-weakening friction provide evidence that the theoretically predicted nonmonotonicity persists in nonsteady, transient frictional dynamics.

  8. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  9. Nonmonotonic belief state frames and reasoning frames

    NARCIS (Netherlands)

    Engelfriet, J.; Herre, H.; Treur, J.

    1995-01-01

    In this paper five levels of specification of nonmonotonic reasoning are distinguished. The notions of semantical frame, belief state frame and reasoning frame are introduced and used as a semantical basis for the first three levels. Moreover, the semantical connections between the levels are

  10. Nonmonotonic critical temperature in superconductor ferromagnet bilayers

    NARCIS (Netherlands)

    Fominov, Ya. V.; Fominov, I.V.; Chtchelkatchev, N.M.; Golubov, Alexandre Avraamovitch

    2002-01-01

    The critical temperature Tc of a superconductor/ferromagnet (SF) bilayer can exhibit nonmonotonic dependence on the thickness df of the F layer. SF systems have been studied for a long time; according to the experimental situation, a ¿dirty¿ limit is often considered which implies that the mean free

  11. Nonmonotonic Skeptical Consequence Relation in Constrained Default Logic

    Directory of Open Access Journals (Sweden)

    Mihaiela Lupea

    2010-12-01

    Full Text Available This paper presents a study of the nonmonotonic consequence relation which models the skeptical reasoning formalised by constrained default logic. The nonmonotonic skeptical consequence relation is defined using the sequent calculus axiomatic system. We study the formal properties desirable for a good nonmonotonic relation: supraclassicality, cut, cautious monotony, cumulativity, absorption, distribution. 

  12. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

    2010-03-24

    These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  13. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Incorporated, San Francisco, CA (United States)

    2010-04-19

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  14. Nonmonotonic low frequency losses in HTSCs

    International Nuclear Information System (INIS)

    Castro, H; Gerber, A; Milner, A

    2007-01-01

    A calorimetric technique has been used in order to study ac-field dissipation in ceramic BSCCO samples at low frequencies between 0.05 and 250 Hz, at temperatures from 65 to 90 K. In contrast to previous studies, where ac losses have been reported with a linear dependence on magnetic field frequency, we find a nonmonotonic function presenting various maxima. Frequencies corresponding to local maxima of dissipation depend on the temperature and the amplitude of the ac magnetic field. Flux creep is argued to be responsible for this behaviour. A simple model connecting the characteristic vortex relaxation times (flux creep) and the location of dissipation maxima versus frequency is proposed

  15. 8th International Workshop on Non-Monotonic Reasoning

    CERN Document Server

    Truszczynski, Mirek

    2000-01-01

    The papers gathered in this collection were presented at the 8th International Workshop on Nonmonotonic Reasoning, NMR2000. The series was started by John McCarthy in 1978. The first international NMR workshop was held at Mohonk Mountain House, New Paltz, New York in June, 1984, and was organized by Ray Reiter and Bonnie Webber. In the last 10 years the area of nonmonotonic reasoning has seen a number of important developments. Significant theoretical advances were made in the understanding of general abstract principles underlying nonmonotonicity. Key results on the expressibility and computational complexity of nonmonotonic logics were established. The role of nonmonotonic reasoning in belief revision, abduction, reasoning about action, planing and uncertainty was further clarified. Several successful NMR systems were built and used in applications such as planning, scheduling, logic programming and constraint satisfaction. The papers in the proceedings reflect these recent advances in the field. They are g...

  16. Nonmonotonic reasoning in description logics. Rational closure for the ABox

    CSIR Research Space (South Africa)

    Casini, G

    2013-07-01

    Full Text Available The introduction of defeasible reasoning in Description Logics has been a main research topic in the field in the last years. Despite the fact that various interesting formalizations of nonmonotonic reasoning for the TBox have been proposed...

  17. Specific non-monotonous interactions increase persistence of ecological networks.

    Science.gov (United States)

    Yan, Chuan; Zhang, Zhibin

    2014-03-22

    The relationship between stability and biodiversity has long been debated in ecology due to opposing empirical observations and theoretical predictions. Species interaction strength is often assumed to be monotonically related to population density, but the effects on stability of ecological networks of non-monotonous interactions that change signs have not been investigated previously. We demonstrate that for four kinds of non-monotonous interactions, shifting signs to negative or neutral interactions at high population density increases persistence (a measure of stability) of ecological networks, while for the other two kinds of non-monotonous interactions shifting signs to positive interactions at high population density decreases persistence of networks. Our results reveal a novel mechanism of network stabilization caused by specific non-monotonous interaction types through either increasing stable equilibrium points or reducing unstable equilibrium points (or both). These specific non-monotonous interactions may be important in maintaining stable and complex ecological networks, as well as other networks such as genes, neurons, the internet and human societies.

  18. Optimization of medium composition for thermostable protease ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ..... Each contour curve represented an infinite number of combinations of two ..... Production in sea-water of.

  19. Purification and Characterization of Thermostable Cellulase from ...

    African Journals Online (AJOL)

    Available online at http://www.tjpr.org ... Methods: Molecular community structure of the newly selected thermophilic bacterial ... Keywords: Thermostable cellulase, Sugarcane bagasse, Purification, Characterization, Hot spring ... Currently, one.

  20. Nonmonotonic Trust Management for P2P Applications

    NARCIS (Netherlands)

    Czenko, M.R.; Tran, H.M.; Doumen, J.M.; Etalle, Sandro; Hartel, Pieter H.; den Hartog, Jeremy

    Community decisions about access control in virtual communities are non-monotonic in nature. This means that they cannot be expressed in current, monotonic trust management languages such as the family of Role Based Trust Management languages (RT). To solve this problem we propose RTo, which adds a

  1. Modelling Embedded Systems by Non-Monotonic Refinement

    NARCIS (Netherlands)

    Mader, Angelika H.; Marincic, J.; Wupper, H.

    2008-01-01

    This paper addresses the process of modelling embedded sys- tems for formal verification. We propose a modelling process built on non-monotonic refinement and a number of guidelines. The outcome of the modelling process is a model, together with a correctness argument that justifies our modelling

  2. Functional analysis of thermostable proteins involved in carbohydrate metabolism

    NARCIS (Netherlands)

    Akerboom, A.P.

    2007-01-01

    Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity,

  3. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Anna Di Fiore

    2015-07-01

    Full Text Available Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

  4. Information flow in layered networks of non-monotonic units

    Science.gov (United States)

    Schittler Neves, Fabio; Martim Schubert, Benno; Erichsen, Rubem, Jr.

    2015-07-01

    Layered neural networks are feedforward structures that yield robust parallel and distributed pattern recognition. Even though much attention has been paid to pattern retrieval properties in such systems, many aspects of their dynamics are not yet well characterized or understood. In this work we study, at different temperatures, the memory activity and information flows through layered networks in which the elements are the simplest binary odd non-monotonic function. Our results show that, considering a standard Hebbian learning approach, the network information content has its maximum always at the monotonic limit, even though the maximum memory capacity can be found at non-monotonic values for small enough temperatures. Furthermore, we show that such systems exhibit rich macroscopic dynamics, including not only fixed point solutions of its iterative map, but also cyclic and chaotic attractors that also carry information.

  5. Modeling non-monotonic properties under propositional argumentation

    Science.gov (United States)

    Wang, Geng; Lin, Zuoquan

    2013-03-01

    In the field of knowledge representation, argumentation is usually considered as an abstract framework for nonclassical logic. In this paper, however, we'd like to present a propositional argumentation framework, which can be used to closer simulate a real-world argumentation. We thereby argue that under a dialectical argumentation game, we can allow non-monotonic reasoning even under classical logic. We introduce two methods together for gaining nonmonotonicity, one by giving plausibility for arguments, the other by adding "exceptions" which is similar to defaults. Furthermore, we will give out an alternative definition for propositional argumentation using argumentative models, which is highly related to the previous reasoning method, but with a simple algorithm for calculation.

  6. Information flow in layered networks of non-monotonic units

    International Nuclear Information System (INIS)

    Neves, Fabio Schittler; Schubert, Benno Martim; Erichsen, Rubem Jr

    2015-01-01

    Layered neural networks are feedforward structures that yield robust parallel and distributed pattern recognition. Even though much attention has been paid to pattern retrieval properties in such systems, many aspects of their dynamics are not yet well characterized or understood. In this work we study, at different temperatures, the memory activity and information flows through layered networks in which the elements are the simplest binary odd non-monotonic function. Our results show that, considering a standard Hebbian learning approach, the network information content has its maximum always at the monotonic limit, even though the maximum memory capacity can be found at non-monotonic values for small enough temperatures. Furthermore, we show that such systems exhibit rich macroscopic dynamics, including not only fixed point solutions of its iterative map, but also cyclic and chaotic attractors that also carry information. (paper)

  7. POLARIZED LINE FORMATION IN NON-MONOTONIC VELOCITY FIELDS

    Energy Technology Data Exchange (ETDEWEB)

    Sampoorna, M.; Nagendra, K. N., E-mail: sampoorna@iiap.res.in, E-mail: knn@iiap.res.in [Indian Institute of Astrophysics, Koramangala, Bengaluru 560034 (India)

    2016-12-10

    For a correct interpretation of the observed spectro-polarimetric data from astrophysical objects such as the Sun, it is necessary to solve the polarized line transfer problems taking into account a realistic temperature structure, the dynamical state of the atmosphere, a realistic scattering mechanism (namely, the partial frequency redistribution—PRD), and the magnetic fields. In a recent paper, we studied the effects of monotonic vertical velocity fields on linearly polarized line profiles formed in isothermal atmospheres with and without magnetic fields. However, in general the velocity fields that prevail in dynamical atmospheres of astrophysical objects are non-monotonic. Stellar atmospheres with shocks, multi-component supernova atmospheres, and various kinds of wave motions in solar and stellar atmospheres are examples of non-monotonic velocity fields. Here we present studies on the effect of non-relativistic non-monotonic vertical velocity fields on the linearly polarized line profiles formed in semi-empirical atmospheres. We consider a two-level atom model and PRD scattering mechanism. We solve the polarized transfer equation in the comoving frame (CMF) of the fluid using a polarized accelerated lambda iteration method that has been appropriately modified for the problem at hand. We present numerical tests to validate the CMF method and also discuss the accuracy and numerical instabilities associated with it.

  8. Thermostable crude endoglucanase produced by Aspergillus ...

    African Journals Online (AJOL)

    Cellulases are used in many industries worldwide and there is an ever increasing need to isolate, produce or develop thermostable cellulases. Manipulation of fermentation techniques in order to obtain desirable product(s) can be one line of action. In this study Aspergillus fumigatus was grown on chopped wheat straw in a ...

  9. Purification and Characterization of Thermostable Cellulase from ...

    African Journals Online (AJOL)

    The thermostable CMCase was purified with ion-exchange and gel filtration chromatography. Results: ... Conclusion: Due to its high temperature stability, the purified XM70-CMCase may be useful for industrial application such as biofuel, animal feed industry, paper industry and clarification of fruit juices. Keywords: ...

  10. Non-monotonic behaviour in relaxation dynamics of image restoration

    International Nuclear Information System (INIS)

    Ozeki, Tomoko; Okada, Masato

    2003-01-01

    We have investigated the relaxation dynamics of image restoration through a Bayesian approach. The relaxation dynamics is much faster at zero temperature than at the Nishimori temperature where the pixel-wise error rate is minimized in equilibrium. At low temperature, we observed non-monotonic development of the overlap. We suggest that the optimal performance is realized through premature termination in the relaxation processes in the case of the infinite-range model. We also performed Markov chain Monte Carlo simulations to clarify the underlying mechanism of non-trivial behaviour at low temperature by checking the local field distributions of each pixel

  11. Nonmonotonic Thermal Casimir Force from Geometry-Temperature Interplay

    International Nuclear Information System (INIS)

    Weber, Alexej; Gies, Holger

    2010-01-01

    The geometry dependence of Casimir forces is significantly more pronounced in the presence of thermal fluctuations due to a generic geometry-temperature interplay. We show that the thermal force for standard sphere-plate or cylinder-plate geometries develops a nonmonotonic behavior already in the simple case of a fluctuating Dirichlet scalar. In particular, the attractive thermal force can increase for increasing distances below a critical temperature. This anomalous behavior is triggered by a reweighting of relevant fluctuations on the scale of the thermal wavelength. The essence of the phenomenon becomes transparent within the worldline picture of the Casimir effect.

  12. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    of fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly...... for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes....... Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...

  13. Theoretical and experimental study of non-monotonous effects

    International Nuclear Information System (INIS)

    Delforge, J.

    1977-01-01

    In recent years, the study of the effects of low dose rates has expanded considerably, especially in connection with current problems concerning the environment and health physics. After having made a precise definition of the different types of non-monotonous effect which may be encountered, for each the main experimental results known are indicated, as well as the principal consequences which may be expected. One example is the case of radiotherapy, where there is a chance of finding irradiation conditions such that the ratio of destructive action on malignant cells to healthy cells is significantly improved. In the second part of the report, the appearance of these phenomena, especially at low dose rates are explained. For this purpose, the theory of transformation systems of P. Delattre is used as a theoretical framework. With the help of a specific example, it is shown that non-monotonous effects are frequently encountered, especially when the overall effect observed is actually the sum of several different elementary effects (e.g. in survival curves, where death may be due to several different causes), or when the objects studied possess inherent kinetics not limited to restoration phenomena alone (e.g. cellular cycle) [fr

  14. Thermostable cellulase from a thermomonospora gene

    Science.gov (United States)

    Wilson, D.B.; Walker, L.P.; Zhang, S.

    1997-10-14

    The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

  15. Thermostability in endoglucanases is fold-specific

    Science.gov (United States)

    2011-01-01

    Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

  16. Thermostability in endoglucanases is fold-specific

    Directory of Open Access Journals (Sweden)

    Wolt Jeffrey D

    2011-02-01

    Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

  17. Approaches for improving thermostability characteristics in cellulases.

    Science.gov (United States)

    Anbar, Michael; Bayer, Edward A

    2012-01-01

    Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Non-monotone positive solutions of second-order linear differential equations: existence, nonexistence and criteria

    Directory of Open Access Journals (Sweden)

    Mervan Pašić

    2016-10-01

    Full Text Available We study non-monotone positive solutions of the second-order linear differential equations: $(p(tx'' + q(t x = e(t$, with positive $p(t$ and $q(t$. For the first time, some criteria as well as the existence and nonexistence of non-monotone positive solutions are proved in the framework of some properties of solutions $\\theta (t$ of the corresponding integrable linear equation: $(p(t\\theta''=e(t$. The main results are illustrated by many examples dealing with equations which allow exact non-monotone positive solutions not necessarily periodic. Finally, we pose some open questions.

  19. Obliquely Propagating Non-Monotonic Double Layer in a Hot Magnetized Plasma

    International Nuclear Information System (INIS)

    Kim, T.H.; Kim, S.S.; Hwang, J.H.; Kim, H.Y.

    2005-01-01

    Obliquely propagating non-monotonic double layer is investigated in a hot magnetized plasma, which consists of a positively charged hot ion fluid and trapped, as well as free electrons. A model equation (modified Korteweg-de Vries equation) is derived by the usual reductive perturbation method from a set of basic hydrodynamic equations. A time stationary obliquely propagating non-monotonic double layer solution is obtained in a hot magnetized-plasma. This solution is an analytic extension of the monotonic double layer and the solitary hole. The effects of obliqueness, external magnetic field and ion temperature on the properties of the non-monotonic double layer are discussed

  20. Surfactants non-monotonically modify the onset of Faraday waves

    Science.gov (United States)

    Strickland, Stephen; Shearer, Michael; Daniels, Karen

    2017-11-01

    When a water-filled container is vertically vibrated, subharmonic Faraday waves emerge once the driving from the vibrations exceeds viscous dissipation. In the presence of an insoluble surfactant, a viscous boundary layer forms at the contaminated surface to balance the Marangoni and Boussinesq stresses. For linear gravity-capillary waves in an undriven fluid, the surfactant-induced boundary layer increases the amount of viscous dissipation. In our analysis and experiments, we consider whether similar effects occur for nonlinear Faraday (gravity-capillary) waves. Assuming a finite-depth, infinite-breadth, low-viscosity fluid, we derive an analytic expression for the onset acceleration up to second order in ɛ =√{ 1 / Re } . This expression allows us to include fluid depth and driving frequency as parameters, in addition to the Marangoni and Boussinesq numbers. For millimetric fluid depths and driving frequencies of 30 to 120 Hz, our analysis recovers prior numerical results and agrees with our measurements of NBD-PC surfactant on DI water. In both case, the onset acceleration increases non-monotonically as a function of Marangoni and Boussinesq numbers. For shallower systems, our model predicts that surfactants could decrease the onset acceleration. DMS-0968258.

  1. Non-monotonic Pre-fixed Points and Learning

    Directory of Open Access Journals (Sweden)

    Stefano Berardi

    2013-08-01

    Full Text Available We consider the problem of finding pre-fixed points of interactive realizers over arbitrary knowledge spaces, obtaining a relative recursive procedure. Knowledge spaces and interactive realizers are an abstract setting to represent learning processes, that can interpret non-constructive proofs. Atomic pieces of information of a knowledge space are stratified into levels, and evaluated into truth values depending on knowledge states. Realizers are then used to define operators that extend a given state by adding and possibly removing atoms: in a learning process states of knowledge change nonmonotonically. Existence of a pre-fixed point of a realizer is equivalent to the termination of the learning process with some state of knowledge which is free of patent contradictions and such that there is nothing to add. In this paper we generalize our previous results in the case of level 2 knowledge spaces and deterministic operators to the case of omega-level knowledge spaces and of non-deterministic operators.

  2. Thermostable enzymes as biocatalysts in the biofuel industry.

    Science.gov (United States)

    Yeoman, Carl J; Han, Yejun; Dodd, Dylan; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

    2010-01-01

    Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Engineering of pectinolytic enzymes for enhanced thermostability

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller

    Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp...... consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous...... as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were...

  4. Non-monotonic field dependence of critical current in composite superconductors

    International Nuclear Information System (INIS)

    Andrianov, V.V.; Baev, V.P.; Ivanov, S.S.

    1982-01-01

    The nonmonotonic field dependence of critical current Im(B/sub a/ in composite superconductors is investigated experimentally for current and field varying simultaneously with final rates I and B/sub a/

  5. Thermostable Alginate degrading enzymes and their methods of use

    NARCIS (Netherlands)

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

    2015-01-01

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

  6. An electronic implementation for Liao's chaotic delayed neuron model with non-monotonous activation function

    International Nuclear Information System (INIS)

    Duan Shukai; Liao Xiaofeng

    2007-01-01

    A new chaotic delayed neuron model with non-monotonously increasing transfer function, called as chaotic Liao's delayed neuron model, was recently reported and analyzed. An electronic implementation of this model is described in detail. At the same time, some methods in circuit design, especially for circuit with time delayed unit and non-monotonously increasing activation unit, are also considered carefully. We find that the dynamical behaviors of the designed circuits are closely similar to the results predicted by numerical experiments

  7. Guest Controlled Nonmonotonic Deep Cavity Cavitand Assembly State Switching.

    Science.gov (United States)

    Tang, Du; Barnett, J Wesley; Gibb, Bruce C; Ashbaugh, Henry S

    2017-11-30

    Octa-acid (OA) and tetra-endo-methyl octa-acid (TEMOA) are water-soluble, deep-cavity cavitands with nanometer-sized nonpolar pockets that readily bind complementary guests, such as n-alkanes. Experimentally, OA exhibits a progression of 1:1 to 2:2 to 2:1 host/guest complexes (X:Y where X is the number of hosts and Y is the number of guests) with increasing alkane chain length from methane to tetradecane. Differing from OA only by the addition of four methyl groups ringing the portal of the pocket, TEMOA exhibits a nonmonotonic progression of assembly states from 1:1 to 2:2 to 1:1 to 2:1 with increasing guest length. Here we present a systematic molecular simulation study to parse the molecular and thermodynamic determinants that distinguish the succession of assembly stoichiometries observed for these similar hosts. Potentials of mean force between hosts and guests, determined via umbrella sampling, are used to characterize association free energies. These free energies are subsequently used in a reaction network model to predict the equilibrium distributions of assemblies. Our models accurately reproduce the experimentally observed trends, showing that TEMOA's endo-methyl units constrict the opening of the binding pocket, limiting the conformations available to bound guests and disrupting the balance between monomeric complexes and dimeric capsules. The success of our simulations demonstrate their utility at interpreting the impact of even simple chemical modifications on supramolecular assembly and highlight their potential to aid bottom-up design.

  8. A discrete wavelet spectrum approach for identifying non-monotonic trends in hydroclimate data

    Science.gov (United States)

    Sang, Yan-Fang; Sun, Fubao; Singh, Vijay P.; Xie, Ping; Sun, Jian

    2018-01-01

    The hydroclimatic process is changing non-monotonically and identifying its trends is a great challenge. Building on the discrete wavelet transform theory, we developed a discrete wavelet spectrum (DWS) approach for identifying non-monotonic trends in hydroclimate time series and evaluating their statistical significance. After validating the DWS approach using two typical synthetic time series, we examined annual temperature and potential evaporation over China from 1961-2013 and found that the DWS approach detected both the warming and the warming hiatus in temperature, and the reversed changes in potential evaporation. Further, the identified non-monotonic trends showed stable significance when the time series was longer than 30 years or so (i.e. the widely defined climate timescale). The significance of trends in potential evaporation measured at 150 stations in China, with an obvious non-monotonic trend, was underestimated and was not detected by the Mann-Kendall test. Comparatively, the DWS approach overcame the problem and detected those significant non-monotonic trends at 380 stations, which helped understand and interpret the spatiotemporal variability in the hydroclimatic process. Our results suggest that non-monotonic trends of hydroclimate time series and their significance should be carefully identified, and the DWS approach proposed has the potential for wide use in the hydrological and climate sciences.

  9. A discrete wavelet spectrum approach for identifying non-monotonic trends in hydroclimate data

    Directory of Open Access Journals (Sweden)

    Y.-F. Sang

    2018-01-01

    Full Text Available The hydroclimatic process is changing non-monotonically and identifying its trends is a great challenge. Building on the discrete wavelet transform theory, we developed a discrete wavelet spectrum (DWS approach for identifying non-monotonic trends in hydroclimate time series and evaluating their statistical significance. After validating the DWS approach using two typical synthetic time series, we examined annual temperature and potential evaporation over China from 1961–2013 and found that the DWS approach detected both the warming and the warming hiatus in temperature, and the reversed changes in potential evaporation. Further, the identified non-monotonic trends showed stable significance when the time series was longer than 30 years or so (i.e. the widely defined climate timescale. The significance of trends in potential evaporation measured at 150 stations in China, with an obvious non-monotonic trend, was underestimated and was not detected by the Mann–Kendall test. Comparatively, the DWS approach overcame the problem and detected those significant non-monotonic trends at 380 stations, which helped understand and interpret the spatiotemporal variability in the hydroclimatic process. Our results suggest that non-monotonic trends of hydroclimate time series and their significance should be carefully identified, and the DWS approach proposed has the potential for wide use in the hydrological and climate sciences.

  10. Lugol’s solution-induced painless thyroiditis

    Directory of Open Access Journals (Sweden)

    Ji Wei Yang

    2017-05-01

    Full Text Available Lugol’s solution is usually employed for a limited period for thyroidectomy preparation in patients with Graves’ disease and for the control of severe thyrotoxicosis and thyroid storm. We describe a rare case of Lugol’s solution-induced painless thyroiditis. In November 2014, a 59-year-old woman was prescribed Lugol’s solution four drops per day for the alleviation of menopausal symptoms. She was referred to our clinic in June 2015 for fatigue, hair loss, and a 20-lb weight loss without thyroid pain or discomfort. Physical examination revealed a normal thyroid gland. On 7 May 2015, laboratory tests revealed a suppressed thyroid-stimulating hormone (TSH 0.01 U/L with elevated free T4 3.31 ng/dL (42.54 pmol/L. Repeat testing on 25 May 2015 showed spontaneous normalization of the free thyroid hormone levels with persistently low TSH 0.10 U/L. Following these results, a family physician prescribed methimazole 10 mg PO TID and very soon after, the TSH concentration rose to >100 U/L along with subnormal free T4 and T3 levels. Methimazole was promptly discontinued, namely within 18 days of its initiation. Over the course of the next few months, the patient spontaneously achieved clinical and biochemical euthyroidism. To our knowledge, this is a unique case of painless thyroiditis induced by Lugol’s solution, which has not been reported before. Lugol’s solution is a short-term medication given for the preparation of thyroidectomy in patients with Graves’ disease and for the control of severe thyrotoxicosis. Iodine excess can cause both hyperthyroidism and hypothyroidism. Rarely, Lugol’s solution can cause acute painless thyroiditis.

  11. Highly Efficient Thermostable DSM Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Inc., San Francisco, CA (United States)

    2011-04-26

    These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  12. Consistent mutational paths predict eukaryotic thermostability

    Directory of Open Access Journals (Sweden)

    van Noort Vera

    2013-01-01

    Full Text Available Abstract Background Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1, we could also characterise the molecular consequences of some of these mutations. Conclusions The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

  13. A Mathematical Model for Non-monotonic Deposition Profiles in Deep Bed Filtration Systems

    DEFF Research Database (Denmark)

    Yuan, Hao; Shapiro, Alexander

    2011-01-01

    A mathematical model for suspension/colloid flow in porous media and non-monotonic deposition is proposed. It accounts for the migration of particles associated with the pore walls via the second energy minimum (surface associated phase). The surface associated phase migration is characterized...... by advection and diffusion/dispersion. The proposed model is able to produce a nonmonotonic deposition profile. A set of methods for estimating the modeling parameters is provided in the case of minimal particle release. The estimation can be easily performed with available experimental information....... The numerical modeling results highly agree with the experimental observations, which proves the ability of the model to catch a non-monotonic deposition profile in practice. An additional equation describing a mobile population behaving differently from the injected population seems to be a sufficient...

  14. Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia; Jørgensen, Henning

    2013-01-01

    the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5...... % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more...... ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation....

  15. Thermostability of biological systems: fundamentals, challenges, and quantification.

    Science.gov (United States)

    He, Xiaoming

    2011-01-01

    This review examines the fundamentals and challenges in engineering/understanding the thermostability of biological systems over a wide temperature range (from the cryogenic to hyperthermic regimen). Applications of the bio-thermostability engineering to either destroy unwanted or stabilize useful biologicals for the treatment of diseases in modern medicine are first introduced. Studies on the biological responses to cryogenic and hyperthermic temperatures for the various applications are reviewed to understand the mechanism of thermal (both cryo and hyperthermic) injury and its quantification at the molecular, cellular and tissue/organ levels. Methods for quantifying the thermophysical processes of the various applications are then summarized accounting for the effect of blood perfusion, metabolism, water transport across cell plasma membrane, and phase transition (both equilibrium and non-equilibrium such as ice formation and glass transition) of water. The review concludes with a summary of the status quo and future perspectives in engineering the thermostability of biological systems.

  16. Thermostability in rubredoxin and its relationship to mechanical rigidity

    Science.gov (United States)

    Rader, A. J.

    2010-03-01

    The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors.

  17. Thermostability in rubredoxin and its relationship to mechanical rigidity

    International Nuclear Information System (INIS)

    Rader, A J

    2010-01-01

    The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors

  18. A Nonmonotone Line Search Filter Algorithm for the System of Nonlinear Equations

    Directory of Open Access Journals (Sweden)

    Zhong Jin

    2012-01-01

    Full Text Available We present a new iterative method based on the line search filter method with the nonmonotone strategy to solve the system of nonlinear equations. The equations are divided into two groups; some equations are treated as constraints and the others act as the objective function, and the two groups are just updated at the iterations where it is needed indeed. We employ the nonmonotone idea to the sufficient reduction conditions and filter technique which leads to a flexibility and acceptance behavior comparable to monotone methods. The new algorithm is shown to be globally convergent and numerical experiments demonstrate its effectiveness.

  19. Comparison of the thermostability of cellulases from various thermophilic fungi

    Energy Technology Data Exchange (ETDEWEB)

    Wojtczak, G; Breuil, C; Yamada, J; Saddler, J N

    1987-10-01

    The cellulase activities of six thermophilic fungi were compared. Although the thermophilic fungi grew at relatively high temperatures (> 45/sup 0/C) the optimum temperatures for assaying the various cellulase activities were only slightly higher than the optimum temperatures for the mesophilic fungi, Trichoderma harzianum. Over prolonged incubation (> 24 h) the thermophilic strains demonstrated a higher hydrolytic potential as a result of the greater thermostability of the cellulase components. Although the extracellular cellulase activities had similar pH and temperature optima, in some cases the thermostability of the extracellular components were considerably lower.

  20. Prediction of Protein Thermostability by an Efficient Neural Network Approach

    Directory of Open Access Journals (Sweden)

    Jalal Rezaeenour

    2016-10-01

    Full Text Available Introduction: Manipulation of protein stability is important for understanding the principles that govern protein thermostability, both in basic research and industrial applications. Various data mining techniques exist for prediction of thermostable proteins. Furthermore, ANN methods have attracted significant attention for prediction of thermostability, because they constitute an appropriate approach to mapping the non-linear input-output relationships and massive parallel computing. Method: An Extreme Learning Machine (ELM was applied to estimate thermal behavior of 1289 proteins. In the proposed algorithm, the parameters of ELM were optimized using a Genetic Algorithm (GA, which tuned a set of input variables, hidden layer biases, and input weights, to and enhance the prediction performance. The method was executed on a set of amino acids, yielding a total of 613 protein features. A number of feature selection algorithms were used to build subsets of the features. A total of 1289 protein samples and 613 protein features were calculated from UniProt database to understand features contributing to the enzymes’ thermostability and find out the main features that influence this valuable characteristic. Results:At the primary structure level, Gln, Glu and polar were the features that mostly contributed to protein thermostability. At the secondary structure level, Helix_S, Coil, and charged_Coil were the most important features affecting protein thermostability. These results suggest that the thermostability of proteins is mainly associated with primary structural features of the protein. According to the results, the influence of primary structure on the thermostabilty of a protein was more important than that of the secondary structure. It is shown that prediction accuracy of ELM (mean square error can improve dramatically using GA with error rates RMSE=0.004 and MAPE=0.1003. Conclusion: The proposed approach for forecasting problem

  1. Non-monotonic reasoning in conceptual modeling and ontology design: A proposal

    CSIR Research Space (South Africa)

    Casini, G

    2013-06-01

    Full Text Available -1 2nd International Workshop on Ontologies and Conceptual Modeling (Onto.Com 2013), Valencia, Spain, 17-21 June 2013 Non-monotonic reasoning in conceptual modeling and ontology design: A proposal Giovanni Casini1 and Alessandro Mosca2 1...

  2. Alternans by non-monotonic conduction velocity restitution, bistability and memory

    International Nuclear Information System (INIS)

    Kim, Tae Yun; Hong, Jin Hee; Heo, Ryoun; Lee, Kyoung J

    2013-01-01

    Conduction velocity (CV) restitution is a key property that characterizes any medium supporting traveling waves. It reflects not only the dynamics of the individual constituents but also the coupling mechanism that mediates their interaction. Recent studies have suggested that cardiac tissues, which have a non-monotonic CV-restitution property, can support alternans, a period-2 oscillatory response of periodically paced cardiac tissue. This study finds that single-hump, non-monotonic, CV-restitution curves are a common feature of in vitro cultures of rat cardiac cells. We also find that the Fenton–Karma model, one of the well-established mathematical models of cardiac tissue, supports a very similar non-monotonic CV restitution in a physiologically relevant parameter regime. Surprisingly, the mathematical model as well as the cell cultures support bistability and show cardiac memory that tends to work against the generation of an alternans. Bistability was realized by adopting two different stimulation protocols, ‘S1S2’, which produces a period-1 wave train, and ‘alternans-pacing’, which favors a concordant alternans. Thus, we conclude that the single-hump non-monotonicity in the CV-restitution curve is not sufficient to guarantee a cardiac alternans, since cardiac memory interferes and the way the system is paced matters. (paper)

  3. Production of thermostable and organic solvent-tolerant alkaline ...

    African Journals Online (AJOL)

    An alkaliphilic bacterium producing organic solvent-tolerant and thermostable alkaline protease was isolated from poultry litter site and identified as Bacillus coagulans PSB-07. Protease production under different submerged fermentation conditions were investigated with the aim of optimizing yield of enzyme. B. coagulans ...

  4. Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

    Science.gov (United States)

    Kumar, Vishal; Dangi, Arun Kumar; Shukla, Pratyoosh

    2018-03-01

    Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase-substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.

  5. Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

    Science.gov (United States)

    van den Brink, Joost; Facun, Kryss; de Vries, Michel; Stielow, J Benjamin

    2015-12-01

    Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  6. Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

    Directory of Open Access Journals (Sweden)

    Siika-aho Matti

    2011-01-01

    Full Text Available Abstract Background Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases, resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties connected with high initial solid loadings in the lignocellulose to ethanol process. Results The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A. Conclusions Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter hydrothermally pretreated wheat straw. Based on temperature profiling studies, Thermoascus aurantiacus EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.

  7. Development of a thermostable microneedle patch for influenza vaccination

    Science.gov (United States)

    Mistilis, Matthew; Bommarius, Andreas S; Prausnitz, Mark R.

    2017-01-01

    The goal of this study is to develop thermostable microneedle patch formulations for influenza vaccine that can be partially or completely removed from the cold chain. During vaccine drying associated with microneedle patch manufacturing, ammonium acetate and HEPES buffer salts stabilized influenza vaccine, surfactants had little effect during drying, drying temperature had weak effects on vaccine stability, and drying on polydimethylsiloxane led to increased stability compared to drying on stainless steel. A number of excipients, mostly polysaccharides and some amino acids, further stabilized the influenza vaccine during drying. Over longer time scales of storage, combinations of stabilizers preserved the most vaccine activity. Finally, dissolving microneedle patches formulated with arginine and calcium heptagluconate had no significant activity loss for all three strains of seasonal influenza vaccine during storage at room temperature for six months. We conclude that appropriately formulated microneedle patches can exhibit remarkable thermostability that could enable storage and distribution of influenza vaccine outside the cold chain. PMID:25448542

  8. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    , such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...... strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self...... immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials....

  9. On-line learning of non-monotonic rules by simple perceptron

    OpenAIRE

    Inoue, Jun-ichi; Nishimori, Hidetoshi; Kabashima, Yoshiyuki

    1997-01-01

    We study the generalization ability of a simple perceptron which learns unlearnable rules. The rules are presented by a teacher perceptron with a non-monotonic transfer function. The student is trained in the on-line mode. The asymptotic behaviour of the generalization error is estimated under various conditions. Several learning strategies are proposed and improved to obtain the theoretical lower bound of the generalization error.

  10. ASPMT(QS): Non-Monotonic Spatial Reasoning with Answer Set Programming Modulo Theories

    OpenAIRE

    Wałęga, Przemysław Andrzej; Bhatt, Mehul; Schultz, Carl

    2015-01-01

    The systematic modelling of \\emph{dynamic spatial systems} [9] is a key requirement in a wide range of application areas such as comonsense cognitive robotics, computer-aided architecture design, dynamic geographic information systems. We present ASPMT(QS), a novel approach and fully-implemented prototype for non-monotonic spatial reasoning ---a crucial requirement within dynamic spatial systems-- based on Answer Set Programming Modulo Theories (ASPMT). ASPMT(QS) consists of a (qualitative) s...

  11. Non-Monotonic Spatial Reasoning with Answer Set Programming Modulo Theories

    OpenAIRE

    Wałęga, Przemysław Andrzej; Schultz, Carl; Bhatt, Mehul

    2016-01-01

    The systematic modelling of dynamic spatial systems is a key requirement in a wide range of application areas such as commonsense cognitive robotics, computer-aided architecture design, and dynamic geographic information systems. We present ASPMT(QS), a novel approach and fully-implemented prototype for non-monotonic spatial reasoning -a crucial requirement within dynamic spatial systems- based on Answer Set Programming Modulo Theories (ASPMT). ASPMT(QS) consists of a (qualitative) spatial re...

  12. The non-monotonic shear-thinning flow of two strongly cohesive concentrated suspensions

    OpenAIRE

    Buscall, Richard; Kusuma, Tiara E.; Stickland, Anthony D.; Rubasingha, Sayuri; Scales, Peter J.; Teo, Hui-En; Worrall, Graham L.

    2014-01-01

    The behaviour in simple shear of two concentrated and strongly cohesive mineral suspensions showing highly non-monotonic flow curves is described. Two rheometric test modes were employed, controlled stress and controlled shear-rate. In controlled stress mode the materials showed runaway flow above a yield stress, which, for one of the suspensions, varied substantially in value and seemingly at random from one run to the next, such that the up flow-curve appeared to be quite irreproducible. Th...

  13. Non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment

    OpenAIRE

    Lagarde, Fabien; Beausoleil, Claire; Belcher, Scott M; Belzunces, Luc P; Emond, Claude; Guerbet, Michel; Rousselle, Christophe

    2015-01-01

    International audience; Experimental studies investigating the effects of endocrine disruptors frequently identify potential unconventional dose-response relationships called non-monotonic dose-response (NMDR) relationships. Standardized approaches for investigating NMDR relationships in a risk assessment context are missing. The aim of this work was to develop criteria for assessing the strength of NMDR relationships. A literature search was conducted to identify published studies that repor...

  14. Internal m=1, n=1 helical mode in a tokamak with nonmonotonic current profile

    International Nuclear Information System (INIS)

    Kuvshinov, B.N.; Mikhajlovskij, A.B.

    1988-01-01

    Internal helical mode in a tokamak with two resonance surfaces, on which storing coefficient reduces to unity is studied theoretically. A general criterion for the investigated perturbations stability is obtained. Dispersion equation, describing both ideal and resistive helical modes, is derived. Analytic calculations for the case of perturbations localized near the tokamak axis are made. It is shown that in the framework of standard ideal hydrodynamics such perturbations are unstable at characteristic nonmonotonous profiles of the current

  15. Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

    Science.gov (United States)

    Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

    2016-04-26

    The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

  16. Multigenerational contaminant exposures produce non-monotonic, transgenerational responses in Daphnia magna

    International Nuclear Information System (INIS)

    Kimberly, David A.; Salice, Christopher J.

    2015-01-01

    Generally, ecotoxicologists rely on short-term tests that assume populations to be static. Conversely, natural populations may be exposed to the same stressors for many generations, which can alter tolerance to the same (or other) stressors. The objective of this study was to improve our understanding of how multigenerational stressors alter life history traits and stressor tolerance. After continuously exposing Daphnia magna to cadmium for 120 days, we assessed life history traits and conducted a challenge at higher temperature and cadmium concentrations. Predictably, individuals exposed to cadmium showed an overall decrease in reproductive output compared to controls. Interestingly, control D. magna were the most cadmium tolerant to novel cadmium, followed by those exposed to high cadmium. Our data suggest that long-term exposure to cadmium alter tolerance traits in a non-monotonic way. Because we observed effects after one-generation removal from cadmium, transgenerational effects may be possible as a result of multigenerational exposure. - Highlights: • Daphnia magna exposed to cadmium for 120 days. • D. magna exposed to cadmium had decreased reproductive output. • Control D. magna were most cadmium tolerant to novel cadmium stress. • Long-term exposure to cadmium alter tolerance traits in a non-monotonic way. • Transgenerational effects observed as a result of multigenerational exposure. - Adverse effects of long-term cadmium exposure persist into cadmium free conditions, as seen by non-monotonic responses when exposed to novel stress one generation removed.

  17. The Impact of Making Vaccines Thermostable in Niger’s Vaccine Supply Chain

    Science.gov (United States)

    Lee, Bruce Y.; Cakouros, Brigid E.; Assi, Tina-Marie; Connor, Diana L.; Welling, Joel; Kone, Souleymane; Djibo, Ali; Wateska, Angela R.; Pierre, Lionel; Brown, Shawn T.

    2012-01-01

    Objective Determine the effects on the vaccine cold chain of making different types of World Health Organization (WHO) Expanded Program on Immunizations (EPI) vaccines thermostable. Methods Utilizing a detailed computational, discrete-event simulation model of the Niger vaccine supply chain, we simulated the impact of making different combinations of the six current EPI vaccines thermostable. Findings Making any EPI vaccine thermostable relieved existing supply chain bottlenecks (especially at the lowest levels), increased vaccine availability of all EPI vaccines, and decreased cold storage and transport capacity utilization. By far, the most substantial impact came from making the pentavalent vaccine thermostable, increasing its own vaccine availability from 87% to 97% and the vaccine availabilities of all other remaining non-thermostable EPI vaccines to over 93%. By contrast, making each of the other vaccines thermostable had considerably less effect on the remaining vaccines, failing to increase the vaccine availabilities of other vaccines to more than 89%. Making tetanus toxoid vaccine along with the pentavalent thermostable further increased the vaccine availability of all EPI vaccines by at least 1–2%. Conclusion Our study shows the potential benefits of making any of Niger’s EPI vaccines thermostable and therefore supports further development of thermostable vaccines. Eliminating the need for refrigerators and freezers should not necessarily be the only benefit and goal of vaccine thermostability. Rather, making even a single vaccine (or some subset of the vaccines) thermostable could free up significant cold storage space for other vaccines, and thereby help alleviate supply chain bottlenecks that occur throughout the world. PMID:22789507

  18. The impact of making vaccines thermostable in Niger's vaccine supply chain.

    Science.gov (United States)

    Lee, Bruce Y; Cakouros, Brigid E; Assi, Tina-Marie; Connor, Diana L; Welling, Joel; Kone, Souleymane; Djibo, Ali; Wateska, Angela R; Pierre, Lionel; Brown, Shawn T

    2012-08-17

    Determine the effects on the vaccine cold chain of making different types of World Health Organization (WHO) Expanded Program on Immunizations (EPI) vaccines thermostable. Utilizing a detailed computational, discrete-event simulation model of the Niger vaccine supply chain, we simulated the impact of making different combinations of the six current EPI vaccines thermostable. Making any EPI vaccine thermostable relieved existing supply chain bottlenecks (especially at the lowest levels), increased vaccine availability of all EPI vaccines, and decreased cold storage and transport capacity utilization. By far, the most substantial impact came from making the pentavalent vaccine thermostable, increasing its own vaccine availability from 87% to 97% and the vaccine availabilities of all other remaining non-thermostable EPI vaccines to over 93%. By contrast, making each of the other vaccines thermostable had considerably less effect on the remaining vaccines, failing to increase the vaccine availabilities of other vaccines to more than 89%. Making tetanus toxoid vaccine along with the pentavalent thermostable further increased the vaccine availability of all EPI vaccines by at least 1-2%. Our study shows the potential benefits of making any of Niger's EPI vaccines thermostable and therefore supports further development of thermostable vaccines. Eliminating the need for refrigerators and freezers should not necessarily be the only benefit and goal of vaccine thermostability. Rather, making even a single vaccine (or some subset of the vaccines) thermostable could free up significant cold storage space for other vaccines, and thereby help alleviate supply chain bottlenecks that occur throughout the world. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

    1992-01-01

    Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

  20. Nonmonotonic and anisotropic magnetoresistance effect in antiferromagnet CaMn2Bi2

    Science.gov (United States)

    Kawaguchi, N.; Urata, T.; Hatano, T.; Iida, K.; Ikuta, H.

    2018-04-01

    We found a large and unique magnetoresistance (MR) effect for CaMn2Bi2 . When the magnetic field was applied along the crystallographic c axis at low temperatures, the resistivity increased with the magnetic field and the MR ratio reached several hundred percent, but then it decreased with further increasing the applied field. In addition, the angle dependence measurement revealed a strong anisotropy. This compound is an antiferromagnetic semiconductor with a narrow band gap, and Mn atoms form a corrugated honeycomb lattice. Therefore, a frustration among the magnetic moments is expected, and we propose that our observations can be understood by a nonmonotonic modulation of magnetic fluctuation under the magnetic field.

  1. Oscillation of Nonlinear Delay Differential Equation with Non-Monotone Arguments

    Directory of Open Access Journals (Sweden)

    Özkan Öcalan

    2017-07-01

    Full Text Available Consider the first-order nonlinear retarded differential equation $$ x^{\\prime }(t+p(tf\\left( x\\left( \\tau (t\\right \\right =0, t\\geq t_{0} $$ where $p(t$ and $\\tau (t$ are function of positive real numbers such that $%\\tau (t\\leq t$ for$\\ t\\geq t_{0},\\ $and$\\ \\lim_{t\\rightarrow \\infty }\\tau(t=\\infty $. Under the assumption that the retarded argument is non-monotone, new oscillation results are given. An example illustrating the result is also given.

  2. Application of non-monotonic logic to failure diagnosis of nuclear power plant

    International Nuclear Information System (INIS)

    Takahashi, M.; Kitamura, M.; Sugiyama, K.

    1989-01-01

    A prototype diagnosis system for nuclear power plants was developed based on Truth Maintenance systems: TMS and Dempster-Shafer probability theory. The purpose of this paper is to establish basic technique for more intelligent, man-computer cooperative diagnosis system. The developed system is capable of carrying out the diagnostic inference under the imperfect observation condition with the help of the proposed belief revision procedure with TMS and the systematic uncertainty treatment with Dempster-Shafer theory. The usefulness and potentiality of the present non-monotonic logic were demonstrated through simulation experiments

  3. Nonmonotonic Behavior of Supermultiplet Pattern Formation in a Noisy Lotka-Volterra System

    International Nuclear Information System (INIS)

    Fiasconaro, A.; Valenti, D.; Spagnolo, B.

    2004-01-01

    The noise-induced pattern formation in a population dynamical model of three interacting species in the coexistence regime is investigated. A coupled map lattice of Lotka-Volterra equations in the presence of multiplicative noise is used to analyze the spatiotemporal evolution. The spatial correlation of the species concentration as a function of time and of the noise intensity is investigated. A nonmonotonic behavior of the area of the patterns as a function of both noise intensity and evolution time is found. (author)

  4. A Nonmonotone Trust Region Method for Nonlinear Programming with Simple Bound Constraints

    International Nuclear Information System (INIS)

    Chen, Z.-W.; Han, J.-Y.; Xu, D.-C.

    2001-01-01

    In this paper we propose a nonmonotone trust region algorithm for optimization with simple bound constraints. Under mild conditions, we prove the global convergence of the algorithm. For the monotone case it is also proved that the correct active set can be identified in a finite number of iterations if the strict complementarity slackness condition holds, and so the proposed algorithm reduces finally to an unconstrained minimization method in a finite number of iterations, allowing a fast asymptotic rate of convergence. Numerical experiments show that the method is efficient

  5. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    Science.gov (United States)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2016-06-28

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  6. Understanding thermostability and pH dependent properties of proteins

    DEFF Research Database (Denmark)

    Galberg, Pernille

    The work performed in this thesis is part of a larger project (“Computational design of stable enzymes”) involving several research teams, which aimed to improve PROPKA (http://propka.ki.ku.dk) and to provide the scientific community with a computational protocol and associated PROPKA program......, which could be used for predicting mutations with expectation of increased thermostability at a certain pH value or a shifted pH activity optimum. The ability of a Bacillus circulans xylanase (BCX) mutant (N35D/A115E) to induce a decrease in pH activity optimum was evaluated by a pH dependent xylanase...

  7. Solute induced relaxation in glassy polymers: Experimental measurements and nonequilibrium thermodynamic model

    International Nuclear Information System (INIS)

    Minelli, Matteo; Doghieri, Ferruccio

    2014-01-01

    Data for kinetics of mass uptake from vapor sorption experiments in thin glassy polymer samples are here interpreted in terms of relaxation times for volume dilation. To this result, both models from non-equilibrium thermodynamics and from mechanics of volume relaxation contribute. Different kind of sorption experiments have been considered in order to facilitate the direct comparison between kinetics of solute induced volume dilation and corresponding data from process driven by pressure or temperature jumps

  8. The effect of the electrical double layer on hydrodynamic lubrication: a non-monotonic trend with increasing zeta potential

    Directory of Open Access Journals (Sweden)

    Dalei Jing

    2017-07-01

    Full Text Available In the present study, a modified Reynolds equation including the electrical double layer (EDL-induced electroviscous effect of lubricant is established to investigate the effect of the EDL on the hydrodynamic lubrication of a 1D slider bearing. The theoretical model is based on the nonlinear Poisson–Boltzmann equation without the use of the Debye–Hückel approximation. Furthermore, the variation in the bulk electrical conductivity of the lubricant under the influence of the EDL is also considered during the theoretical analysis of hydrodynamic lubrication. The results show that the EDL can increase the hydrodynamic load capacity of the lubricant in a 1D slider bearing. More importantly, the hydrodynamic load capacity of the lubricant under the influence of the EDL shows a non-monotonic trend, changing from enhancement to attenuation with a gradual increase in the absolute value of the zeta potential. This non-monotonic hydrodynamic lubrication is dependent on the non-monotonic electroviscous effect of the lubricant generated by the EDL, which is dominated by the non-monotonic electrical field strength and non-monotonic electrical body force on the lubricant. The subject of the paper is the theoretical modeling and the corresponding analysis.

  9. Non-monotonic effect of growth temperature on carrier collection in SnS solar cells

    International Nuclear Information System (INIS)

    Chakraborty, R.; Steinmann, V.; Mangan, N. M.; Brandt, R. E.; Poindexter, J. R.; Jaramillo, R.; Mailoa, J. P.; Hartman, K.; Polizzotti, A.; Buonassisi, T.; Yang, C.; Gordon, R. G.

    2015-01-01

    We quantify the effects of growth temperature on material and device properties of thermally evaporated SnS thin-films and test structures. Grain size, Hall mobility, and majority-carrier concentration monotonically increase with growth temperature. However, the charge collection as measured by the long-wavelength contribution to short-circuit current exhibits a non-monotonic behavior: the collection decreases with increased growth temperature from 150 °C to 240 °C and then recovers at 285 °C. Fits to the experimental internal quantum efficiency using an opto-electronic model indicate that the non-monotonic behavior of charge-carrier collection can be explained by a transition from drift- to diffusion-assisted components of carrier collection. The results show a promising increase in the extracted minority-carrier diffusion length at the highest growth temperature of 285 °C. These findings illustrate how coupled mechanisms can affect early stage device development, highlighting the critical role of direct materials property measurements and simulation

  10. Subtractive, divisive and non-monotonic gain control in feedforward nets linearized by noise and delays.

    Science.gov (United States)

    Mejias, Jorge F; Payeur, Alexandre; Selin, Erik; Maler, Leonard; Longtin, André

    2014-01-01

    The control of input-to-output mappings, or gain control, is one of the main strategies used by neural networks for the processing and gating of information. Using a spiking neural network model, we studied the gain control induced by a form of inhibitory feedforward circuitry-also known as "open-loop feedback"-, which has been experimentally observed in a cerebellum-like structure in weakly electric fish. We found, both analytically and numerically, that this network displays three different regimes of gain control: subtractive, divisive, and non-monotonic. Subtractive gain control was obtained when noise is very low in the network. Also, it was possible to change from divisive to non-monotonic gain control by simply modulating the strength of the feedforward inhibition, which may be achieved via long-term synaptic plasticity. The particular case of divisive gain control has been previously observed in vivo in weakly electric fish. These gain control regimes were robust to the presence of temporal delays in the inhibitory feedforward pathway, which were found to linearize the input-to-output mappings (or f-I curves) via a novel variability-increasing mechanism. Our findings highlight the feedforward-induced gain control analyzed here as a highly versatile mechanism of information gating in the brain.

  11. Subtractive, divisive and non-monotonic gain control in feedforward nets linearized by noise and delays

    Directory of Open Access Journals (Sweden)

    Jorge F Mejias

    2014-02-01

    Full Text Available The control of input-to-output mappings, or gain control, is one of the main strategies used by neural networks for the processing and gating of information. Using a spiking neural network model, we studied the gain control induced by a form of inhibitory feedforward circuitry — also known as ’open-loop feedback’ —, which has been experimentally observed in a cerebellum-like structure in weakly electric fish. We found, both analytically and numerically, that this network displays three different regimes of gain control: subtractive, divisive, and non-monotonic. Subtractive gain control was obtained when noise is very low in the network. Also, it was possible to change from divisive to non-monotonic gain control by simply modulating the strength of the feedforward inhibition, which may be achieved via long-term synaptic plasticity. The particular case of divisive gain control has been previously observed in vivo in weakly electric fish. These gain control regimes were robust to the presence of temporal delays in the inhibitory feedforward pathway, which were found to linearize the input-to-output mappings (or f-I curves via a novel variability-increasing mechanism. Our findings highlight the feedforward-induced gain control analyzed here as a highly versatile mechanism of information gating in the brain.

  12. Non-monotonic wetting behavior of chitosan films induced by silver nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Praxedes, A.P.P.; Webler, G.D.; Souza, S.T. [Instituto de Física, Universidade Federal de Alagoas, 57072-970 Maceió, AL (Brazil); Ribeiro, A.S. [Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, 57072-970 Maceió, AL (Brazil); Fonseca, E.J.S. [Instituto de Física, Universidade Federal de Alagoas, 57072-970 Maceió, AL (Brazil); Oliveira, I.N. de, E-mail: italo@fis.ufal.br [Instituto de Física, Universidade Federal de Alagoas, 57072-970 Maceió, AL (Brazil)

    2016-05-01

    Highlights: • The addition of silver nanoparticles modifies the morphology of chitosan films. • Metallic nanoparticles can be used to control wetting properties of chitosan films. • The contact angle shows a non-monotonic dependence on the silver concentration. - Abstract: The present work is devoted to the study of structural and wetting properties of chitosan-based films containing silver nanoparticles. In particular, the effects of silver concentration on the morphology of chitosan films are characterized by different techniques, such as atomic force microscopy (AFM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). By means of dynamic contact angle measurements, we study the modification on surface properties of chitosan-based films due to the addition of silver nanoparticles. The results are analyzed in the light of molecular-kinetic theory which describes the wetting phenomena in terms of statistical dynamics for the displacement of liquid molecules in a solid substrate. Our results show that the wetting properties of chitosan-based films are high sensitive to the fraction of silver nanoparticles, with the equilibrium contact angle exhibiting a non-monotonic behavior.

  13. Induction and Confirmation Theory: An Approach based on a Paraconsistent Nonmonotonic Logic

    Directory of Open Access Journals (Sweden)

    Ricardo Sousa Silvestre

    2010-12-01

    Full Text Available This paper is an effort to realize and explore the connections that exist between nonmonotonic logic and confirmation theory. We pick up one of the most wide-spread nonmonotonic formalisms – default logic – and analyze to what extent and under what adjustments it could work as a logic of induction in the philosophical sense. By making use of this analysis, we extend default logic so as to make it able to minimally perform the task of a logic of induction, having as a result a system which we believe has interesting properties from the standpoint of theory of confirmation. It is for instance able to represent chains of inductive rules as well as to reason paraconsistently on the conclusions obtained from them. We then use this logic to represent some traditional ideas concerning confirmation theory, in particular the ones proposed by Carl Hempel in his classical paper “Studies in the Logic of Confirmation” of 1945 and the ones incorporated in the so-called abductive and hy-pothetico-deductive models.

  14. Induction and Confirmation Theory: An Approach based on a Paraconsistent Nonmonotonic Logic

    Directory of Open Access Journals (Sweden)

    Ricardo Sousa Silvestre

    2011-05-01

    Full Text Available This paper is an effort to realize and explore the connections that exist between nonmonotonic logic and confirmation theory. We pick up one of the most wide-spread nonmonotonic formalisms – default logic – and analyze to what extent and under what adjustments it could work as a logic of induction in the philosophical sense. By making use of this analysis, we extend default logic so as to make it able to minimally perform the task of a logic of induction, having as a result a system which we believe has interesting properties from the standpoint of theory of confirmation. It is for instance able to represent chains of inductive rules as well as to reason paraconsistently on the conclusions obtained from them. We then use this logic to represent some traditional ideas concerning confirmation theory, in particular the ones proposed by Carl Hempel in his classical paper "Studies in the Logic of Confirmation" of 1945 and the ones incorporated in the so-called abductive and hy-pothetico-deductive models.

  15. Large Airborne Full Tensor Gradient Data Inversion Based on a Non-Monotone Gradient Method

    Science.gov (United States)

    Sun, Yong; Meng, Zhaohai; Li, Fengting

    2018-03-01

    Following the development of gravity gradiometer instrument technology, the full tensor gravity (FTG) data can be acquired on airborne and marine platforms. Large-scale geophysical data can be obtained using these methods, making such data sets a number of the "big data" category. Therefore, a fast and effective inversion method is developed to solve the large-scale FTG data inversion problem. Many algorithms are available to accelerate the FTG data inversion, such as conjugate gradient method. However, the conventional conjugate gradient method takes a long time to complete data processing. Thus, a fast and effective iterative algorithm is necessary to improve the utilization of FTG data. Generally, inversion processing is formulated by incorporating regularizing constraints, followed by the introduction of a non-monotone gradient-descent method to accelerate the convergence rate of FTG data inversion. Compared with the conventional gradient method, the steepest descent gradient algorithm, and the conjugate gradient algorithm, there are clear advantages of the non-monotone iterative gradient-descent algorithm. Simulated and field FTG data were applied to show the application value of this new fast inversion method.

  16. Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

    Science.gov (United States)

    Malau, N. D.; Sianturi, M.

    2017-03-01

    Molecular dynamics methods we have applied as a tool in designing thermostable Aspergillus niger Xylanase, by examining Root Mean Square Deviation (RMSD) and The Stability of the Secondary Structure of enzymes structure at its optimum temperature and compare with its high temperature behavior. As RMSD represents structural fluctuation at a particular temperature, a better understanding of this factor will suggest approaches to bioengineer these enzymes to enhance their thermostability. In this work molecular dynamic simulations of Aspergillus niger xylanase (ANX) have been carried at 400K (optimum catalytic temperature) for 2.5 ns and 500K (ANX reported inactive temperature) for 2.5 ns. Analysis have shown that the Root Mean Square Deviation (RMSD) significant increase at higher temperatures compared at optimum temperature and some of the secondary structures of ANX that have been damaged at high temperature. Structural analysis revealed that the fluctuations of the α-helix and β-sheet regions are larger at higher temperatures compared to the fluctuations at optimum temperature.

  17. Potential and utilization of thermophiles and thermostable enzymes in biorefining

    Directory of Open Access Journals (Sweden)

    Karlsson Eva

    2007-03-01

    Full Text Available Abstract In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

  18. Thermostability promotes the cooperative function of split adenylate kinases.

    Science.gov (United States)

    Nguyen, Peter Q; Liu, Shirley; Thompson, Jeremy C; Silberg, Jonathan J

    2008-05-01

    Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >10(3)-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

  19. A novel non-thermostable deuterolysin from Aspergillus oryzae.

    Science.gov (United States)

    Maeda, Hiroshi; Katase, Toru; Sakai, Daisuke; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Abe, Keietsu; Yamagata, Youhei

    2016-09-01

    Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.

  20. Thermal effects on the enhanced ductility in non-monotonic uniaxial tension of DP780 steel sheet

    Science.gov (United States)

    Majidi, Omid; Barlat, Frederic; Korkolis, Yannis P.; Fu, Jiawei; Lee, Myoung-Gyu

    2016-11-01

    To understand the material behavior during non-monotonic loading, uniaxial tension tests were conducted in three modes, namely, the monotonic loading, loading with periodic relaxation and periodic loading-unloadingreloading, at different strain rates (0.001/s to 0.01/s). In this study, the temperature gradient developing during each test and its contribution to increasing the apparent ductility of DP780 steel sheets were considered. In order to assess the influence of temperature, isothermal uniaxial tension tests were also performed at three temperatures (298 K, 313 K and 328 K (25 °C, 40 °C and 55 °C)). A digital image correlation system coupled with an infrared thermography was used in the experiments. The results show that the non-monotonic loading modes increased the apparent ductility of the specimens. It was observed that compared with the monotonic loading, the temperature gradient became more uniform when a non-monotonic loading was applied.

  1. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    Science.gov (United States)

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

  2. An electronic implementation for Liao's chaotic delayed neuron model with non-monotonous activation function

    Energy Technology Data Exchange (ETDEWEB)

    Duan Shukai [Department of Computer Science and Engineering, Chongqing University, Chongqing 400044 (China); School of Electronic and Information Engineering, Southwest University, Chongqing 400715 (China)], E-mail: duansk@swu.edu.cn; Liao Xiaofeng [Department of Computer Science and Engineering, Chongqing University, Chongqing 400044 (China)], E-mail: xfliao@cqu.edu.cn

    2007-09-10

    A new chaotic delayed neuron model with non-monotonously increasing transfer function, called as chaotic Liao's delayed neuron model, was recently reported and analyzed. An electronic implementation of this model is described in detail. At the same time, some methods in circuit design, especially for circuit with time delayed unit and non-monotonously increasing activation unit, are also considered carefully. We find that the dynamical behaviors of the designed circuits are closely similar to the results predicted by numerical experiments.

  3. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria

    2015-03-13

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption by the ferritin. Thermostable ferritin proved to be an excellent system for rapid phosphate and arsenate removal from aqueous solutions down to residual concentrations at the picomolar level. These very low concentrations make thermostable ferritin a potential tool to considerably mitigate industrial biofouling by phosphate limitation or to remove arsenate from drinking water.

  4. Stair-Step Particle Flux Spectra on the Lunar Surface: Evidence for Nonmonotonic Potentials?

    Science.gov (United States)

    Collier, Michael R.; Newheart, Anastasia; Poppe, Andrew R.; Hills, H. Kent; Farrell, William M.

    2016-01-01

    We present examples of unusual "stair-step" differential flux spectra observed by the Apollo 14 Suprathermal Ion Detector Experiment on the lunar dayside surface in Earth's magnetotail. These spectra exhibit a relatively constant differential flux below some cutoff energy and then drop off precipitously, by about an order of magnitude or more, at higher energies. We propose that these spectra result from photoions accelerated on the lunar dayside by nonmonotonic potentials (i.e.,potentials that do not decay to zero monotonically) and present a model for the expected differential flux. The energy of the cutoff and the magnitude of the differential flux are related to the properties of the local space environment and are consistent with the observed flux spectra. If this interpretation is correct, these surface-based ion observations provide a unique perspective that both complements and enhances the conclusions obtained by remote-sensing orbiter observations on the Moon's exospheric and electrostatic properties.

  5. Quantitative non-monotonic modeling of economic uncertainty by probability and possibility distributions

    DEFF Research Database (Denmark)

    Schjær-Jacobsen, Hans

    2012-01-01

    uncertainty can be calculated. The possibility approach is particular well suited for representation of uncertainty of a non-statistical nature due to lack of knowledge and requires less information than the probability approach. Based on the kind of uncertainty and knowledge present, these aspects...... to the understanding of similarities and differences of the two approaches as well as practical applications. The probability approach offers a good framework for representation of randomness and variability. Once the probability distributions of uncertain parameters and their correlations are known the resulting...... are thoroughly discussed in the case of rectangular representation of uncertainty by the uniform probability distribution and the interval, respectively. Also triangular representations are dealt with and compared. Calculation of monotonic as well as non-monotonic functions of variables represented...

  6. Non-monotonic resonance in a spatially forced Lengyel-Epstein model

    Energy Technology Data Exchange (ETDEWEB)

    Haim, Lev [Physics Department, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel); Department of Oncology, Soroka University Medical Center, Beer-Sheva 84101 (Israel); Hagberg, Aric [Center for Nonlinear Studies, Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States); Meron, Ehud [Physics Department, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel); Department of Solar Energy and Environmental Physics, BIDR, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben-Gurion 84990 (Israel)

    2015-06-15

    We study resonant spatially periodic solutions of the Lengyel-Epstein model modified to describe the chlorine dioxide-iodine-malonic acid reaction under spatially periodic illumination. Using multiple-scale analysis and numerical simulations, we obtain the stability ranges of 2:1 resonant solutions, i.e., solutions with wavenumbers that are exactly half of the forcing wavenumber. We show that the width of resonant wavenumber response is a non-monotonic function of the forcing strength, and diminishes to zero at sufficiently strong forcing. We further show that strong forcing may result in a π/2 phase shift of the resonant solutions, and argue that the nonequilibrium Ising-Bloch front bifurcation can be reversed. We attribute these behaviors to an inherent property of forcing by periodic illumination, namely, the increase of the mean spatial illumination as the forcing amplitude is increased.

  7. Condition-based inspection/replacement policies for non-monotone deteriorating systems with environmental covariates

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Xuejing [Universite de Technologie de Troyes, Institut Charles Delaunay and STMR UMR CNRS 6279, 12 rue Marie Curie, 10010 Troyes (France); School of mathematics and statistics, Lanzhou University, Lanzhou 730000 (China); Fouladirad, Mitra, E-mail: mitra.fouladirad@utt.f [Universite de Technologie de Troyes, Institut Charles Delaunay and STMR UMR CNRS 6279, 12 rue Marie Curie, 10010 Troyes (France); Berenguer, Christophe [Universite de Technologie de Troyes, Institut Charles Delaunay and STMR UMR CNRS 6279, 12 rue Marie Curie, 10010 Troyes (France); Bordes, Laurent [Universite de Pau et des Pays de l' Adour, LMA UMR CNRS 5142, 64013 PAU Cedex (France)

    2010-08-15

    The aim of this paper is to discuss the problem of modelling and optimising condition-based maintenance policies for a deteriorating system in presence of covariates. The deterioration is modelled by a non-monotone stochastic process. The covariates process is assumed to be a time-homogenous Markov chain with finite state space. A model similar to the proportional hazards model is used to show the influence of covariates on the deterioration. In the framework of the system under consideration, an appropriate inspection/replacement policy which minimises the expected average maintenance cost is derived. The average cost under different conditions of covariates and different maintenance policies is analysed through simulation experiments to compare the policies performances.

  8. Condition-based inspection/replacement policies for non-monotone deteriorating systems with environmental covariates

    International Nuclear Information System (INIS)

    Zhao Xuejing; Fouladirad, Mitra; Berenguer, Christophe; Bordes, Laurent

    2010-01-01

    The aim of this paper is to discuss the problem of modelling and optimising condition-based maintenance policies for a deteriorating system in presence of covariates. The deterioration is modelled by a non-monotone stochastic process. The covariates process is assumed to be a time-homogenous Markov chain with finite state space. A model similar to the proportional hazards model is used to show the influence of covariates on the deterioration. In the framework of the system under consideration, an appropriate inspection/replacement policy which minimises the expected average maintenance cost is derived. The average cost under different conditions of covariates and different maintenance policies is analysed through simulation experiments to compare the policies performances.

  9. Non-monotonic spatial distribution of the interstellar dust in astrospheres: finite gyroradius effect

    Science.gov (United States)

    Katushkina, O. A.; Alexashov, D. B.; Izmodenov, V. V.; Gvaramadze, V. V.

    2017-02-01

    High-resolution mid-infrared observations of astrospheres show that many of them have filamentary (cirrus-like) structure. Using numerical models of dust dynamics in astrospheres, we suggest that their filamentary structure might be related to specific spatial distribution of the interstellar dust around the stars, caused by a gyrorotation of charged dust grains in the interstellar magnetic field. Our numerical model describes the dust dynamics in astrospheres under an influence of the Lorentz force and assumption of a constant dust charge. Calculations are performed for the dust grains with different sizes separately. It is shown that non-monotonic spatial dust distribution (viewed as filaments) appears for dust grains with the period of gyromotion comparable with the characteristic time-scale of the dust motion in the astrosphere. Numerical modelling demonstrates that the number of filaments depends on charge-to-mass ratio of dust.

  10. Non-monotonicity and divergent time scale in Axelrod model dynamics

    Science.gov (United States)

    Vazquez, F.; Redner, S.

    2007-04-01

    We study the evolution of the Axelrod model for cultural diversity, a prototypical non-equilibrium process that exhibits rich dynamics and a dynamic phase transition between diversity and an inactive state. We consider a simple version of the model in which each individual possesses two features that can assume q possibilities. Within a mean-field description in which each individual has just a few interaction partners, we find a phase transition at a critical value qc between an active, diverse state for q < qc and a frozen state. For q lesssim qc, the density of active links is non-monotonic in time and the asymptotic approach to the steady state is controlled by a time scale that diverges as (q-qc)-1/2.

  11. Nonmonotonic magnetoresistance of a two-dimensional viscous electron-hole fluid in a confined geometry

    Science.gov (United States)

    Alekseev, P. S.; Dmitriev, A. P.; Gornyi, I. V.; Kachorovskii, V. Yu.; Narozhny, B. N.; Titov, M.

    2018-02-01

    Ultrapure conductors may exhibit hydrodynamic transport where the collective motion of charge carriers resembles the flow of a viscous fluid. In a confined geometry (e.g., in ultra-high-quality nanostructures), the electronic fluid assumes a Poiseuille-type flow. Applying an external magnetic field tends to diminish viscous effects leading to large negative magnetoresistance. In two-component systems near charge neutrality, the hydrodynamic flow of charge carriers is strongly affected by the mutual friction between the two constituents. At low fields, the magnetoresistance is negative, however, at high fields the interplay between electron-hole scattering, recombination, and viscosity results in a dramatic change of the flow profile: the magnetoresistance changes its sign and eventually becomes linear in very high fields. This nonmonotonic magnetoresistance can be used as a fingerprint to detect viscous flow in two-component conducting systems.

  12. Non-monotonic relationships between emotional arousal and memory for color and location.

    Science.gov (United States)

    Boywitt, C Dennis

    2015-01-01

    Recent research points to the decreased diagnostic value of subjective retrieval experience for memory accuracy for emotional stimuli. While for neutral stimuli rich recollective experiences are associated with better context memory than merely familiar memories this association appears questionable for emotional stimuli. The present research tested the implicit assumption that the effect of emotional arousal on memory is monotonic, that is, steadily increasing (or decreasing) with increasing arousal. In two experiments emotional arousal was manipulated in three steps using emotional pictures and subjective retrieval experience as well as context memory were assessed. The results show an inverted U-shape relationship between arousal and recognition memory but for context memory and retrieval experience the relationship was more complex. For frame colour, context memory decreased linearly while for spatial location it followed the inverted U-shape function. The complex, non-monotonic relationships between arousal and memory are discussed as possible explanations for earlier divergent findings.

  13. Assessing dose-response relationships for endocrine disrupting chemicals (EDCs): a focus on non-monotonicity.

    Science.gov (United States)

    Zoeller, R Thomas; Vandenberg, Laura N

    2015-05-15

    The fundamental principle in regulatory toxicology is that all chemicals are toxic and that the severity of effect is proportional to the exposure level. An ancillary assumption is that there are no effects at exposures below the lowest observed adverse effect level (LOAEL), either because no effects exist or because they are not statistically resolvable, implying that they would not be adverse. Chemicals that interfere with hormones violate these principles in two important ways: dose-response relationships can be non-monotonic, which have been reported in hundreds of studies of endocrine disrupting chemicals (EDCs); and effects are often observed below the LOAEL, including all environmental epidemiological studies examining EDCs. In recognition of the importance of this issue, Lagarde et al. have published the first proposal to qualitatively assess non-monotonic dose response (NMDR) relationships for use in risk assessments. Their proposal represents a significant step forward in the evaluation of complex datasets for use in risk assessments. Here, we comment on three elements of the Lagarde proposal that we feel need to be assessed more critically and present our arguments: 1) the use of Klimisch scores to evaluate study quality, 2) the concept of evaluating study quality without topical experts' knowledge and opinions, and 3) the requirement of establishing the biological plausibility of an NMDR before consideration for use in risk assessment. We present evidence-based logical arguments that 1) the use of the Klimisch score should be abandoned for assessing study quality; 2) evaluating study quality requires experts in the specific field; and 3) an understanding of mechanisms should not be required to accept observable, statistically valid phenomena. It is our hope to contribute to the important and ongoing debate about the impact of NMDRs on risk assessment with positive suggestions.

  14. Thermostating extended Lagrangian Born-Oppenheimer molecular dynamics.

    Science.gov (United States)

    Martínez, Enrique; Cawkwell, Marc J; Voter, Arthur F; Niklasson, Anders M N

    2015-04-21

    Extended Lagrangian Born-Oppenheimer molecular dynamics is developed and analyzed for applications in canonical (NVT) simulations. Three different approaches are considered: the Nosé and Andersen thermostats and Langevin dynamics. We have tested the temperature distribution under different conditions of self-consistent field (SCF) convergence and time step and compared the results to analytical predictions. We find that the simulations based on the extended Lagrangian Born-Oppenheimer framework provide accurate canonical distributions even under approximate SCF convergence, often requiring only a single diagonalization per time step, whereas regular Born-Oppenheimer formulations exhibit unphysical fluctuations unless a sufficiently high degree of convergence is reached at each time step. The thermostated extended Lagrangian framework thus offers an accurate approach to sample processes in the canonical ensemble at a fraction of the computational cost of regular Born-Oppenheimer molecular dynamics simulations.

  15. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria; Paravidino, Monica; Vrouwenvelder, Johannes S.; Wolterbeek, Hubert Th.; van Loosdrecht, Mark C.M.; Hagen, Wilfred R.

    2015-01-01

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption

  16. Low dose effects and non-monotonic dose responses for endocrine active chemicals: Science to practice workshop: Workshop summary

    DEFF Research Database (Denmark)

    Beausoleil, Claire; Ormsby, Jean-Nicolas; Gies, Andreas

    2013-01-01

    A workshop was held in Berlin September 12–14th 2012 to assess the state of the science of the data supporting low dose effects and non-monotonic dose responses (“low dose hypothesis”) for chemicals with endocrine activity (endocrine disrupting chemicals or EDCs). This workshop consisted of lectu...

  17. Exogenous cellulases of thermophilic micromycetes. Pt. 2. Thermostability of enzyme preparations

    Energy Technology Data Exchange (ETDEWEB)

    Kvesitadze, G; Gogilashvili, L; Svanidze, R; Buachidze, T; Chirgadze, L; Nizharadze, D

    1986-01-01

    The ability of a large number of higher fungi to form extracellular cellulases is investigated. Some representatives of these fungi grow at 40-50/sup 0/C, and form extracellular cellulases exceeding cellulases of mesophilic fungi in thermostability. It is shown that cellulases of higher thermophilic fungi differ by their thermostability. The temperature optimum of cellulase action of higher fungi occurs within 60-62/sup 0/C.

  18. Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

    Directory of Open Access Journals (Sweden)

    Anuradha Balan

    2012-01-01

    Full Text Available Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v; yeast extract 1.25% (w/v; NaCl 0.45% (w/v olive oil 0.1% (v/v with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16 and olive oil with optimal activity (100% compared to other substrates.

  19. Semiparametric approach for non-monotone missing covariates in a parametric regression model

    KAUST Repository

    Sinha, Samiran

    2014-02-26

    Missing covariate data often arise in biomedical studies, and analysis of such data that ignores subjects with incomplete information may lead to inefficient and possibly biased estimates. A great deal of attention has been paid to handling a single missing covariate or a monotone pattern of missing data when the missingness mechanism is missing at random. In this article, we propose a semiparametric method for handling non-monotone patterns of missing data. The proposed method relies on the assumption that the missingness mechanism of a variable does not depend on the missing variable itself but may depend on the other missing variables. This mechanism is somewhat less general than the completely non-ignorable mechanism but is sometimes more flexible than the missing at random mechanism where the missingness mechansim is allowed to depend only on the completely observed variables. The proposed approach is robust to misspecification of the distribution of the missing covariates, and the proposed mechanism helps to nullify (or reduce) the problems due to non-identifiability that result from the non-ignorable missingness mechanism. The asymptotic properties of the proposed estimator are derived. Finite sample performance is assessed through simulation studies. Finally, for the purpose of illustration we analyze an endometrial cancer dataset and a hip fracture dataset.

  20. Multistability and gluing bifurcation to butterflies in coupled networks with non-monotonic feedback

    International Nuclear Information System (INIS)

    Ma Jianfu; Wu Jianhong

    2009-01-01

    Neural networks with a non-monotonic activation function have been proposed to increase their capacity for memory storage and retrieval, but there is still a lack of rigorous mathematical analysis and detailed discussions of the impact of time lag. Here we consider a two-neuron recurrent network. We first show how supercritical pitchfork bifurcations and a saddle-node bifurcation lead to the coexistence of multiple stable equilibria (multistability) in the instantaneous updating network. We then study the effect of time delay on the local stability of these equilibria and show that four equilibria lose their stability at a certain critical value of time delay, and Hopf bifurcations of these equilibria occur simultaneously, leading to multiple coexisting periodic orbits. We apply centre manifold theory and normal form theory to determine the direction of these Hopf bifurcations and the stability of bifurcated periodic orbits. Numerical simulations show very interesting global patterns of periodic solutions as the time delay is varied. In particular, we observe that these four periodic solutions are glued together along the stable and unstable manifolds of saddle points to develop a butterfly structure through a complicated process of gluing bifurcations of periodic solutions

  1. Non-monotonic dose dependence of the Ge- and Ti-centres in quartz

    International Nuclear Information System (INIS)

    Woda, C.; Wagner, G.A.

    2007-01-01

    The dose response of the Ge- and Ti-centres in quartz is studied over a large dose range. After an initial signal increase in the low dose range, both defects show a pronounced decrease in signal intensities for high doses. The model by Euler and Kahan [1987. Radiation effects and anelastic loss in germanium-doped quartz. Phys. Rev. B 35 (9), 4351-4359], in which the signal drop is explained by an enhanced trapping of holes at the electron trapping site, is critically discussed. A generalization of the model is then developed, following similar considerations by Lawless et al. [2005. A model for non-monotonic dose dependence of thermoluminescence (TL). J. Phys. Condens. Matter 17, 737-753], who explained a signal drop in TL by an enhanced recombination rate with electrons at the recombination centre. Finally, an alternative model for the signal decay is given, based on the competition between single and double electron capture at the electron trapping site. From the critical discussion of the different models it is concluded that the double electron capture mechanism is the most probable effect for the dose response

  2. Non-monotonic reorganization of brain networks with Alzheimer’s disease progression

    Directory of Open Access Journals (Sweden)

    Hyoungkyu eKim

    2015-06-01

    Full Text Available Background: Identification of stage-specific changes in brain network of patients with Alzheimer’s disease (AD is critical for rationally designed therapeutics that delays the progression of the disease. However, pathological neural processes and their resulting changes in brain network topology with disease progression are not clearly known. Methods: The current study was designed to investigate the alterations in network topology of resting state fMRI among patients in three different clinical dementia rating (CDR groups (i.e., CDR = 0.5, 1, 2 and amnestic mild cognitive impairment (aMCI and age-matched healthy subject groups. We constructed cost networks from these 5 groups and analyzed their network properties using graph theoretical measures.Results: The topological properties of AD brain networks differed in a non-monotonic, stage-specific manner. Interestingly, local and global efficiency and betweenness of the network were rather higher in the aMCI and AD (CDR 1 groups than those of prior stage groups. The number, location, and structure of rich-clubs changed dynamically as the disease progressed.Conclusions: The alterations in network topology of the brain are quite dynamic with AD progression, and these dynamic changes in network patterns should be considered meticulously for efficient therapeutic interventions of AD.

  3. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Non-Monotonic Survival of Staphylococcus aureus with Respect to Ciprofloxacin Concentration Arises from Prophage-Dependent Killing of Persisters

    Directory of Open Access Journals (Sweden)

    Elizabeth L. Sandvik

    2015-11-01

    Full Text Available Staphylococcus aureus is a notorious pathogen with a propensity to cause chronic, non-healing wounds. Bacterial persisters have been implicated in the recalcitrance of S. aureus infections, and this motivated us to examine the persistence of S. aureus to ciprofloxacin, a quinolone antibiotic. Upon treatment of exponential phase S. aureus with ciprofloxacin, we observed that survival was a non-monotonic function of ciprofloxacin concentration. Maximal killing occurred at 1 µg/mL ciprofloxacin, which corresponded to survival that was up to ~40-fold lower than that obtained with concentrations ≥ 5 µg/mL. Investigation of this phenomenon revealed that the non-monotonic response was associated with prophage induction, which facilitated killing of S. aureus persisters. Elimination of prophage induction with tetracycline was found to prevent cell lysis and persister killing. We anticipate that these findings may be useful for the design of quinolone treatments.

  5. How do people learn from negative evidence? Non-monotonic generalizations and sampling assumptions in inductive reasoning.

    Science.gov (United States)

    Voorspoels, Wouter; Navarro, Daniel J; Perfors, Amy; Ransom, Keith; Storms, Gert

    2015-09-01

    A robust finding in category-based induction tasks is for positive observations to raise the willingness to generalize to other categories while negative observations lower the willingness to generalize. This pattern is referred to as monotonic generalization. Across three experiments we find systematic non-monotonicity effects, in which negative observations raise the willingness to generalize. Experiments 1 and 2 show that this effect emerges in hierarchically structured domains when a negative observation from a different category is added to a positive observation. They also demonstrate that this is related to a specific kind of shift in the reasoner's hypothesis space. Experiment 3 shows that the effect depends on the assumptions that the reasoner makes about how inductive arguments are constructed. Non-monotonic reasoning occurs when people believe the facts were put together by a helpful communicator, but monotonicity is restored when they believe the observations were sampled randomly from the environment. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Mapping axonal density and average diameter using non-monotonic time-dependent gradient-echo MRI

    DEFF Research Database (Denmark)

    Nunes, Daniel; Cruz, Tomás L; Jespersen, Sune N

    2017-01-01

    available in the clinic, or extremely long acquisition schemes to extract information from parameter-intensive models. In this study, we suggest that simple and time-efficient multi-gradient-echo (MGE) MRI can be used to extract the axon density from susceptibility-driven non-monotonic decay in the time...... the quantitative results are compared against ground-truth histology, they seem to reflect the axonal fraction (though with a bias, as evident from Bland-Altman analysis). As well, the extra-axonal fraction can be estimated. The results suggest that our model is oversimplified, yet at the same time evidencing......-dependent signal. We show, both theoretically and with simulations, that a non-monotonic signal decay will occur for multi-compartmental microstructures – such as axons and extra-axonal spaces, which we here used in a simple model for the microstructure – and that, for axons parallel to the main magnetic field...

  7. Nonmonotonic behaviour of superconducting critical temperature of Nb/CuNi bilayers with a nanometer range of layer thickness

    International Nuclear Information System (INIS)

    Morari, R.; Antropov, E.; Socrovisciuc, A.; Prepelitsa, A.; Zdravkov, V.I.; Tagirov, L.R.; Kupriyanov, M.Yu.; Sidorenko, A.S.

    2009-01-01

    Present work reports the result of the proximity effect investigation for superconducting Nb/CuNi-bilayers with the thickness of the ferromagnetic layer (Cu x Ni 1-x ) being in the sub-nanometer range. It was found a non-monotonic behavior of the critical temperature T c , i.e. its growth with the increasing of the ferromagnetic layer thickness dF, for the series of the samples with constant thickness of Nb layer, (d Nb = const). (authors)

  8. Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

    Directory of Open Access Journals (Sweden)

    Lei Jia

    Full Text Available Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG and melting temperature change (dTm were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models.

  9. Non-monotonic probability of thermal reversal in thin-film biaxial nanomagnets with small energy barriers

    Directory of Open Access Journals (Sweden)

    N. Kani

    2017-05-01

    Full Text Available The goal of this paper is to investigate the short time-scale, thermally-induced probability of magnetization reversal for an biaxial nanomagnet that is characterized with a biaxial magnetic anisotropy. For the first time, we clearly show that for a given energy barrier of the nanomagnet, the magnetization reversal probability of an biaxial nanomagnet exhibits a non-monotonic dependence on its saturation magnetization. Specifically, there are two reasons for this non-monotonic behavior in rectangular thin-film nanomagnets that have a large perpendicular magnetic anisotropy. First, a large perpendicular anisotropy lowers the precessional period of the magnetization making it more likely to precess across the x^=0 plane if the magnetization energy exceeds the energy barrier. Second, the thermal-field torque at a particular energy increases as the magnitude of the perpendicular anisotropy increases during the magnetization precession. This non-monotonic behavior is most noticeable when analyzing the magnetization reversals on time-scales up to several tens of ns. In light of the several proposals of spintronic devices that require data retention on time-scales up to 10’s of ns, understanding the probability of magnetization reversal on the short time-scales is important. As such, the results presented in this paper will be helpful in quantifying the reliability and noise sensitivity of spintronic devices in which thermal noise is inevitably present.

  10. Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756

    Science.gov (United States)

    The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-direc...

  11. Interacting opinion and disease dynamics in multiplex networks: Discontinuous phase transition and nonmonotonic consensus times

    Science.gov (United States)

    Velásquez-Rojas, Fátima; Vazquez, Federico

    2017-05-01

    Opinion formation and disease spreading are among the most studied dynamical processes on complex networks. In real societies, it is expected that these two processes depend on and affect each other. However, little is known about the effects of opinion dynamics over disease dynamics and vice versa, since most studies treat them separately. In this work we study the dynamics of the voter model for opinion formation intertwined with that of the contact process for disease spreading, in a population of agents that interact via two types of connections, social and contact. These two interacting dynamics take place on two layers of networks, coupled through a fraction q of links present in both networks. The probability that an agent updates its state depends on both the opinion and disease states of the interacting partner. We find that the opinion dynamics has striking consequences on the statistical properties of disease spreading. The most important is that the smooth (continuous) transition from a healthy to an endemic phase observed in the contact process, as the infection probability increases beyond a threshold, becomes abrupt (discontinuous) in the two-layer system. Therefore, disregarding the effects of social dynamics on epidemics propagation may lead to a misestimation of the real magnitude of the spreading. Also, an endemic-healthy discontinuous transition is found when the coupling q overcomes a threshold value. Furthermore, we show that the disease dynamics delays the opinion consensus, leading to a consensus time that varies nonmonotonically with q in a large range of the model's parameters. A mean-field approach reveals that the coupled dynamics of opinions and disease can be approximately described by the dynamics of the voter model decoupled from that of the contact process, with effective probabilities of opinion and disease transmission.

  12. Non-Monotonic Relation Between Noise Exposure Severity and Neuronal Hyperactivity in the Auditory Midbrain

    Directory of Open Access Journals (Sweden)

    Lara Li Hesse

    2016-08-01

    Full Text Available The occurrence of tinnitus can be linked to hearing loss in the majority of cases, but there is nevertheless a large degree of unexplained heterogeneity in the relation between hearing loss and tinnitus. Part of the problem might be that hearing loss is usually quantified in terms of increased hearing thresholds, which only provides limited information about the underlying cochlear damage. Moreover, noise exposure that does not cause hearing threshold loss can still lead to hidden hearing loss (HHL, i.e. functional deafferentation of auditory nerve fibres (ANFs through loss of synaptic ribbons in inner hair cells. Whilst it is known that increased hearing thresholds can trigger increases in spontaneous neural activity in the central auditory system, i.e. a putative neural correlate of tinnitus, the central effects of HHL have not yet been investigated. Here, we exposed mice to octave-band noise at 100 and 105 dB SPL, to generate HHL and permanent increases of hearing thresholds, respectively. Deafferentation of ANFs was confirmed through measurement of auditory brainstem responses and cochlear immunohistochemistry. Acute extracellular recordings from the auditory midbrain (inferior colliculus demonstrated increases in spontaneous neuronal activity (a putative neural correlate of tinnitus in both groups. Surprisingly the increase in spontaneous activity was most pronounced in the mice with HHL, suggesting that the relation between hearing loss and neuronal hyperactivity might be more complex than currently understood. Our computational model indicated that these differences in neuronal hyperactivity could arise from different degrees of deafferentation of low-threshold ANFs in the two exposure groups.Our results demonstrate that HHL is sufficient to induce changes in central auditory processing, and they also indicate a non-monotonic relationship between cochlear damage and neuronal hyperactivity, suggesting an explanation for why tinnitus might

  13. Structurally stabilized organosilane-templated thermostable mesoporous titania.

    Science.gov (United States)

    Amoli, Vipin; Tiwari, Rashmi; Dutta, Arghya; Bhaumik, Asim; Sinha, Anil Kumar

    2014-01-13

    Structurally thermostable mesoporous anatase TiO2 (m-TiO2) nanoparticles, uniquely decorated with atomically dispersed SiO2, is reported for the first time. The inorganic Si portion of the novel organosilane template, used as a mesopores-directing agent, is found to be incorporated in the pore walls of the titania aggregates, mainly as isolated sites. This is evident by transmission electron microscopy and high-angle annular dark field scanning transmission electron microscopy, combined with electron dispersive X-ray spectroscopy. This type of unique structure provides exceptional stability to this new material against thermal collapse of the mesoporous structure, which is reflected in its high surface area (the highest known for anatase titania), even after high-temperature (550 °C) calcination. Control of crystallite size, pore diameter, and surface area is achieved by varying the molar ratios of the titanium precursor and the template during synthesis. These mesoporous materials retain their porosity and high surface area after template removal and further NaOH/HCl treatment to remove silica. We investigate their performance for dye-sensitized solar cells (DSSCs) with bilayer TiO2 electrodes, which are prepared by applying a coating of m-TiO2 onto a commercial titania (P25) film. The high surface area of the upper mesoporous layer in the P25-m-TiO2 DSSC significantly increases the dye loading ability of the photoanode. The photocurrent and fill factor for the DSSC with the bilayer TiO2 electrode are greatly improved. The large increase in photocurrent current (ca. 56%) in the P25-m-TiO2 DSSC is believed to play a significant role in achieving a remarkable increase in the photovoltaic efficiency (60%) of the device, compared to DSSCs with a monolayer of P25 as the electrode. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. A comparative molecular dynamics study on thermostability of human and chicken prion proteins

    International Nuclear Information System (INIS)

    Ji, Hong-Fang; Zhang, Hong-Yu

    2007-01-01

    To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP C and CkPrP C ), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrP C is comparable with that of CkPrP C , which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP C

  15. A Binomial Modeling Approach for Upscaling Colloid Transport Under Unfavorable Attachment Conditions: Emergent Prediction of Nonmonotonic Retention Profiles

    Science.gov (United States)

    Hilpert, Markus; Johnson, William P.

    2018-01-01

    We used a recently developed simple mathematical network model to upscale pore-scale colloid transport information determined under unfavorable attachment conditions. Classical log-linear and nonmonotonic retention profiles, both well-reported under favorable and unfavorable attachment conditions, respectively, emerged from our upscaling. The primary attribute of the network is colloid transfer between bulk pore fluid, the near-surface fluid domain (NSFD), and attachment (treated as irreversible). The network model accounts for colloid transfer to the NSFD of downgradient grains and for reentrainment to bulk pore fluid via diffusion or via expulsion at rear flow stagnation zones (RFSZs). The model describes colloid transport by a sequence of random trials in a one-dimensional (1-D) network of Happel cells, which contain a grain and a pore. Using combinatorial analysis that capitalizes on the binomial coefficient, we derived from the pore-scale information the theoretical residence time distribution of colloids in the network. The transition from log-linear to nonmonotonic retention profiles occurs when the conditions underlying classical filtration theory are not fulfilled, i.e., when an NSFD colloid population is maintained. Then, nonmonotonic retention profiles result potentially both for attached and NSFD colloids. The concentration maxima shift downgradient depending on specific parameter choice. The concentration maxima were also shown to shift downgradient temporally (with continued elution) under conditions where attachment is negligible, explaining experimentally observed downgradient transport of retained concentration maxima of adhesion-deficient bacteria. For the case of zero reentrainment, we develop closed-form, analytical expressions for the shape, and the maximum of the colloid retention profile.

  16. In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop1[OPEN

    Science.gov (United States)

    Shivhare, Devendra

    2017-01-01

    To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca. Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. PMID:28546437

  17. Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer

    DEFF Research Database (Denmark)

    Huus, Kasper; Havelund, Svend; Olsen, Helle B

    2006-01-01

    The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-stat...

  18. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

    Science.gov (United States)

    Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE...

  19. CHARACTERIZATION OF A NEW BACILLUS-STEAROTHERMOPHILUS ISOLATE - A HIGHLY THERMOSTABLE ALPHA-AMYLASE-PRODUCING STRAIN

    NARCIS (Netherlands)

    WIND, RD; BUITELAAR, RM; EGGINK, G; HUIZING, HJ; DIJKHUIZEN, L

    A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known alpha-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable alpha-amylase. The half-time of inactivation of this

  20. Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress.

    Science.gov (United States)

    Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W; Zhu, Genhai

    2007-10-01

    Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.

  1. Not so monofunctional-a case of thermostable Thermobifida fusca catalase with peroxidase activity

    NARCIS (Netherlands)

    Lončar, Nikola; Fraaije, Marco W

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and

  2. Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

    NARCIS (Netherlands)

    Winter, Remko T.; Heuts, Dominic P. H. M.; Rijpkema, Egon M. A.; van Bloois, Edwin; Wijma, Hein J.; Fraaije, Marco W.

    We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene

  3. Evaluating the value proposition for improving vaccine thermostability to increase vaccine impact in low and middle-income countries.

    Science.gov (United States)

    Karp, Christopher L; Lans, Deborah; Esparza, José; Edson, Eleanore B; Owen, Katey E; Wilson, Christopher B; Heaton, Penny M; Levine, Orin S; Rao, Raja

    2015-07-09

    The need to keep vaccines cold in the face of high ambient temperatures and unreliable access to electricity is a challenge that limits vaccine coverage in low and middle-income countries (LMICs). Greater vaccine thermostability is generally touted as the obvious solution. Despite conventional wisdom, comprehensive analysis of the value proposition for increasing vaccine thermostability has been lacking. Further, while significant investments have been made in increasing vaccine thermostability in recent years, no vaccine products have been commercialized as a result. We analyzed the value proposition for increasing vaccine thermostability, grounding the analysis in specific vaccine use cases (e.g., use in routine immunization [RI] programs, or in campaigns) and in the broader context of cold chain technology and country level supply chain system design. The results were often surprising. For example, cold chain costs actually represent a relatively small fraction of total vaccine delivery system costs. Further, there are critical, vaccine use case-specific temporal thresholds that need to be overcome for significant benefits to be reaped from increasing vaccine thermostability. We present a number of recommendations deriving from this analysis that suggest a rational path toward unlocking the value (maximizing coverage, minimizing total system costs) of increased vaccine thermostability, including: (1) the full range of thermostability of existing vaccines should be defined and included in their labels; (2) for new vaccines, thermostability goals should be addressed up-front at the level of the target product profile; (3) improving cold chain infrastructure and supply chain system design is likely to have the largest impact on total system costs and coverage in the short term-and will influence the degree of thermostability required in the future; (4) in the long term, there remains value in monitoring the emergence of disruptive technologies that could remove the

  4. Permanence of a Semi-Ratio-Dependent Predator-Prey System with Nonmonotonic Functional Response and Time Delay

    Directory of Open Access Journals (Sweden)

    Xuepeng Li

    2009-01-01

    Full Text Available Sufficient conditions for permanence of a semi-ratio-dependent predator-prey system with nonmonotonic functional response and time delay ̇1(=1([1(−11(1(−(−12(2(/(2+21(],  ̇2(=2([2(−21(2(/1(], are obtained, where 1( and 2( stand for the density of the prey and the predator, respectively, and ≠0 is a constant. (≥0 stands for the time delays due to negative feedback of the prey population.

  5. On the connection between nonmonotonic taste behavior and molecular conformation in solution: The case of rebaudioside-A

    Science.gov (United States)

    Chopade, Prashant D.; Sarma, Bipul; Santiso, Erik E.; Simpson, Jeffrey; Fry, John C.; Yurttas, Nese; Biermann, Kari L.; Chen, Jie; Trout, Bernhardt L.; Myerson, Allan S.

    2015-12-01

    The diterpene steviol glycoside, rebaudioside A, is a natural high potency non-caloric sweetener extracted from the leaves of Stevia rebaudiana. This compound shows a parabolic change in sweet taste intensity with temperature which contrasts with the general finding for other synthetic or natural sweeteners whose sweet taste increases with temperature. The nonmonotonic taste behavior was determined by sensory analysis using large taste panels. The conformational landscape of rebaudioside A was established at a range of temperatures by means of nuclear magnetic resonance and molecular dynamics simulation. The relationship between various conformations and the observed sweetness of rebaudioside A is described.

  6. On the connection between nonmonotonic taste behavior and molecular conformation in solution: The case of rebaudioside-A

    International Nuclear Information System (INIS)

    Chopade, Prashant D.; Sarma, Bipul; Santiso, Erik E.; Chen, Jie; Trout, Bernhardt L.; Myerson, Allan S.; Simpson, Jeffrey; Fry, John C.; Biermann, Kari L.; Yurttas, Nese

    2015-01-01

    The diterpene steviol glycoside, rebaudioside A, is a natural high potency non-caloric sweetener extracted from the leaves of Stevia rebaudiana. This compound shows a parabolic change in sweet taste intensity with temperature which contrasts with the general finding for other synthetic or natural sweeteners whose sweet taste increases with temperature. The nonmonotonic taste behavior was determined by sensory analysis using large taste panels. The conformational landscape of rebaudioside A was established at a range of temperatures by means of nuclear magnetic resonance and molecular dynamics simulation. The relationship between various conformations and the observed sweetness of rebaudioside A is described

  7. On the connection between nonmonotonic taste behavior and molecular conformation in solution: The case of rebaudioside-A

    Energy Technology Data Exchange (ETDEWEB)

    Chopade, Prashant D.; Sarma, Bipul; Santiso, Erik E.; Chen, Jie; Trout, Bernhardt L.; Myerson, Allan S., E-mail: myerson@mit.edu [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 66-568, Cambridge, Massachusetts 02139 (United States); Simpson, Jeffrey [Department of Chemistry Instrumentation Facility, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 18-0090, Cambridge, Massachusetts 02139 (United States); Fry, John C.; Biermann, Kari L. [Connect Consulting, 6 Hollands Field, Horsham RH123HQ (United Kingdom); Yurttas, Nese [Cargill, Inc., Global Food Technology, 2301 Crosby Road, Wayzata, Minnesota 55391 (United States)

    2015-12-28

    The diterpene steviol glycoside, rebaudioside A, is a natural high potency non-caloric sweetener extracted from the leaves of Stevia rebaudiana. This compound shows a parabolic change in sweet taste intensity with temperature which contrasts with the general finding for other synthetic or natural sweeteners whose sweet taste increases with temperature. The nonmonotonic taste behavior was determined by sensory analysis using large taste panels. The conformational landscape of rebaudioside A was established at a range of temperatures by means of nuclear magnetic resonance and molecular dynamics simulation. The relationship between various conformations and the observed sweetness of rebaudioside A is described.

  8. Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass

    Directory of Open Access Journals (Sweden)

    Aditya eBhalla

    2015-06-01

    Full Text Available AbstractEfficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylo-oligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70ºC, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70ºC, respectively. At 70ºC, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, CellicHTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70ºC. High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

  9. Multistability of neural networks with discontinuous non-monotonic piecewise linear activation functions and time-varying delays.

    Science.gov (United States)

    Nie, Xiaobing; Zheng, Wei Xing

    2015-05-01

    This paper is concerned with the problem of coexistence and dynamical behaviors of multiple equilibrium points for neural networks with discontinuous non-monotonic piecewise linear activation functions and time-varying delays. The fixed point theorem and other analytical tools are used to develop certain sufficient conditions that ensure that the n-dimensional discontinuous neural networks with time-varying delays can have at least 5(n) equilibrium points, 3(n) of which are locally stable and the others are unstable. The importance of the derived results is that it reveals that the discontinuous neural networks can have greater storage capacity than the continuous ones. Moreover, different from the existing results on multistability of neural networks with discontinuous activation functions, the 3(n) locally stable equilibrium points obtained in this paper are located in not only saturated regions, but also unsaturated regions, due to the non-monotonic structure of discontinuous activation functions. A numerical simulation study is conducted to illustrate and support the derived theoretical results. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R

    Directory of Open Access Journals (Sweden)

    Hongde An

    2015-11-01

    Conclusions: This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi.

  11. Screening of strains with the high activity and thermostability nattokinase by {sup 60}Co γ-ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Shuying, Li; Ying, Nie; Huan, Du; Xuanming, Tang [Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing (China); Zhonglin, Zhao [College of Sciences, Henan Agricultural University, Zhengzhou (China); Xin, Ma [Agricultural Information Institute, Chinese Academic of Agricultural Sciences, Beijing (China); Yan, Li [School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou (China)

    2013-06-15

    In this study, Bacillus natto was irradiated by {sup 60}Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy {sup 60}Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

  12. Screening of strains with the high activity and thermostability nattokinase by "6"0Co γ-ray irradiation

    International Nuclear Information System (INIS)

    Li Shuying; Nie Ying; Du Huan; Tang Xuanming; Zhao Zhonglin; Ma Xin; Li Yan

    2013-01-01

    In this study, Bacillus natto was irradiated by "6"0Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy "6"0Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

  13. FireProt: web server for automated design of thermostable proteins

    Science.gov (United States)

    Musil, Milos; Stourac, Jan; Brezovsky, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Martinek, Tomas

    2017-01-01

    Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot. PMID:28449074

  14. Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface

    DEFF Research Database (Denmark)

    Madsen, Jan Busk; Pakkanen, Kirsi I.; Lee, Seunghwan

    2014-01-01

    Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as charac......Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature...... as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic...

  15. First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus.

    Science.gov (United States)

    Skouri-Gargouri, Houda; Gargouri, Ali

    2008-11-01

    A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.

  16. An optimized formulation of a thermostable spray dried virus-like particles vaccine against human papillomavirus

    Science.gov (United States)

    Saboo, Sugandha; Tumban, Ebenezer; Peabody, Julianne; Wafula, Denis; Peabody, David S.; Chackerian, Bryce; Muttil, Pavan

    2016-01-01

    Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP) based vaccine for Human Papillomavirus (HPV) infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multi-component excipient system and by optimizing the spray drying parameters using a half-factorial design approach. Dry powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry powder VLPs that were stored at 37°C for more than a year elicited high anti-L2 IgG antibody titers. Spray dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where ~84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage. PMID:27019231

  17. MOLECULAR CLONING AND CHARACTERIZATION OF NOVEL THERMOSTABLE LIPASE FROM SHEWANELLA PUTREFACIENS AND USING ENZYMATIC BIODIESEL PRODUCTION

    Directory of Open Access Journals (Sweden)

    Fahri Akbas

    2015-02-01

    Full Text Available A novel thermostable lipase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized and used in biodiesel production. Enzyme characterization was carried out by enzyme assay, SDS-PAGE and other biochemical reactions. The recombinant lipase was found to have a molecular mass of 29 kDa and exhibited lipase activity when Tween 80 was used as the substrate. The purified enzyme showed maximum activity at pH 5.0 and at 80°C. The recombinant lipase was used for the transesterification of canola oil and waste oil. The enzyme retains 50% of its activity at 90°C for 30 minutes. It is also able to retain 20% of its activity even at 100 °C for 20 minutes. These properties of the obtained new recombinant thermostable lipase make it promising as a biocatalyst for industrial processes.

  18. In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

    Science.gov (United States)

    Shivhare, Devendra; Mueller-Cajar, Oliver

    2017-07-01

    To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice ( Oryza sativa ) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. © 2017 American Society of Plant Biologists. All Rights Reserved.

  19. Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

    Science.gov (United States)

    Lee, Yeongjoon; Kwak, Chulhee; Jeong, Ki-Woong; Durai, Prasannavenkatesh; Ryu, Kyoung-Seok; Kim, Eun-Hee; Cheong, Chaejoon; Ahn, Hee-Chul; Kim, Hak Jun; Kim, Yangmee

    2018-05-18

    Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five β-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10 -7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s -1 ), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

  20. Design and Testing of a Thermostable Platform for Multimerization of Single Domain Antibodies

    Science.gov (United States)

    2012-08-01

    H.J. Properties , production, and applications of camelid single domain antibody fragments. Appl. Microbiol. Biot. 2007, 77, 13‒22. 2. Goldman...Conway, J.; Sherwood, L.J.; Fech, M.; Vo, B.; Liu, J.L.; Hayhurst, A. Thermostable llama single domain antibodies for detection of Botulinum A...antiparallel coiled-coil inserted. J. Mol. Bio. 2001, 306, 25‒35. 9. Liu, J.L.; Anderson, G.P.; Goldman, E.R. Isolation of anti- toxin single domain

  1. A stable route to high-{beta}{sub p} plasmas with non-monotonic q-profiles

    Energy Technology Data Exchange (ETDEWEB)

    Soeldner, F X; Baranov, Y; Bhatnagar, V P; Bickley, A J; Challis, C D; Fischer, B; Gormezano, C; Huysmans, G T.A.; Kerner, W; Rimini, F; Sips, A C.C.; Springmann, R; Taroni, A [Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking; Goedbloed, J P; Holties, H A [Institute for Plasmas Physics, Nieuwegein (Netherlands); Parail, V V; Pereverzev, G V [Kurchatov Institute of Atomic Energy, Moscow (Russian Federation)

    1994-07-01

    Steady-state operation of tokamak reactors seems feasible in so-called Advanced Scenarios with high bootstrap current in high-beta{sub p} operation. The stabilization of such discharges with noninductive profile control will be attempted on JET in pursuit of previous high bootstrap current studies. Results of modelling studies of full noninductive current drive scenarios in JET and ITER are presented. Fast Waves (FW), Lower Hybrid (LH) Waves and Neutral Beam Injection (NBI) are used for heating and current drive, alternatively or in combination. A stable route to nonmonotonic q-profiles has been found with a specific ramp-up scenario which combines LH-current drive (LHCD) and a fast Ohmic ramp-up. A hollow current profile with deep shear reversal over the whole central region is thereby formed in an early low-beta phase and frozen in by additional heating. (authors). 5 refs., 4 figs.

  2. Complex, non-monotonic dose-response curves with multiple maxima: Do we (ever) sample densely enough?

    Science.gov (United States)

    Cvrčková, Fatima; Luštinec, Jiří; Žárský, Viktor

    2015-01-01

    We usually expect the dose-response curves of biological responses to quantifiable stimuli to be simple, either monotonic or exhibiting a single maximum or minimum. Deviations are often viewed as experimental noise. However, detailed measurements in plant primary tissue cultures (stem pith explants of kale and tobacco) exposed to varying doses of sucrose, cytokinins (BA or kinetin) or auxins (IAA or NAA) revealed that growth and several biochemical parameters exhibit multiple reproducible, statistically significant maxima over a wide range of exogenous substance concentrations. This results in complex, non-monotonic dose-response curves, reminiscent of previous reports of analogous observations in both metazoan and plant systems responding to diverse pharmacological treatments. These findings suggest the existence of a hitherto neglected class of biological phenomena resulting in dose-response curves exhibiting periodic patterns of maxima and minima, whose causes remain so far uncharacterized, partly due to insufficient sampling frequency used in many studies.

  3. Laser induced non-monotonic degradation in short-circuit current of triple-junction solar cells

    Science.gov (United States)

    Dou, Peng-Cheng; Feng, Guo-Bin; Zhang, Jian-Min; Song, Ming-Ying; Zhang, Zhen; Li, Yun-Peng; Shi, Yu-Bin

    2018-06-01

    In order to study the continuous wave (CW) laser radiation effects and mechanism of GaInP/GaAs/Ge triple-junction solar cells (TJSCs), 1-on-1 mode irradiation experiments were carried out. It was found that the post-irradiation short circuit current (ISC) of the TJSCs initially decreased and then increased with increasing of irradiation laser power intensity. To explain this phenomenon, a theoretical model had been established and then verified by post-damage tests and equivalent circuit simulations. Conclusion was drawn that laser induced alterations in the surface reflection and shunt resistance were the main causes for the observed non-monotonic decrease in the ISC of the TJSCs.

  4. Investigation on de-trapping mechanisms related to non-monotonic kink pattern in GaN HEMT devices

    Directory of Open Access Journals (Sweden)

    Chandan Sharma

    2017-08-01

    Full Text Available This article reports an experimental approach to analyze the kink effect phenomenon which is usually observed during the GaN high electron mobility transistor (HEMT operation. De-trapping of charge carriers is one of the prominent reasons behind the kink effect. The commonly observed non-monotonic behavior of kink pattern is analyzed under two different device operating conditions and it is found that two different de-trapping mechanisms are responsible for a particular kink behavior. These different de-trapping mechanisms are investigated through a time delay analysis which shows the presence of traps with different time constants. Further voltage sweep and temperature analysis corroborates the finding that different de-trapping mechanisms play a role in kink behavior under different device operating conditions.

  5. Investigation on de-trapping mechanisms related to non-monotonic kink pattern in GaN HEMT devices

    Science.gov (United States)

    Sharma, Chandan; Laishram, Robert; Amit, Rawal, Dipendra Singh; Vinayak, Seema; Singh, Rajendra

    2017-08-01

    This article reports an experimental approach to analyze the kink effect phenomenon which is usually observed during the GaN high electron mobility transistor (HEMT) operation. De-trapping of charge carriers is one of the prominent reasons behind the kink effect. The commonly observed non-monotonic behavior of kink pattern is analyzed under two different device operating conditions and it is found that two different de-trapping mechanisms are responsible for a particular kink behavior. These different de-trapping mechanisms are investigated through a time delay analysis which shows the presence of traps with different time constants. Further voltage sweep and temperature analysis corroborates the finding that different de-trapping mechanisms play a role in kink behavior under different device operating conditions.

  6. Search for scalar-tensor gravity theories with a non-monotonic time evolution of the speed-up factor

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, A [Dept Fisica, Universidad de Murcia, E30071-Murcia (Spain); Serna, A [Dept Fisica, Computacion y Comunicaciones, Universidad Miguel Hernandez, E03202-Elche (Spain); Alimi, J-M [Lab. de l' Univers et de ses Theories (LUTH, CNRS FRE2462), Observatoire de Paris-Meudon, F92195-Meudon (France)

    2002-08-21

    We present a method to detect, in the framework of scalar-tensor gravity theories, the existence of stationary points in the time evolution of the speed-up factor. An attractive aspect of this method is that, once the particular scalar-tensor theory has been specified, the stationary points are found through a simple algebraic equation which does not contain any integration. By applying this method to the three classes of scalar-tensor theories defined by Barrow and Parsons, we have found several new cosmological models with a non-monotonic evolution of the speed-up factor. The physical interest of these models is that, as previously shown by Serna and Alimi, they predict the observed primordial abundance of light elements for a very wide range of baryon density. These models are then consistent with recent CMB and Lyman-{alpha} estimates of the baryon content of the universe.

  7. Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

    Science.gov (United States)

    Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

    2015-09-01

    Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

  8. Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05

    Directory of Open Access Journals (Sweden)

    Nur Syazwani Mohtar

    2016-12-01

    Full Text Available The glycogen branching enzyme (EC 2.4.1.18, which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

  9. 'THERMOST' for analysing thermo-structural behaviour of LWR fuel rods under PCI conditions

    International Nuclear Information System (INIS)

    Nuno, H.; Ogawa, S.; Kobayashi, H.

    1983-01-01

    As a method for evaluating fuel rod performance under power ramping or load following operations, the combined FROST/ THERMOST system has been developed and brought into practical use. FROST was presented at the IAEA Blackpool Meeting in 1978, and THERMOST is the subject of this paper. The major purpose of THERMOST is to analyse very detailed thermal and structural fuel behaviour in a rather localised part of the fuel rod whereas FROST deals with whole rod general performance. The code handles two-dimensional thermal and structural analyses simultaneously by using a finite element method, in axial section or in lateral section. It consists of a fundamental FEM system of generalised constitution, and a surrounding subroutine system which characterises fuel behaviour, such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer element (six kinds), and structural analysis by axisymmetric ring and lateral plane element (six kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping conditions is presented with some in-pile test data. (author)

  10. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    Science.gov (United States)

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  11. Expression of Acidothermus cellulolyticus thermostable cellulases in tobacco and rice plants

    Directory of Open Access Journals (Sweden)

    Xiran Jiang

    2017-01-01

    Full Text Available The production of cellulases in plants is an economical method for the conversion of lignocellulosic biomass into fuels. Herein we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (E1 and exoglucanase (Gux1, in tobacco and rice. To evaluate the expression of these recombinant cellulases, we expressed the full-length E1, the catalytic domains of E1 (E1cd and Gux1 (Gux1cd, as well as an E1–Gux1cd fusion enzyme in various subcellular compartments. In the case of tobacco, transgenic plants that expressed apoplast-localized E1 showed the highest level of activity, about three times higher than those that expressed the cytosolic E1. In the case of rice, the level of cellulase-specific activity in the transgenic plants ranged from 11 to 20 nmol 4-methylumbelliferone min−1 mg−1 total soluble protein. The recombinant cellulases exhibited good thermostability below 70 °C. Furthermore, transgenic rice leaves that were stored at room temperature for a month lost about 20% of the initial cellulase activity. Taken together, the results suggested that heterologous expression of thermostable cellulases in plants may be a viable option for biomass conversion.

  12. Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

    Science.gov (United States)

    Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

    2016-10-01

    To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

  13. Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

    Science.gov (United States)

    Reddy, Chinreddy Subramanyam; Achary, V Mohan Murali; Manna, Mrinalini; Singh, Jitender; Kaul, Tanushri; Reddy, Malireddy K

    2015-03-01

    The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

  14. Effect of Pulse Polarity on Thresholds and on Non-monotonic Loudness Growth in Cochlear Implant Users.

    Science.gov (United States)

    Macherey, Olivier; Carlyon, Robert P; Chatron, Jacques; Roman, Stéphane

    2017-06-01

    Most cochlear implants (CIs) activate their electrodes non-simultaneously in order to eliminate electrical field interactions. However, the membrane of auditory nerve fibers needs time to return to its resting state, causing the probability of firing to a pulse to be affected by previous pulses. Here, we provide new evidence on the effect of pulse polarity and current level on these interactions. In experiment 1, detection thresholds and most comfortable levels (MCLs) were measured in CI users for 100-Hz pulse trains consisting of two consecutive biphasic pulses of the same or of opposite polarity. All combinations of polarities were studied: anodic-cathodic-anodic-cathodic (ACAC), CACA, ACCA, and CAAC. Thresholds were lower when the adjacent phases of the two pulses had the same polarity (ACCA and CAAC) than when they were different (ACAC and CACA). Some subjects showed a lower threshold for ACCA than for CAAC while others showed the opposite trend demonstrating that polarity sensitivity at threshold is genuine and subject- or electrode-dependent. In contrast, anodic (CAAC) pulses always showed a lower MCL than cathodic (ACCA) pulses, confirming previous reports. In experiments 2 and 3, the subjects compared the loudness of several pulse trains differing in current level separately for ACCA and CAAC. For 40 % of the electrodes tested, loudness grew non-monotonically as a function of current level for ACCA but never for CAAC. This finding may relate to a conduction block of the action potentials along the fibers induced by a strong hyperpolarization of their central processes. Further analysis showed that the electrodes showing a lower threshold for ACCA than for CAAC were more likely to yield a non-monotonic loudness growth. It is proposed that polarity sensitivity at threshold reflects the local neural health and that anodic asymmetric pulses should preferably be used to convey sound information while avoiding abnormal loudness percepts.

  15. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    Science.gov (United States)

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  16. A New Result Concerning the Solvability of a Class of General Systems of Variational Equations with Nonmonotone Operators: Applications to Dirichlet and Neumann Nonlinear Problems

    Directory of Open Access Journals (Sweden)

    Luisa Toscano

    2016-01-01

    Full Text Available A new result of solvability for a wide class of systems of variational equations depending on parameters and governed by nonmonotone operators is found in a Banach real and reflexive space with applications to Dirichlet and Neumann problems related to nonlinear elliptic systems.

  17. Recovery of avirulent, thermostable Newcastle disease virus strain NDV4-C from cloned cDNA and stable expression of an inserted foreign gene

    NARCIS (Netherlands)

    Zhang, X.; Liu, H.; Liu, P.; Peeters, B.P.H.; Zhao, C.; Kong, X.

    2013-01-01

    A reverse genetics system for thermostable Newcastle disease virus (NDV) is not currently available. In this study, we developed a reverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery of NDV4-C was achieved by using either T7 RNA polymerase or cellular RNA

  18. A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

    Science.gov (United States)

    Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

    2018-01-01

    We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement.

    Science.gov (United States)

    Farnoosh, Gholamreza; Khajeh, Khosro; Latifi, Ali Mohammad; Aghamollaei, Hossein

    2016-12-01

    The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, delta G inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

  20. Protein thermostability prediction within homologous families using temperature-dependent statistical potentials.

    Directory of Open Access Journals (Sweden)

    Fabrizio Pucci

    Full Text Available The ability to rationally modify targeted physical and biological features of a protein of interest holds promise in numerous academic and industrial applications and paves the way towards de novo protein design. In particular, bioprocesses that utilize the remarkable properties of enzymes would often benefit from mutants that remain active at temperatures that are either higher or lower than the physiological temperature, while maintaining the biological activity. Many in silico methods have been developed in recent years for predicting the thermodynamic stability of mutant proteins, but very few have focused on thermostability. To bridge this gap, we developed an algorithm for predicting the best descriptor of thermostability, namely the melting temperature Tm, from the protein's sequence and structure. Our method is applicable when the Tm of proteins homologous to the target protein are known. It is based on the design of several temperature-dependent statistical potentials, derived from datasets consisting of either mesostable or thermostable proteins. Linear combinations of these potentials have been shown to yield an estimation of the protein folding free energies at low and high temperatures, and the difference of these energies, a prediction of the melting temperature. This particular construction, that distinguishes between the interactions that contribute more than others to the stability at high temperatures and those that are more stabilizing at low T, gives better performances compared to the standard approach based on T-independent potentials which predict the thermal resistance from the thermodynamic stability. Our method has been tested on 45 proteins of known Tm that belong to 11 homologous families. The standard deviation between experimental and predicted Tm's is equal to 13.6°C in cross validation, and decreases to 8.3°C if the 6 worst predicted proteins are excluded. Possible extensions of our approach are discussed.

  1. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

    Science.gov (United States)

    Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

    2016-11-01

    Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Identification of thermostable β-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger

    DEFF Research Database (Denmark)

    Pedersen, Mads; Lauritzen, Henrik Klitgaard; Frisvad, Jens Christian

    2007-01-01

    Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable beta......-xylosidases. The beta-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75 degrees C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60 degrees C. At 75 degrees C, these values were 38 and 44%, respectively. Whereas A. niger...

  3. Thermal Degradation Mechanism of a Thermostable Polyester Stabilized with an Open-Cage Oligomeric Silsesquioxane

    Directory of Open Access Journals (Sweden)

    Yolanda Bautista

    2017-12-01

    Full Text Available A polyester composite was prepared through the polymerization of an unsaturated ester resin with styrene and an open-cage oligomeric silsesquioxane with methacrylate groups. The effect of the open-cage oligomeric silsesquioxane on the thermal stability of the thermostable polyester was studied using both thermogravimetric analysis and differential thermal analysis. The results showed that the methacryl oligomeric silsesquioxane improved the thermal stability of the polyester. The decomposition mechanism of the polyester/oligomer silsesquioxane composite was proposed by Fourier transform infrared spectroscopy (FTIR analysis of the volatiles.

  4. Methods of hydrolyzing a cellulose using halophilic, thermostable and ionic liquids tolerant cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2018-01-09

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  5. Evaluations on power ramp data of PWR fuels by FROST and THERMOST codes

    International Nuclear Information System (INIS)

    Murai, K.; Ogawa, S.; Nuno, H.; Kondo, Y.

    1987-01-01

    An evaluation is presented of power ramp data of Mitsubishi's PWR fuel rods tested in R-2, Studsvik, which was analysed by FROST and THERMOST codes. The analyses give good predictions for measured diameter changes and on-power rod elongations. The work indicates that FROST is capable of analysing both radial and axial pellet-cladding mechanism interaction (PCMI) appropriately, and that predicted states of PCMI (i.e. stress and strain which cannot be measured directly) are considered to be reliable. The ramp data used in the present analyses were obtained in two joint programmes with five Japanese PWR utilities (KEPCO, KYEPCO, SEPCO, HEPCO, and JAPCO). (UK)

  6. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    -silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified...

  7. Non-monotonic piezoresistive behaviour of graphene nanoplatelet (GNP-polymer composite flexible films prepared by solvent casting

    Directory of Open Access Journals (Sweden)

    S. Makireddi

    2017-07-01

    Full Text Available Graphene-polymer nanocomposite films show good piezoresistive behaviour and it is reported that the sensitivity increases either with the increased sheet resistance or decreased number density of the graphene fillers. A little is known about this behaviour near the percolation region. In this study, graphene nanoplatelet (GNP/poly (methyl methacrylate (PMMA flexible films are fabricated via solution casting process at varying weight percent of GNP. Electrical and piezoresistive behaviour of these films is studied as a function of GNP concentration. Piezoresistive strain sensitivity of the films is measured by affixing the film to an aluminium specimen which is subjected to monotonic uniaxial tensile load. The change in resistance of the film with strain is monitored using a four probe. An electrical percolation threshold at 3 weight percent of GNP is observed. We report non-monotonic piezoresistive behaviour of these films as a function GNP concentration. We observe an increase in gauge factor (GF with unstrained resistance of the films up to a critical resistance corresponding to percolation threshold. Beyond this limit the GF decreases with unstrained resistance.

  8. Marangoni instability in a thin film heated from below: Effect of nonmonotonic dependence of surface tension on temperature

    Science.gov (United States)

    Sarma, Rajkumar; Mondal, Pranab Kumar

    2018-04-01

    We investigate Marangoni instability in a thin liquid film resting on a substrate of low thermal conductivity and separated from the surrounding gas phase by a deformable free surface. Considering a nonmonotonic variation of surface tension with temperature, here we analytically derive the neutral stability curve for the monotonic and oscillatory modes of instability (for both the long-wave and short-wave perturbations) under the framework of linear stability analysis. For the long-wave instability, we derive a set of amplitude equations using the scaling k ˜(Bi) 1 /2 , where k is the wave number and Bi is the Biot number. Through this investigation, we demonstrate that for such a fluid layer upon heating from below, both monotonic and oscillatory instability can appear for a certain range of the dimensionless parameters, viz., Biot number (Bi ) , Galileo number (Ga ) , and inverse capillary number (Σ ) . Moreover, we unveil, through this study, the influential role of the above-mentioned parameters on the stability of the system and identify the critical values of these parameters above which instability initiates in the liquid layer.

  9. Problems of increasing of thermostability of highly permeable Ni-Zn ferrites and relative materials for telecommunications

    Energy Technology Data Exchange (ETDEWEB)

    Gonchar, A. E-mail: letyuk@mail.ru; Andreev, V.; Letyuk, L.; Shishkanov, A.; Maiorov, V

    2003-01-01

    The work considers ways of increasing of thermostability of ferrites of the basic systems NiO-ZnO-Fe{sub 2}O{sub 3} and MgO-ZnO-Fe{sub 2}O{sub 3} and relative materials for telecommunication. Sufficient results in increasing of the thermostability were achieved by doping Cu ions and controlling rejection of Fe{sub 2}O{sub 3} content from equimolar composition. These results allow to increase the Curie temperature to 130-140 deg. C for Ni-Zn ferrites with initial permeability 2000.

  10. Raman D-band in the irradiated graphene: Origin of the non-monotonous dependence of its intensity with defect concentration

    International Nuclear Information System (INIS)

    Codorniu Pujals, Daniel

    2013-01-01

    Raman spectroscopy is one of the most used experimental techniques in studying irradiated carbon nanostructures, in particular graphene, due to its high sensibility to the presence of defects in the crystalline lattice. Special attention has been given to the variation of the intensity of the Raman D-band of graphene with the concentration of defects produced by irradiation. Nowadays, there are enough experimental evidences about the non-monotonous character of that dependence, but the explanation of this behavior is still controversial. In the present work we developed a simplified mathematical model to obtain a functional relationship between these two magnitudes and showed that the non-monotonous dependence is intrinsic to the nature of the D-band and that it is not necessarily linked to amorphization processes. The obtained functional dependence was used to fit experimental data taken from other authors. The determination coefficient of the fitting was 0.96.

  11. Cytotoxicity of binary mixtures of human pharmaceuticals in a fish cell line: approaches for non-monotonic concentration-response relationships.

    Science.gov (United States)

    Bain, Peter A; Kumar, Anupama

    2014-08-01

    Predicting the effects of mixtures of environmental micropollutants is a priority research area. In this study, the cytotoxicity of ten pharmaceuticals to the rainbow trout cell line RTG-2 was determined using the neutral red uptake assay. Fluoxetine (FL), propranolol (PPN), and diclofenac (DCF) were selected for further study as binary mixtures. Biphasic concentration-response relationships were observed in cells exposed to FL and PPN. In the case of PPN, microscopic examination revealed lysosomal swelling indicative of direct uptake and accumulation of the compound. Three equations describing non-monotonic concentration-response relationships were evaluated and one was found to consistently provide more accurate estimates of the median and 10% effect concentrations compared with a sigmoidal concentration-response model. Predictive modeling of the effects of binary mixtures of FL, PPN, and DCF was undertaken using an implementation of the concentration addition (CA) conceptual model incorporating non-monotonic concentration-response relationships. The cytotoxicity of the all three binary combinations could be adequately predicted using CA, suggesting that the toxic mode of action in RTG-2 cells is unrelated to the therapeutic mode of action of these compounds. The approach presented here is widely applicable to the study of mixture toxicity in cases where non-monotonic concentration-response relationships are observed. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  12. The ''THERMOST'' for analysing thermo-structural behaviour of LWR fuel rod under PCI conditions

    International Nuclear Information System (INIS)

    Nuno, H.; Ogawa, S.; Kobayashi, H.

    1983-01-01

    As one of the methods for evaluating the fuel rod performances under power ramping or load following operations, the combined ''FROST'' and ''THERMOST'' system has been developed and being brought into practical use. The former had already been presented at Blackpool Meeting in 1978, and the latter is going to be presented in this paper. The major purpose of the THERMOST is to analyse very detailed thermal and structural fuel behaviours in a rather localized part of fuel rod whereas the FROST deals with whole-rod-wide general performances. The code handles 2-dimensional thermal and structural analyses simultaneously by using finite element method, in axial section wide or in lateral section wide. It consists of a fundamental FEM system of generalized constitution and its surrounding subroutine system which characterizes fuel behaviours such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer elements (6 kinds) and structural analysis by axisymmetric ring and lateral plane elements (6 kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping condition is presented with some inpile test data. (author)

  13. Molecular Dynamics Simulation of Barnase: Contribution of Noncovalent Intramolecular Interaction to Thermostability

    Directory of Open Access Journals (Sweden)

    Zhiguo Chen

    2013-01-01

    Full Text Available Bacillus amyloliquefaciens ribonuclease Barnase (RNase Ba is a 12 kD (kilodalton small extracellular ribonuclease. It has broad application prospects in agriculture, clinical medicine, pharmaceutical, and so forth. In this work, the thermal stability of Barnase has been studied using molecular dynamics simulation at different temperatures. The present study focuses on the contribution of noncovalent intramolecular interaction to protein stability and how they affect the thermal stability of the enzyme. Profiles of root mean square deviation and root mean square fluctuation identify thermostable and thermosensitive regions of Barnase. Analyses of trajectories in terms of secondary structure content, intramolecular hydrogen bonds and salt bridge interactions indicate distinct differences in different temperature simulations. In the simulations, Four three-member salt bridge networks (Asp8-Arg110-Asp12, Arg83-Asp75-Arg87, Lys66-Asp93-Arg69, and Asp54-Lys27-Glu73 have been identified as critical salt bridges for thermostability which are maintained stably at higher temperature enhancing stability of three hydrophobic cores. The study may help enlighten our knowledge of protein structural properties, noncovalent interactions which can stabilize secondary peptide structures or promote folding, and also help understand their actions better. Such an understanding is required for designing efficient enzymes with characteristics for particular applications at desired working temperatures.

  14. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  15. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    International Nuclear Information System (INIS)

    Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

    2006-01-01

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

  16. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

  17. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

    International Nuclear Information System (INIS)

    Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

    2012-01-01

    A thermostable penicillin G acylase from A. faecalis has been crystallized in two space groups: C222 1 and P4 1 2 1 2. X-ray diffraction data were collected to 3.3 and 3.5 Å resolution, respectively. The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222 1 , with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P4 1 2 1 2, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme

  18. Thermostable 𝜶-Amylase Activity from Thermophilic Bacteria Isolated from Bora Hot Spring, Central Sulawesi

    Science.gov (United States)

    Gazali, F. M.; Suwastika, I. N.

    2018-03-01

    α-Amylase is one of the most important enzyme in biotechnology field, especially in industrial application. Thermostability of α-Amylase produced by thermophilic bacteria improves industrial process of starch degradation in starch industry. The present study were concerned to the characterization of α-Amylase activity from indigenous thermophilic bacteria isolated from Bora hot spring, Central Sulawesi. There were 18 isolates which had successfully isolated from 90°C sediment samples of Bora hot spring and 13 of them showed amylolytic activity. The α-Amylase activity was measured qualitatively at starch agar and quantitatively based on DNS (3,5-Dinitrosalicylic acid) methods, using maltose as standard solution. Two isolates (out of 13 amylolytic bacteria), BR 002 and BR 015 showed amylolytic index of 0.8 mm and 0.5 mm respectively, after being incubated at 55°C in the 0.002% Starch Agar Medium. The α-Amylase activity was further characterized quantitatively which includes the optimum condition of pH and temperature of α-Amylase crude enzyme from each isolate. To our knowledge, this is the first report on isolation and characterization of a thermostable α-Amylase from thermophilic bacteria isolated from Central Sulawesi particularly from Bora hot spring.

  19. Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability

    International Nuclear Information System (INIS)

    Bhuiya, Mohammad Wadud; Suryadi, Jimmy; Zhou, Zholi; Brown, Bernard Andrew II

    2013-01-01

    The crystal structure of A. pernix L7Ae is reported, providing insight into the extreme thermostability of this protein. Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383 K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56 Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the mesophilic counterpart mammalian 15.5 kD protein. The primary stabilizing feature in the Ap L7Ae protein appears to be the large number of ion pairs and extensive ion-pair network that connects secondary-structural elements. To our knowledge, Ap L7Ae is among the most thermostable single-domain monomeric proteins presently observed

  20. Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

    Science.gov (United States)

    Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

    2014-04-10

    Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    Science.gov (United States)

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  2. Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

    Science.gov (United States)

    Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

    2017-05-01

    To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

  3. Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573.

    Science.gov (United States)

    Boukhris, Ines; Farhat-Khemakhem, Ameny; Blibech, Monia; Bouchaala, Kameleddine; Chouayekh, Hichem

    2015-09-01

    The extracellular phytase produced by the Bacillus amyloliquefaciens US573 strain, isolated from geothermal soil located in Southern Tunisia was purified and characterized. This calcium-dependent and bile-stable enzyme (PHY US573) was optimally active at pH 7.5 and 70 °C. It showed a good stability at pH ranging from 4 to 10, and especially, an exceptional thermostability as it recovered 50 and 62% of activity after heating for 10 min at 100 and 90 °C, respectively. In addition, PHY US573 was found to be extremely salt-tolerant since it preserved 80 and 95% of activity in the presence of 20 g/l of NaCl and LiCl, respectively. The gene corresponding to PHY US573 was cloned. It encodes a 383 amino acids polypeptide exhibiting 99% identity with the highly thermostable phytases from Bacillus sp. MD2 and B. amyloliquefaciens DS11 (3 and 5 residues difference, respectively), suggesting the existence of common molecular determinants responsible for their remarkable heat stability. Overall, our findings illustrated that in addition to its high potential for application in feed industry, the salt tolerance of the PHY US573 phytase, may represent an exciting new avenue for improvement of phosphorus-use efficiency of salt-tolerant plants in soils with high salt and phytate content. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Enhanced Thermostability of Arabidopsis Rubisco Activase Improves Photosynthesis and Growth Rates under Moderate Heat Stress[OA

    Science.gov (United States)

    Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M.; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W.; Zhu, Genhai

    2007-01-01

    Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Δrca) line. In a long-term growth test at either constant 26°C or daily 4-h 30°C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions. PMID:17933901

  5. Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.

    Science.gov (United States)

    Chen, Ana; Li, Yamei; Nie, Jianqi; McNeil, Brian; Jeffrey, Laura; Yang, Yankun; Bai, Zhonghu

    2015-10-01

    Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. INCREASING THE THERMOSTABILITY OF THE NEUTRAL PROTEINASE OF BACILLUS-STEAROTHERMOPHILUS BY IMPROVEMENT OF INTERNAL HYDROGEN-BONDING

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERZEE, [No Value; VANDENBURG, B; VENEMA, G

    1992-01-01

    In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of

  7. Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2015-05-01

    Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

  8. Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution.

    Science.gov (United States)

    Wu, Dongxia; Ran, Tinting; Wang, Weiwu; Xu, Dongqing

    2016-01-01

    Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated from Serratia sp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin from Serratia sp. FS14 was solved to a crystallographic R factor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189-198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

  9. Virus-like particle nanoreactors: programmed en capsulation of the thermostable CelB glycosidase inside the P22 capsid

    NARCIS (Netherlands)

    Patterson, D.P.; Schwarz, B.; El-Boubbou, K.; Oost, van der J.; Prevelige, P.E.; Douglas, T.

    2012-01-01

    Self-assembling biological systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically

  10. Time-dependent, non-monotonic response of warm convective cloud fields to changes in aerosol loading

    Directory of Open Access Journals (Sweden)

    G. Dagan

    2017-06-01

    Full Text Available Large eddy simulations (LESs with bin microphysics are used here to study cloud fields' sensitivity to changes in aerosol loading and the time evolution of this response. Similarly to the known response of a single cloud, we show that the mean field properties change in a non-monotonic trend, with an optimum aerosol concentration for which the field reaches its maximal water mass or rain yield. This trend is a result of competition between processes that encourage cloud development versus those that suppress it. However, another layer of complexity is added when considering clouds' impact on the field's thermodynamic properties and how this is dependent on aerosol loading. Under polluted conditions, rain is suppressed and the non-precipitating clouds act to increase atmospheric instability. This results in warming of the lower part of the cloudy layer (in which there is net condensation and cooling of the upper part (net evaporation. Evaporation at the upper part of the cloudy layer in the polluted simulations raises humidity at these levels and thus amplifies the development of the next generation of clouds (preconditioning effect. On the other hand, under clean conditions, the precipitating clouds drive net warming of the cloudy layer and net cooling of the sub-cloud layer due to rain evaporation. These two effects act to stabilize the atmospheric boundary layer with time (consumption of the instability. The evolution of the field's thermodynamic properties affects the cloud properties in return, as shown by the migration of the optimal aerosol concentration toward higher values.

  11. Canonical single field slow-roll inflation with a non-monotonic tensor-to-scalar ratio

    Energy Technology Data Exchange (ETDEWEB)

    Germán, Gabriel [Rudolf Peierls Centre for Theoretical Physics, University of Oxford, 1 Keble Road, Oxford, OX1 3NP (United Kingdom); Herrera-Aguilar, Alfredo [Instituto de Física, Benemérita Universidad Autónoma de Puebla, Apdo. postal J-48, CP 72570, Puebla, Pue., México (Mexico); Hidalgo, Juan Carlos [Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Apdo. postal 48-3, 62251 Cuernavaca, Morelos, México (Mexico); Sussman, Roberto A., E-mail: gabriel@fis.unam.mx, E-mail: aherrera@ifuap.buap.mx, E-mail: hidalgo@fis.unam.mx, E-mail: sussman@nucleares.unam.mx [Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Apdo. postal 70-543, 04510 México D. F., México (Mexico)

    2016-05-01

    We take a pragmatic, model independent approach to single field slow-roll canonical inflation by imposing conditions, not on the potential, but on the slow-roll parameter ε(φ) and its derivatives ε'(φ) and ε''(φ), thereby extracting general conditions on the tensor-to-scalar ratio r and the running n {sub sk} at φ {sub H} where the perturbations are produced, some 50–60 e -folds before the end of inflation. We find quite generally that for models where ε(φ) develops a maximum, a relatively large r is most likely accompanied by a positive running while a negligible tensor-to-scalar ratio implies negative running. The definitive answer, however, is given in terms of the slow-roll parameter ξ{sub 2}(φ). To accommodate a large tensor-to-scalar ratio that meets the limiting values allowed by the Planck data, we study a non-monotonic ε(φ) decreasing during most part of inflation. Since at φ {sub H} the slow-roll parameter ε(φ) is increasing, we thus require that ε(φ) develops a maximum for φ > φ {sub H} after which ε(φ) decrease to small values where most e -folds are produced. The end of inflation might occur trough a hybrid mechanism and a small field excursion Δφ {sub e} ≡ |φ {sub H} −φ {sub e} | is obtained with a sufficiently thin profile for ε(φ) which, however, should not conflict with the second slow-roll parameter η(φ). As a consequence of this analysis we find bounds for Δφ {sub e} , r {sub H} and for the scalar spectral index n {sub sH} . Finally we provide examples where these considerations are explicitly realised.

  12. Identification and characterization of a novel thermostable pyrethroid-hydrolyzing enzyme isolated through metagenomic approach

    Directory of Open Access Journals (Sweden)

    Fan Xinjiong

    2012-03-01

    Full Text Available Abstract Background Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present. Results In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3 in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (772.9 U/mg. The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate. Conclusion This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most

  13. Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

    Science.gov (United States)

    Bu, Yifan; Cui, Yinglu; Peng, Ying; Hu, Meirong; Tian, Yu'e; Tao, Yong; Wu, Bian

    2018-04-01

    Xylanases, which cleave the β-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

  14. Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus.

    Science.gov (United States)

    Raghunath, Pendru

    2014-01-01

    Vibrio parahaemolyticus is the leading cause of seafood borne bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. However, not all strains of V. parahaemolyticus are pathogenic. The thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. However, about 10% of clinical strains do not contain tdh and/or trh. Environmental isolates of V. parahaemolyticus lacking tdh and/or trh are also highly cytotoxic to human gastrointestinal cells. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist. This mini review aims at discussing the possible roles of tdh and trh genes in clinical and environmental isolates of V. parahaemolyticus.

  15. Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

    Science.gov (United States)

    Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

    2018-03-01

    Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

  16. Chemoenzymatic Synthesis of Oligo(L-cysteine) for Use as a Thermostable Bio-Based Material.

    Science.gov (United States)

    Ma, Yinan; Sato, Ryota; Li, Zhibo; Numata, Keiji

    2016-01-01

    Oligomerization of thiol-unprotected L-cysteine ethyl ester (Cys-OEt) catalyzed by proteinase K in aqueous solution has been used to synthesize oligo(L-cysteine) (OligoCys) with a well-defined chemical structure and relatively large degree of polymerization (DP) up to 16-17 (average 8.8). By using a high concentration of Cys-OEt, 78.0% free thiol content was achieved. The thermal properties of OligoCys are stable, with no glass transition until 200 °C, and the decomposition temperature could be increased by oxidation. Chemoenzymatically synthesized OligoCys has great potential for use as a thermostable bio-based material with resistance to oxidation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

    Science.gov (United States)

    Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir; Bischoff, Kenneth M; Sani, Rajesh K

    2013-01-01

    Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Localization and Instability in Sheared Granular Materials: Role of Pore Fluids and Non-monotonic Rate Dependent Rheology

    Science.gov (United States)

    Ma, X.; Elbanna, A. E.; Kothari, K.

    2017-12-01

    Fault zone dynamics hold the key to resolving many outstanding geophysical problems including the heat flow paradox, discrepancy between fault static and dynamic strength, and energy partitioning. Most fault zones that generate tectonic events are gouge filled and fluid saturated posing the need for formulating gouge-specific constitutive models that capture spatially heterogeneous compaction and dilation, non-monotonic rate dependence, and transition between localized and distributed deformation. In this presentation, we focus primarily on elucidating microscopic underpinnings for shear banding and stick-slip instabilities in sheared saturated granular materials and explore their implications for earthquake dynamics. We use a non-equilibrium thermodynamics model, the Shear Transformation Zone theory, to investigate the dynamics of strain localization and its connection to stability of sliding in the presence and absence of pore fluids. We also consider the possible influence of self-induced mechanical vibrations as well as the role of external acoustic vibrations as analogue for triggering by a distant event. For the dry case, our results suggest that at low and intermediate strain rates, persistent shear bands develop only in the absence of vibrations. Vibrations tend to fluidize the granular network and de-localize slip at these rates. Stick-slip is only observed for rough grains and it is confined to the shear band. At high strain rates, stick-slip disappears and the different systems exhibit similar stress-slip response. Changing the vibration intensity, duration or time of application alters the system response and may cause long-lasting rheological changes. The presence of pore fluids modifies the stick slip pattern and may lead to both loss and development of slip instability depending on the value of the confining pressure, imposed strain rate and hydraulic parameters. We analyze these observations in terms of possible transitions between rate

  19. Non-monotonic swelling of surface grafted hydrogels induced by pH and/or salt concentration

    Science.gov (United States)

    Longo, Gabriel S.; Olvera de la Cruz, Monica; Szleifer, I.

    2014-09-01

    We use a molecular theory to study the thermodynamics of a weak-polyacid hydrogel film that is chemically grafted to a solid surface. We investigate the response of the material to changes in the pH and salt concentration of the buffer solution. Our results show that the pH-triggered swelling of the hydrogel film has a non-monotonic dependence on the acidity of the bath solution. At most salt concentrations, the thickness of the hydrogel film presents a maximum when the pH of the solution is increased from acidic values. The quantitative details of such swelling behavior, which is not observed when the film is physically deposited on the surface, depend on the molecular architecture of the polymer network. This swelling-deswelling transition is the consequence of the complex interplay between the chemical free energy (acid-base equilibrium), the electrostatic repulsions between charged monomers, which are both modulated by the absorption of ions, and the ability of the polymer network to regulate charge and control its volume (molecular organization). In the absence of such competition, for example, for high salt concentrations, the film swells monotonically with increasing pH. A deswelling-swelling transition is similarly predicted as a function of the salt concentration at intermediate pH values. This reentrant behavior, which is due to the coupling between charge regulation and the two opposing effects triggered by salt concentration (screening electrostatic interactions and charging/discharging the acid groups), is similar to that found in end-grafted weak polyelectrolyte layers. Understanding how to control the response of the material to different stimuli, in terms of its molecular structure and local chemical composition, can help the targeted design of applications with extended functionality. We describe the response of the material to an applied pressure and an electric potential. We present profiles that outline the local chemical composition of the

  20. Thermostability and photophysical properties of mixed-ligand carboxylate/benzimidazole Zn(II)-coordination polymers

    International Nuclear Information System (INIS)

    Barros, Bráulio Silva; Chojnacki, Jaroslaw; Macêdo Soares, Antonia Alice; Kulesza, Joanna; Lourenço da Luz, Leonis; Júnior, Severino Alves

    2015-01-01

    The reaction between Zn(NO 3 ) 2 ·6H 2 O or Zn(CH 3 COO) 2 ·2H 2 O and isophthalic acid (1,3-H 2 bdc) in the presence of benzimidazole (Hbzim) in dimethylformamide (DMF)/ethanol (EtOH)/H 2 O solvent mixture at room temperature yielded two structurally different coordination polymers: [Zn 2 (1,3-bdc) 2 (Hbzim) 2 ] (1) and [Zn 2 (1,3-bdc)(bzim) 2 ] (2). (1) is a 2D-layered framework with a molecule of benzimidazole coordinated to the Zn center, whereas (2) is a 3D framework with benzimidazolate species acting as a co-ligand and bridging two Zn(II) ions. Reactions performed at 90 °C led to the formation of coordination polymers structurally similar to (2), independently of the Zn(II) source used. In the absence of benzimidazole, the reaction between ZnAc 2 .2H 2 O and 1,3-H 2 bdc at 90 °C resulted in the formation of (3), a 3D coordination polymer Zn(HCOO) 3 (Me 2 NH 2 + ). It was observed that the thermostability and the photophysical properties of (1) and (2) are strongly dependent on the coordination modes and packing of benzimidazole in the solid state. These materials present photoluminescence in the wide range of the spectrum, from UV to IR. A full understanding of a physical process occurring in these intriguing systems, including complete energy level diagrams with possible transitions were provided. - Graphical abstract: Display Omitted - Highlights: • Structurally different Zn(II)-coordination polymers were prepared. • The formation of frameworks was counter anion and temperature dependent. • Photoluminescence in the wide range of the spectrum, from UV to IR was observed. • Thermostability and luminescence depended on bzim packing in the structure

  1. Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

    Science.gov (United States)

    Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

    2016-01-01

    Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

  2. Enzimas termoestáveis: fontes, produção e aplicação industrial Thermostable enzymes: sources, production and industrial applications

    Directory of Open Access Journals (Sweden)

    Eleni Gomes

    2007-02-01

    Full Text Available REVIEW: Living organisms encountered in hostile environments that are characterized by extreme temperatures rely on novel molecular mechanisms to enhance the thermal stability of their proteins, nucleic acids, lipids and cell membranes. Proteins isolated from thermophilic organisms usually exhibit higher intrinsic thermal stabilities than their counterparts isolated from mesophilic organisms. Although the molecular basis of protein thermostability is only partially understood, structural studies have suggested that the factors that may contribute to enhance protein thermostability mainly include hydrophobic packing, enhanced secondary structure propensity, helix dipole stabilization, absence of residues sensitive to oxidation or deamination, and increased electrostatic interactions. Thermostable enzymes such as amylases, xylanases and pectinases isolated from thermophilic organisms are potentially of interest in the optimization of industrial processes due to their enhanced stability. In the present review, an attempt is made to delineate the structural factors that increase enzyme thermostability and to document the research results in the production of these enzymes.

  3. Engineering of an Extremely Thermostable Alpha/Beta Barrel Scaffold to Serve as a High Affinity Molecular Recognition Element for Use in Sensor Applications

    Science.gov (United States)

    2015-12-23

    Molecular Recognition Element For Use in Sensor Applications Report Title The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol ...SECURITY CLASSIFICATION OF: The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol dehydrogenase D (AdhD) from Pyrococcus...furiosus) to bind an explosive molecule, RDX. The enzyme naturally catalyzes the nicotinamide cofactor-dependent oxidation or reduction of alcohols

  4. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    Science.gov (United States)

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P phytase to be used as an animal feed supplement.

  5. Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain

    International Nuclear Information System (INIS)

    Roy, Sumana; Choudhury, Debi; Chakrabarti, Chandana; Biswas, Sampa; Dattagupta, J. K.

    2011-01-01

    The crystallization of the precursor of a thermostable variant of papain and the collection of diffraction data to 2.6 Å resolution are reported. The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2 1 , with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 Å 3 Da −1 , corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful

  6. Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

    Directory of Open Access Journals (Sweden)

    Koh Eunhee

    2007-07-01

    Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

  7. Thermostability of Multidomain Proteins: Elongation Factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and Their Chimeric Forms

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Hůlková, Marta; Maloň, Petr; Kepková, M.; Jonák, Jiří

    2004-01-01

    Roč. 13, č. 1 (2004), s. 89-99 ISSN 0961-8368 R&D Projects: GA AV ČR IPP1050128; GA ČR GA204/98/0863; GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : elongation factor EF-Tu, thermostability, chimeric protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.116, year: 2004

  8. Multistability of memristive Cohen-Grossberg neural networks with non-monotonic piecewise linear activation functions and time-varying delays.

    Science.gov (United States)

    Nie, Xiaobing; Zheng, Wei Xing; Cao, Jinde

    2015-11-01

    The problem of coexistence and dynamical behaviors of multiple equilibrium points is addressed for a class of memristive Cohen-Grossberg neural networks with non-monotonic piecewise linear activation functions and time-varying delays. By virtue of the fixed point theorem, nonsmooth analysis theory and other analytical tools, some sufficient conditions are established to guarantee that such n-dimensional memristive Cohen-Grossberg neural networks can have 5(n) equilibrium points, among which 3(n) equilibrium points are locally exponentially stable. It is shown that greater storage capacity can be achieved by neural networks with the non-monotonic activation functions introduced herein than the ones with Mexican-hat-type activation function. In addition, unlike most existing multistability results of neural networks with monotonic activation functions, those obtained 3(n) locally stable equilibrium points are located both in saturated regions and unsaturated regions. The theoretical findings are verified by an illustrative example with computer simulations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Annealing temperature dependent non-monotonic d{sup 0} ferromagnetism in pristine In{sub 2}O{sub 3} nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Haiming; Xing, Pengfei, E-mail: pfxing@tju.edu.cn; Yao, Dongsheng; Wu, Ping

    2017-05-01

    Cubic bixbyite In{sub 2}O{sub 3} nanoparticles with room temperature d{sup 0} ferromagnetism were prepared by sol-gel method with the air annealing temperature ranging from 500 to 900 °C. X-ray diffraction, X-ray photoelectron spectroscopy, Raman-scattering and photoluminescence were carried out to demonstrate the presence of oxygen vacancies. The lattice constant, the atomic ratio of crystal O and In, the Raman peak at 369 cm{sup −1}, the PL emission peak at 396 nm and the saturation magnetization of d{sup 0} ferromagnetism all had a consistent non-monotonic change with the increasing annealing temperature. With further considering the relation between the grain size and the distribution of oxygen vacancies, we think that d{sup 0} ferromagnetism in our samples is directly related with the singly charged oxygen vacancies at the surface of In{sub 2}O{sub 3} nanoparticles. - Highlights: • Effect of air-annealing temperature on the d{sup 0} ferromagnetism of pure In{sub 2}O{sub 3}. • Oxygen-deficiency states of all samples were detected by Raman scattering and PL. • Ferromagnetism changes non-monotonically with the increasing annealing temperature. • d{sup 0} ferromagnetism in our In{sub 2}O{sub 3} nanoparticles is related with the surface V{sub O}{sup +}.

  10. Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

    Science.gov (United States)

    Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

    2015-03-01

    Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.

    Science.gov (United States)

    Liu, Yi; Cui, Wenjing; Liu, Zhongmei; Cui, Youtian; Xia, Yuanyuan; Kobayashi, Michihiko; Zhou, Zhemin

    2014-09-01

    Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

    Science.gov (United States)

    Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

    2017-06-19

    Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

  13. Development of thermostable Peste des Petits Ruminants (PPR) virus vaccine and assessment of molecular changes in the F gene

    International Nuclear Information System (INIS)

    Palaniswami, K.S.; Thangavelu, A.; Velmurugan, R.

    2005-01-01

    Two Indian PPRV isolates were subjected to thermal hardening procedures to increase the proportion of temperature-resistant virions. Initial infectivity loss was compensated by titre increases on subsequent cell passages at 37 deg C. The immunogenicity of 'thermostable' viruses was assessed by virulent PPRV challenge and for safety by host animal inoculation and antibodies assessment. Vaccine viruses were not found using PCR on ocular and nasal swabs, although virus nucleic acid and antigens were demonstrated in spleen and lymph nodes by FAT and PCR. One vaccine strain (MIB187(T)) giving 100% protection (tested on only a few animals) was freeze dried and the minimum protective dose calculated. Changes in the virus genome after thermo-adaptation were examined using RT-PCR to amplify portions of the F gene, and three base changes were observed in the thermostable PPR strain (compared with the F gene sequence of the Nigerian PPRV strain). At room temperature, the titre and potency of the thermo-adapted vaccine remained constant up to one month at the10 5.5 TCID 50 level, and was 10 4.5 TCID 50 /100 μl after two months. Field trials with over 40 000 doses of the thermostable vaccine under various environmental conditions have given serum neutralization titres exceeding 2 3 and are assumed protective. (author)

  14. Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability.

    Science.gov (United States)

    Snider, John L; Oosterhuis, Derrick M; Collins, Guy D; Pilon, Cristiane; Fitzsimons, Toby R

    2013-03-15

    Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T15, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400h) leaf water potentials (ΨMD), leaf-air vapor pressure deficit (VPD), actual quantum yield (ΦPSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T15 was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (ΨMD=-3.1MPa), and ETR was either unaffected or increased under water-stress. Additionally, T15 for PHY 499 was ∼5°C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20-30 days Tmax≥35°C), and showed an increase in T15 with higher average Tmax. When average T15 values for each location and sample date were plotted versus average daily Tmax, strong, positive relationships (r(2) from .954 to .714) were observed between Tmax and T15. For all locations T15 was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between

  15. Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

    Science.gov (United States)

    Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

    2014-07-01

    The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

  16. VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

    Science.gov (United States)

    Mendes, Juliano S; Santiago, André da S; Toledo, Marcelo A S; Rosselli-Murai, Luciana K; Favaro, Marianna T P; Santos, Clelton A; Horta, Maria Augusta C; Crucello, Aline; Beloti, Lilian L; Romero, Fabian; Tasic, Ljubica; de Souza, Alessandra A; de Souza, Anete P

    2015-01-01

    Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

  17. On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes.

    Science.gov (United States)

    Maier, Manfred; Radtke, Carsten P; Hubbuch, Jürgen; Niemeyer, Christof M; Rabe, Kersten S

    2018-05-04

    The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three-dimensional (3D) printing techniques offer high-throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose-based inks for a simple temperature-controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D-printed parts for a continuous, two-step sequential biotransformation in a fluidic setup. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A thermostable serralysin inhibitor from marine bacterium Flavobacterium sp. YS-80-122

    Science.gov (United States)

    Liang, Pengjuan; Li, Shangyong; Wang, Kun; Wang, Fang; Xing, Mengxin; Hao, Jianhua; Sun, Mi

    2018-03-01

    Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, LupI, shows <40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64 μmol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100°C for 1-60 min followed by incubation at 0°C. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.

  19. Thermostable Bacteriocin BL8 from Bacillus licheniformis isolated from marine sediment.

    Science.gov (United States)

    Smitha, S; Bhat, S G

    2013-03-01

    To isolate and characterize bacteriocin, BL8, from the bacteria identified as Bacillus licheniformis from marine environment. One-hundred and twelve bacterial isolates from sediment and water samples collected off the coast of Cochin, India, were screened for antibacterial activity. Strain BTHT8, identified as Bacillus licheniformis, inhibited the growth of Gram-positive test organisms. The active component labelled as bacteriocin BL8 was partially purified by ammonium sulphate fractionation and was subjected to glycine SDS-PAGE. The band exhibiting antimicrobial activity was electroeluted and analysed using MALDI-TOF mass spectrometry, and the molecular mass was determined as 1.4 kDa. N-terminal amino acid sequencing of BL8 gave a 13 amino acid sequence stretch. Bacteriocin BL8 was stable even after boiling at 100 °C for 30 min and over a wide pH range of 1-12. A novel, pH-tolerant and thermostable bacteriocin BL8, active against the tested Gram-positive bacteria, was isolated from Bacillus licheniformis. This study reports a stable, low molecular weight bacteriocin from Bacillus licheniformis. This bacteriocin can be used to address two important applications: as a therapeutic agent and as a biopreservative in food processing industry. © 2012 The Society for Applied Microbiology.

  20. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

    Directory of Open Access Journals (Sweden)

    Shi Gui-Yang

    2009-10-01

    Full Text Available Abstract Background Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. Results A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. Conclusion This production level of BLA by B. licheniformis CBBD302(pHY-amyL is amongst the highest levels in Gram-positive bacteria reported so far.

  1. Geometric Simulation Approach for Grading and Assessing the Thermostability of CALBs

    Directory of Open Access Journals (Sweden)

    B. Senthilkumar

    2016-01-01

    Full Text Available Candida antarctica lipase B (CALB is a known stable and highly active enzyme used widely in biodiesel synthesis. In this work, the stability of native (4K6G and mutant (4K5Q CALB was studied through various structural parameters using conformational sampling approach. The contours of polar surface area and surface area of mutant CALB were 11357.67 Å2 and 30007.4 Å2, respectively, showing an enhanced stability compared to native CALB with a statistically significant P value of < 0.0001. Moreover, simulated thermal denaturation of CALB, a process involving dilution of hydrogen bond, significantly shielded against different intervals of energy application in mutant CALB revealing its augmentation of structural rigidity against native CALB. Finally, computational docking analysis showed an increase in the binding affinity of CALB and its substrate (triglyceride in mutant CALB with Atomic Contact Energy (ACE of −91.23 kcal/mol compared to native CALB (ACE of −70.3 kcal/mol. The computational observations proposed that the use of mutant CALB (4K5Q could serve as a best template for production of biodiesel in the future. Additionally, it can also be used as a template to identify efficient thermostable lipases through further mutations.

  2. Construction and engineering of a thermostable self-sufficient cytochrome P450

    Energy Technology Data Exchange (ETDEWEB)

    Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  3. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Science.gov (United States)

    Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

    2015-11-01

    There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  4. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Directory of Open Access Journals (Sweden)

    David Bednar

    2015-11-01

    Full Text Available There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  5. VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

    Directory of Open Access Journals (Sweden)

    Juliano S Mendes

    Full Text Available Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC, a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c and nonpathogenic (XfJ1a12 strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

  6. Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes.

    Science.gov (United States)

    Annamalai, Neelamegam; Rajeswari, Mayavan Veeramuthu; Elayaraja, Sivaramasamy; Balasubramanian, Thangavel

    2013-04-15

    An extracellular thermostable, haloalkaline cellulase by bioconversion of lignocellulosic wastes from Bacillus halodurans CAS 1 was purified to homogeneity with recovery of 12.54% and purity fold 7.96 with the molecular weight of 44 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 60°C, 9.0 and 30% and it retained 80% of activity even at 80°C, 12 and 35% respectively. The activity was greatly inhibited by EDTA, indicating that it was a metalloenzyme and significant inhibition by PMSF revealed that serine residue was essential for catalytic activity. The purified cellulase hydrolyzed CMC, cellobiose and xylan, but not avicel, cellulose and PNPG. Furthermore, the cellulase was highly stable in the presence of detergents and organic solvents such as acetone, n-hexane and acetonitrile. Thus, the purified cellulase from B. halodurans utilizing lignocellulosic biomass could be greatly useful to develop industrial processes. Published by Elsevier Ltd.

  7. Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant.

    Science.gov (United States)

    Rahman, D Y; Sarian, F D; van Wijk, A; Martinez-Garcia, M; van der Maarel, M J E C

    2017-01-01

    The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae might provide an alternative source of phycocyanin. Cyanidioschyzon merolae belongs to the order Cyanidiophyceae of the phylum Rhodophyta. Its natural habitat are sulfuric hot springs and geysers found near volcanic areas in, e.g., Yellowstone National Park in the USA and in Java, Indonesia. It grows optimally at a pH between 0.5 and 3.0 and at temperatures up to 56 °C. The low pH at which C . merolae grows minimizes the risk of microbial contamination and could limit production loss. As C . merolae lacks a cell wall, phycocyanin with a high purity number of 9.9 could be extracted by an osmotic shock using a simple ultrapure water extraction followed by centrifugation. The denaturation midpoint at pH 5 was 83 °C, being considerably higher than the A . platensis phycocyanin (65 °C). The C . merolae phycocyanin was relatively stable at pH 4 and 5 up to 80 °C. The high thermostability at slightly acidic pH makes the C . merolae phycocyanin an interesting alternative to A . platensis phycocyanin as a natural blue food colorant.

  8. Reconstitution of β-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

    International Nuclear Information System (INIS)

    Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.

    2006-01-01

    CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards β-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 o C. In this system, β-carotene was hydroxylated to β-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low β-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated β-carotene at 3- and also 3'-positions, resulting in β-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol β-cryptoxanthin produced per nmol P450 per min

  9. Construction and engineering of a thermostable self-sufficient cytochrome P450

    International Nuclear Information System (INIS)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-01-01

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  10. Not so monofunctional--a case of thermostable Thermobifida fusca catalase with peroxidase activity.

    Science.gov (United States)

    Lončar, Nikola; Fraaije, Marco W

    2015-03-01

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

  11. Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

    Science.gov (United States)

    Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

    2017-11-01

    Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae.

    Science.gov (United States)

    Wu, Dingxin; Wang, Linchun; Li, Yuwei; Zhao, Shumiao; Peng, Nan; Liang, Yunxiang

    2016-02-01

    An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

  13. The effect of different stabilizers on the thermostability of electron beam crosslinked polyethylene in hot water

    International Nuclear Information System (INIS)

    Hassanpour, S.; Khoylou, F.

    2003-01-01

    Plastic pipes owing to their flexibility, great lengths, easier handling and absence of corrosion have been used for hot-water installations. Crosslinked high-density polyethylene is one of the best materials, being used for this purpose. The useful lifetime of unstabilized polyethylene is predicted to vary from a few months in hot water (30-40 deg. C) to almost two years in cool water (0-10 deg. C). Polyethylene was mixed with different types of stabilizers, in order to increase its durability. The samples were irradiated at 100-150 kGy. The amount of gel fraction and the changes in mechanical properties were measured. Irradiated samples were immersed in hot water for 1000 h. The thermostability of the specimens and the existence of antioxidants were measured by the induction time technique using differential scanning calorimetry at different time intervals. Furthermore, the changes in chemical structure and mechanical properties of the samples during their immersion in hot water were determined

  14. Nonmonotonous changes of thymus nuclei lipid metabolism upon chronic gamma-radiation of rats at a dose-rate of 3 c Gy/Day

    International Nuclear Information System (INIS)

    Kulagina, T.P.; Kolomijtseva, I.K.; Moiseeva, S.A.; Kuzin, A.M.

    2000-01-01

    The dynamics of changes in the thymus nuclei lipid metabolism under chronic gamma-radiation in low doses with the dose rate of 3 cGy/day is studied. It is shown, that at the 25 cGy dose rate there takes place activation of exchange in the fatly-acid part of the phospholipid molecule with simultaneous activation of the cholesterol and fatty acids synthesis. The synthesis of cholesterol and fatty acids at 50 cGy remains activated, whereas metabolism of the fatty-acid part of the phospholipids molecule is sharply depressed. The identified changes reveal the similarity with the processes, proceeding by the apoptose induction. At the same time the dynamics of the thymocyte nuclei lipid exchange in the process of adaptation to the long radiation effect as nonmonotonous metabolic response to low dose impact is characterized for the first time [ru

  15. Non-monotonic behavior of electron temperature in argon inductively coupled plasma and its analysis via novel electron mean energy equation

    Science.gov (United States)

    Zhao, Shu-Xia

    2018-03-01

    In this work, the behavior of electron temperature against the power in argon inductively coupled plasma is investigated by a fluid model. The model properly reproduces the non-monotonic variation of temperature with power observed in experiments. By means of a novel electron mean energy equation proposed for the first time in this article, this electron temperature behavior is interpreted. In the overall considered power range, the skin effect of radio frequency electric field results in localized deposited power density, responsible for an increase of electron temperature with power by means of one parameter defined as power density divided by electron density. At low powers, the rate fraction of multistep and Penning ionizations of metastables that consume electron energy two times significantly increases with power, which dominates over the skin effect and consequently leads to the decrease of temperature with power. In the middle power regime, a transition region of temperature is given by the competition between the ionizing effect of metastables and the skin effect of electric field. The power location where the temperature alters its trend moves to the low power end as increasing the pressure due to the lack of metastables. The non-monotonic curve of temperature is asymmetric at the short chamber due to the weak role of skin effect in increasing the temperature and tends symmetric when axially prolonging the chamber. Still, the validity of the fluid model in this prediction is estimated and the role of neutral gas heating is guessed. This finding is helpful for people understanding the different trends of temperature with power in the literature.

  16. Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

    Directory of Open Access Journals (Sweden)

    Chengtuo Niu

    Full Text Available 1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

  17. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Shalini RAI

    2014-03-01

    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  18. Evaluation of enhanced thermostability and operational stability of carbonic anhydrase from Micrococcus species.

    Science.gov (United States)

    Bhattacharya, Abhishek; Shrivastava, Ankita; Sharma, Anjana

    2013-06-01

    Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0-8.0 and 35-45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5-10.0 and 35-55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T(1/20) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC(50) values and inhibitory constant measurement (K(i)). The K(m) and V(max) for M. lylae CA (2.31 mM; 769.23 μmol/mg/min) and M. luteus CA (2.0 mM; 1,000 μmol/mg/min) were calculated from Lineweaver-Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO(2) sequestration.

  19. Production, Purification, and Characterization of Thermostable α-Amylase Produced by Bacillus licheniformis Isolate AI20

    Directory of Open Access Journals (Sweden)

    Yasser R. Abdel-Fattah

    2013-01-01

    Full Text Available An optimization strategy, based on statistical experimental design, is employed to enhance the production of thermostable α-amylase by a thermotolerant B. licheniformis AI20 isolate. Using one variant at time (OVAT method, starch, yeast extract, and CaCl2 were observed to influence the enzyme production significantly. Thereafter, the response surface methodology (RSM was adopted to acquire the best process conditions among the selected variables, where a three-level Box-Behnken design was employed to create a polynomial quadratic model correlating the relationship between the three variables and α-amylase activity. The optimal combination of the major constituents of media for α-amylase production was 1.0% starch, 0.75% yeast extract, and 0.02% CaCl2. The predicted optimum α-amylase activity was 384 U/mL/min, which is two folds more than the basal medium conditions. The produced α-amylase was purified through various chromatographic techniques. The estimated enzyme molecular mass was 55 kDa and the α-amylase had an optimal temperature and pH of 60–80°C and 6–7.5, respectively. Values of Vmax and Km for the purified enzyme were 454 mU/mg and 0.709 mg/mL. The α-amylase enzyme showed great stability against different solvents. Additionally, the enzyme activity was slightly inhibited by detergents, sodium dodecyl sulphate (SDS, or chelating agents such as EDTA and EGTA. On the other hand, great enzyme stability against different divalent metal ions was observed at 0.1 mM concentration, but 10 mM of Cu2+ or Zn2+ reduced the enzyme activity by 25 and 55%, respectively.

  20. Studies for methods to improve thermostability of the functionalized butadiene styrene rubbers

    Directory of Open Access Journals (Sweden)

    A. L. Rumyantseva

    2018-01-01

    Full Text Available It is well known that the tire performance properties can deteriorate in the processes of production, processing, storage and operation. One of the reasons for that is a series of processes occurring in the polymer under the influence of different factors: thermal, mechanical or chemical. This problem is particularly relevant for functionalized polymers, as functional groups can interact with each other, causing side cross linking reactions that lead to a deterioration of consumer properties of the products. The main purpose of this work was to study influence of several key factors on the thermostability of functionalized rubbers in order to find a solution: different types of antioxidants, rubber polymerizate stripping conditions and rubber processing. In accordance with the problem, solutions were found and work was carried out in several directions: changing the pH of the medium in the rubber stripping and using antioxidants containing carbonyl groups located in ?-positions to methylene groups, namely Irganox 1520 and Irganox 1076. As an evaluation factor, thermal treatment was selected in two modes: at 100 °C for 48 hours and after extruder at 130 °C for 5 minutes + 100 °C for 48 hours. At the same time, the following parameters were determined: molecular weight characteristics and Mooney viscosity of the starting polymers and after thermal aging. During the experiments, it was found that the acidity of the medium in the water degasser does not affect the crosslinking of the functionalized rubber during storage. In addition, a study was made of the effect of the type of antioxidant and its quantity on the thermal stability of functionalized styrene butadiene rubbers, as well as the study of the effect of the content of the modifying agent on the thermal stability of the product. It has been found that the use, as antioxidants, of carbonyl compounds containing a methylene group at the ?-position, leads to inhibition of the cross

  1. Cloning, purification and characterization of a thermostable β-galactosidase from Bacillus licheniformis strain KG9.

    Science.gov (United States)

    Matpan Bekler, F; Stougaard, P; Güven, K; Gül Güven, R; Acer, Ö

    2015-06-28

    A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

  2. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil.

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    Sandeep Kaur Saggu

    Full Text Available Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.

  3. Crystal structure of a thermostable Old Yellow Enzyme from Thermus scotoductus SA-01

    International Nuclear Information System (INIS)

    Opperman, Diederik J.; Sewell, Bryan T.; Litthauer, Derek; Isupov, Mikhail N.; Littlechild, Jennifer A.; Heerden, Esta van

    2010-01-01

    Recent characterization of the chromate reductase (CrS) from the thermophile Thermus scotoductus SA-01 revealed this enzyme to be related to the Old Yellow Enzyme (OYE) family. Here, we report the structure of a thermostable OYE homolog in its holoform at 2.2 A as well as its complex with p-hydroxybenzaldehyde (pHBA). The enzyme crystallized as octamers with the monomers showing a classical TIM barrel fold which upon dimerization yields the biologically active form of the protein. A sulfate ion is bound above the si-side of the non-covalently bound FMN cofactor in the oxidized solved structure but is displaced upon pHBA binding. The active-site architecture is highly conserved as with other members of this enzyme family. The pHBA in the CrS complex is positioned by hydrogen bonding to the two conserved catalytic-site histidines. The most prominent structural difference between CrS and other OYE homologs is the size of the 'capping domain'. Thermostabilization of the enzyme is achieved in part through increased proline content within loops and turns as well as increased intersubunit interactions through hydrogen bonding and complex salt bridge networks. CrS is able to reduce the C=C bonds of α,β-unsaturated carbonyl compounds with a preference towards cyclic substrates however no activity was observed towards β-substituted substrates. Mutational studies have confirmed the role of Tyr177 as the proposed proton donor although reduction could still occur at a reduced rate when this residue was mutated to phenylalanine.

  4. Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

    Directory of Open Access Journals (Sweden)

    Paulo S. Monteiro

    2015-03-01

    Full Text Available An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

  5. Partial Purification and Characterization of a Thermostable β-Mannanase from Aspergillus foetidus

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    Juliana da Conceição Infante de Marco

    2015-10-01

    Full Text Available An extracellular β-mannanase was isolated from samples of crude extract of the mesophilic fungus Aspergillus foetidus grown on soybean husk as a carbon source. The induction profile showed that β-mannanase reached a maximum activity level (2.0 IU/mL on the 15th day of cultivation. The enzyme was partially purified by ultrafiltration and gel filtration chromatography procedures and was named Man 58. Sodium dodecyl sulfate-polyacrilamide electrophoresis and zymogram analysis of Man 58 showed two bands of approximately 43 and 45 kDa with β-mannanase activity. Ultrafiltration showed that β-mannanase activity was only detected in the concentrated sample. Man 58 was most active at 60 °C and at pH 4.0. It was thermostable in the temperature range of 40–60 °C for eleven days, and the half-life at 70 °C was ten days. Man 58 showed Km and Vmax values of 3.29 mg/mL and 1.76 IU/mL respectively, with locust bean gum as a substrate. It was mostly activated by FeSO4 and CoCl2 and inhibited by MgSO4, FeCl3, CuSO4, MgCl2, ZnCl2, ZnSO4, CaCl2, CuCl2, KCl and ethylenediaminetetraacetic acid (EDTA. Phenolic compounds did not inhibit the enzyme. On the other hand, auto-hydrolysis liquor showed an inhibitory effect on Man 58 activity.

  6. Experimental study on the thermostable property of aramid fiber reinforced PE-RT pipes

    Directory of Open Access Journals (Sweden)

    Guoquan Qi

    2015-11-01

    Full Text Available Flexible composite pipes are advantageous in ultra high strength, high modulus, pH and corrosion resistance and light weight, but there are still some hidden safety troubles because they are poorer in thermostable capacity. Therefore, test samples of flexible composite pipes were prepared with high-temperature polythene (PE-RT as the neck bush and aramid fiber as the reinforcement layer. Experimental study was conducted by using HPHT vessel and differential thermal scanner for different working conditions, different temperatures, whole-pipe pressure-bearing capacity and 1000 h viability. It is shown by the environmental compatibility test that high temperature has little effect on the weight, Vicat softening temperature, mechanical properties and structures of neck bush PE-RT, but exerts an obvious effect on the tensility and whole-pipe water pressure blasting of the reinforcement aramid fiber. Besides, the drop of whole-pipe pressure-bearing capacity is caused by deformation and breaking of aramid fibers when the reinforcement layer is under the force of internal pressure. Finally, disorientation and crystallization of molecular thermal motion occur with the rise of temperature, so amorphous orientation reduces, crystallinity factor and crystalline orientation factor increase gradually, thus, disorientation of macromolecular chains increases and tensile strength decreases. It is concluded that this type of flexible composite pipe can smoothly pass 1000 h viability test. And it is recommended that it be used in the situations with temperature not higher than 95 °C and internal pressure not higher than 4 MPa.

  7. Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

    Science.gov (United States)

    Monteiro, Paulo S.; Guimarães, Valéria M.; de Melo, Ricardo R.; de Rezende, Sebastião T.

    2015-01-01

    An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 5 s −1 and 4.7 × 10 6 s −1 .M −1 , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg 2+ , Cd 2+ , K + and Ca 2+ , and it was drastically inhibited by F − . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment. PMID:26221114

  8. Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

    2014-01-01

    Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

  9. Draft genome sequence of a thermostable, alkaliphilic α-amylase and protease producing Bacillus amyloliquefaciens strain KCP2.

    Science.gov (United States)

    Prajapati, Vimalkumar S; Ray, Sanket; Narayan, Jitendra; Joshi, Chaitanya C; Patel, Kamlesh C; Trivedi, Ujjval B; Patel, R M

    2017-12-01

    Bacillus amyloliquefaciens strain KCP2 was isolated from municipal food waste samples collected in Vallabh Vidyanagar, Gujarat, India. Strain KCP2 is noteworthy due to its ability to produce a thermostable, alkaliphilic α-amylase and a protease. These enzymes have importance in several industrial processes including bread making, brewing, starch processing, pharmacy, and textile industries. Whole genome sequencing of strain KCP2 showed that the estimated genome size was 3.9 Mb, the G + C content was 46%, and it coded for 4113 genes.

  10. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  11. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    Science.gov (United States)

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  12. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

    Directory of Open Access Journals (Sweden)

    Jiivittha Veno

    2017-11-01

    Full Text Available In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386 was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.

  13. Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Narayanan, Sai Shyam; Sokkar, Pandian; Ramachandran, Murugesan; Nampoothiri, Kesavan Madhavan

    2011-07-01

    Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    International Nuclear Information System (INIS)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert

    2013-01-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site

  15. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    Energy Technology Data Exchange (ETDEWEB)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610 (United States)

    2013-05-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

  16. Design considerations and analysis planning of a phase 2a proof of concept study in rheumatoid arthritis in the presence of possible non-monotonicity

    Directory of Open Access Journals (Sweden)

    Feng Liu

    2017-10-01

    Full Text Available Abstract Background It is important to quantify the dose response for a drug in phase 2a clinical trials so the optimal doses can then be selected for subsequent late phase trials. In a phase 2a clinical trial of new lead drug being developed for the treatment of rheumatoid arthritis (RA, a U-shaped dose response curve was observed. In the light of this result further research was undertaken to design an efficient phase 2a proof of concept (PoC trial for a follow-on compound using the lessons learnt from the lead compound. Methods The planned analysis for the Phase 2a trial for GSK123456 was a Bayesian Emax model which assumes the dose-response relationship follows a monotonic sigmoid “S” shaped curve. This model was found to be suboptimal to model the U-shaped dose response observed in the data from this trial and alternatives approaches were needed to be considered for the next compound for which a Normal dynamic linear model (NDLM is proposed. This paper compares the statistical properties of the Bayesian Emax model and NDLM model and both models are evaluated using simulation in the context of adaptive Phase 2a PoC design under a variety of assumed dose response curves: linear, Emax model, U-shaped model, and flat response. Results It is shown that the NDLM method is flexible and can handle a wide variety of dose-responses, including monotonic and non-monotonic relationships. In comparison to the NDLM model the Emax model excelled with higher probability of selecting ED90 and smaller average sample size, when the true dose response followed Emax like curve. In addition, the type I error, probability of incorrectly concluding a drug may work when it does not, is inflated with the Bayesian NDLM model in all scenarios which would represent a development risk to pharmaceutical company. The bias, which is the difference between the estimated effect from the Emax and NDLM models and the simulated value, is comparable if the true dose response

  17. Design considerations and analysis planning of a phase 2a proof of concept study in rheumatoid arthritis in the presence of possible non-monotonicity.

    Science.gov (United States)

    Liu, Feng; Walters, Stephen J; Julious, Steven A

    2017-10-02

    It is important to quantify the dose response for a drug in phase 2a clinical trials so the optimal doses can then be selected for subsequent late phase trials. In a phase 2a clinical trial of new lead drug being developed for the treatment of rheumatoid arthritis (RA), a U-shaped dose response curve was observed. In the light of this result further research was undertaken to design an efficient phase 2a proof of concept (PoC) trial for a follow-on compound using the lessons learnt from the lead compound. The planned analysis for the Phase 2a trial for GSK123456 was a Bayesian Emax model which assumes the dose-response relationship follows a monotonic sigmoid "S" shaped curve. This model was found to be suboptimal to model the U-shaped dose response observed in the data from this trial and alternatives approaches were needed to be considered for the next compound for which a Normal dynamic linear model (NDLM) is proposed. This paper compares the statistical properties of the Bayesian Emax model and NDLM model and both models are evaluated using simulation in the context of adaptive Phase 2a PoC design under a variety of assumed dose response curves: linear, Emax model, U-shaped model, and flat response. It is shown that the NDLM method is flexible and can handle a wide variety of dose-responses, including monotonic and non-monotonic relationships. In comparison to the NDLM model the Emax model excelled with higher probability of selecting ED90 and smaller average sample size, when the true dose response followed Emax like curve. In addition, the type I error, probability of incorrectly concluding a drug may work when it does not, is inflated with the Bayesian NDLM model in all scenarios which would represent a development risk to pharmaceutical company. The bias, which is the difference between the estimated effect from the Emax and NDLM models and the simulated value, is comparable if the true dose response follows a placebo like curve, an Emax like curve, or log

  18. Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger.

    Science.gov (United States)

    Li, Lijun; Liao, Hui; Yang, Yan; Gong, Jianye; Liu, Jianan; Jiang, Zedong; Zhu, Yanbing; Xiao, Anfeng; Ni, Hui

    2018-06-01

    To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

    Science.gov (United States)

    Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-12-01

    We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

  20. Non-linear, non-monotonic effect of nano-scale roughness on particle deposition in absence of an energy barrier: Experiments and modeling

    Science.gov (United States)

    Jin, Chao; Glawdel, Tomasz; Ren, Carolyn L.; Emelko, Monica B.

    2015-12-01

    Deposition of colloidal- and nano-scale particles on surfaces is critical to numerous natural and engineered environmental, health, and industrial applications ranging from drinking water treatment to semi-conductor manufacturing. Nano-scale surface roughness-induced hydrodynamic impacts on particle deposition were evaluated in the absence of an energy barrier to deposition in a parallel plate system. A non-linear, non-monotonic relationship between deposition surface roughness and particle deposition flux was observed and a critical roughness size associated with minimum deposition flux or “sag effect” was identified. This effect was more significant for nanoparticles (<1 μm) than for colloids and was numerically simulated using a Convective-Diffusion model and experimentally validated. Inclusion of flow field and hydrodynamic retardation effects explained particle deposition profiles better than when only the Derjaguin-Landau-Verwey-Overbeek (DLVO) force was considered. This work provides 1) a first comprehensive framework for describing the hydrodynamic impacts of nano-scale surface roughness on particle deposition by unifying hydrodynamic forces (using the most current approaches for describing flow field profiles and hydrodynamic retardation effects) with appropriately modified expressions for DLVO interaction energies, and gravity forces in one model and 2) a foundation for further describing the impacts of more complicated scales of deposition surface roughness on particle deposition.

  1. Loschmidt echo for local perturbations: non-monotonic cross-over from the Fermi-golden-rule to the escape-rate regime

    International Nuclear Information System (INIS)

    Goussev, Arseni; Waltner, Daniel; Richter, Klaus; Jalabert, Rodolfo A

    2008-01-01

    We address the sensitivity of quantum mechanical time evolution by considering the time decay of the Loschmidt echo (LE) (or fidelity) for local perturbations of the Hamiltonian. Within a semiclassical approach, we derive analytical expressions for the LE decay for chaotic systems for the whole range from weak to strong local perturbations and identify different decay regimes which complement those known for the case of global perturbations. For weak perturbations, a Fermi-golden-rule (FGR)-type behavior is recovered. For strong perturbations, the escape-rate regime is reached, where the LE decays exponentially with a rate independent of the perturbation strength. The transition between the FGR regime and the escape-rate regime is non-monotonic, i.e. the rate of the exponential time-decay of the LE oscillates as a function of the perturbation strength. We further perform extensive quantum mechanical calculations of the LE based on numerical wave packet evolution, which strongly support our semiclassical theory. Finally, we discuss in some detail possible experimental realizations for observing the predicted behavior of the LE

  2. Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution.

    Science.gov (United States)

    Jiang, Huang; Zhang, Shaowei; Gao, Haofeng; Hu, Nan

    2016-01-22

    In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

  3. A CLASS OF NONMONOTONE TRUST REGION ALGORITHMS FOR LINEARLY CONSTRAINED OPTIMIZATION%线性约束优化的一类非单调信赖域算法

    Institute of Scientific and Technical Information of China (English)

    葛恒武; 陈中文

    2002-01-01

    We present a class of nonmonotone trust region algorithms for linearly constrained optimization in this paper.The algorithm may adjust automatically the scope of the monotonicity by the degree that the quadratic model is "trusted".Under the suitable conditions,it is proved that any limit point of the infinite sequence generated by the algorithm is the Kuhn-Tucker point of the primal problem.Finally,some numerical results show that the new algorithm is very effective.

  4. Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

    Science.gov (United States)

    Ryu, Se-Ah; Kim, Chang Sup; Kim, Hye-Jung; Baek, Dae Heoun; Oh, Deok-Kun

    2003-01-01

    D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

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

    DEFF Research Database (Denmark)

    Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

    2017-01-01

    processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase......Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production...... was estimated to be 77 kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation...

  6. Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus

    Science.gov (United States)

    2013-01-01

    Background Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. Results A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose. Conclusions The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization. PMID:24053641

  7. Effect of thermostable α-amylase injection on mechanical and physiochemical properties for saccharification of extruded corn starch.

    Science.gov (United States)

    Myat, Lin; Ryu, Gi-Hyung

    2014-01-30

    In industry, a jet cooker is used to gelatinize starch by mixing the starch slurry with steam under pressure at 100-175 °C. A higher degree of starch hydrolysis in an extruder is possible with glucoamylase. Unfortunately, it is difficult to carry out liquefaction and saccharification in parallel, because the temperature of gelatinization will be too high and will inactivate glucoamylase. Since the temperature for liquefaction and saccharification is different, it is hard to change the temperature from high (required for liquefaction) to low (required for saccharification). The industrial gelatinization process is usually carried out with 30-35% (w/w) dry solids starch slurry. Conventional jet cookers cannot be used any more at high substrate concentrations owing to higher viscosity. In this study, therefore, corn starch was extruded at different melt temperatures to overcome these limitations and to produce the highest enzyme-accessible starch extrudates. Significant effects on physical properties (water solubility index, water absorption index and color) and chemical properties (reducing sugar and % increase in reducing sugar after saccharification) were achieved by addition of thermostable α-amylase at melt temperatures of 115 and 135 °C. However, there was no significant effect on % increase in reducing sugar of extruded corn starch at 95 °C. The results show the great potential of extrusion with thermostable α-amylase injection at 115 and 135 °C as an effective pretreatment for breaking down starch granules, because of the significant increase (P < 0.05) in % reducing sugar and enzyme-accessible extrudates for saccharification yield. © 2013 Society of Chemical Industry.

  8. Nonmonotonic Temperature Dependence of the Pressure-Dependent Reaction Rate Constant and Kinetic Isotope Effect of Hydrogen Radical Reaction with Benzene Calculated by Variational Transition-State Theory.

    Science.gov (United States)

    Zhang, Hui; Zhang, Xin; Truhlar, Donald G; Xu, Xuefei

    2017-11-30

    The reaction between H and benzene is a prototype for reactions of radicals with aromatic hydrocarbons. Here we report calculations of the reaction rate constants and the branching ratios of the two channels of the reaction (H addition and H abstraction) over a wide temperature and pressure range. Our calculations, obtained with an accurate potential energy surface, are based on variational transition-state theory for the high-pressure limit of the addition reaction and for the abstraction reaction and on system-specific quantum Rice-Ramsperger-Kassel theory calibrated by variational transition-state theory for pressure effects on the addition reaction. The latter is a very convenient way to include variational effects, corner-cutting tunneling, and anharmonicity in falloff calculations. Our results are in very good agreement with the limited experimental data and show the importance of including pressure effects in the temperature interval where the mechanism changes from addition to abstraction. We found a negative temperature effect of the total reaction rate constants at 1 atm pressure in the temperature region where experimental data are missing and accurate theoretical data were previously missing as well. We also calculated the H + C 6 H 6 /C 6 D 6 and D + C 6 H 6 /C 6 D 6 kinetic isotope effects, and we compared our H + C 6 H 6 results to previous theoretical data for H + toluene. We report a very novel nonmonotonic dependence of the kinetic isotope effect on temperature. A particularly striking effect is the prediction of a negative temperature dependence of the total rate constant over 300-500 K wide temperature ranges, depending on the pressure but generally in the range from 600 to 1700 K, which includes the temperature range of ignition in gasoline engines, which is important because aromatics are important components of common fuels.

  9. Environmentally-relevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles.

    Science.gov (United States)

    Brodeur, Julie C; Sassone, Alina; Hermida, Gladys N; Codugnello, Nadia

    2013-06-01

    Despite of the various studies reporting on the subject, anticipating the impacts of the widely-used herbicide atrazine on anuran tadpoles metamorphosis remains complex as increases or decreases of larval period duration are almost as frequently reported as an absence of effect. The aim of the present study was to examine the effects of environmentally-relevant concentrations of atrazine (0.1, 1, 10, 100, and 1000μg/L) on the timings of metamorphosis and body size at metamorphosis in the common South American toad, Rhinella arenarum (Anura: bufonidae). None of the atrazine concentrations tested significantly altered survival. Low atrazine concentrations in the range of 1-100μg/L were found to accelerate developmental rate in a non-monotonic U-shaped concentration-response relationship. This observed acceleration of the metamorphic process occurred entirely between stages 25 and 39; treated tadpoles proceeding through metamorphosis as control animals beyond this point. Together with proceeding through metamorphosis at a faster rate, tadpoles exposed to atrazine concentrations in the range of 1-100μg/L furthermore transformed into significantly larger metamorphs than controls, the concentration-response curve taking the form of an inverted U in this case. The no observed effect concentration (NOEC) was 0.1μg atrazine/L for both size at metamorphosis and timings of metamorphosis. Tadpoles exposed to 100μg/L 17β-estradiol presented the exact same alterations of developmental rate and body size as those treated with 1, 10 and 100μg/L of atrazine. Elements of the experimental design that facilitated the detection of alterations of metamorphosis at low concentrations of atrazine are discussed, together with the ecological significance of those findings. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Some remarks on non-monotonic effects at low radiation intensities, on the problem of extrapolating doses between high and low intensities and on the problem of thresholds

    International Nuclear Information System (INIS)

    Delattre, P.

    1983-01-01

    On the basis of a general descriptive framework which takes into account the intensity factor and the time distribution of radiation, a detailed justification for which is to be found in earlier publications, the three fundamental problems mentioned in the title of this paper can be approached in a new way. If the biological effect e for a given dose D delivered at different radiation intensities phi is studied, we find that the curve e=f(phi) can exhibit non-monotonic shapes. This type of phenomenon is known in pharmacology and toxicology and may well exist also for low- or medium-intensity radiation effects. Extrapolation of the effects of a given dose between high and low radiation intensities phi is usually carried out by means of an empirical linear or linear-quadratic formulation. This procedure is insufficiently justified from a theoretical point of view. It is shown here that the effects can be written in the form e=k(phi)D and that the factor of proportionality k(phi) is a generally very complicated function of phi. Hence, the usual extrapolation procedures cannot deal with certain ranges of values of phi within which the effects observed at a given dose may be greater than when the dose is delivered at higher intensity. The problem of thresholds is actually far more difficult than the current literature on the subject would suggest. It is shown here, on the basis of considerations of qualitative dynamics, that several types of threshold must be defined, starting with a threshold for the radiation intensity phi. All these thresholds are interrelated hierarchically in fairly complex ways which must be studied case by case. These results show that it is illusory to attempt to define a universal notion of threshold in terms of dose. The conceptual framework used in the proposed approach proves also to be very illuminating for other studies in progress, particularly in the investigation of phenomena associated with ageing and carcinogenesis. (author)

  11. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

    Directory of Open Access Journals (Sweden)

    Salleh Abu

    2009-04-01

    Full Text Available Abstract Background Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia. Results A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v of (AB600 = 0.5 inoculum size, in a culture medium (pH 7.0 and incubated for 24 h at 37°C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg. The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60°C, respectively. Conclusion Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic

  12. Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

    Science.gov (United States)

    Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

    2018-01-01

    We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans.

    Science.gov (United States)

    Jiang, Wei; Wang, Ya-Li; Fang, Bai-Shan

    2018-05-09

    As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40-70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

  14. Development of soluble and immobilized biocatalysts based on a recombinant thermostable ß-fructosidase enabling complete sucrose inversion at pasteurization temperatures

    OpenAIRE

    Menéndez, Carmen; Martínez, Duniesky; Trujillo, Luis E; Ramírez, Ricardo; Sobrino, Alina; Cutiño-Ávila, Bessy V; Basabe, Liliana; del Monte-Martínez, Alberto; Pérez, Enrique R; Hernández, Lázaro

    2014-01-01

    Biocatalysts for the industrial production of invert sugar are preferred to stably operate at high sucrose concentrations and pasteurization temperatures. Thermotoga maritima ß-fructosidase (BfrA) is more thermostable and less susceptible to substrate inhibition than the current commercial invertase from Saccharomyces cerevisiae. In this research, the non-saccharolytic host Pichia pastoris was engineered for BfrA production. Fed-batch fermentation of the recombinant yeast for 72 h using cane ...

  15. Thermo-stable carbon nanotube-TiO_2 nanocompsite as electron highways in dye-sensitized solar cell produced by bio-nano-process

    International Nuclear Information System (INIS)

    Inoue, Ippei; Yasueda, Hisashi; Yamauchi, Hirofumi; Okamoto, Naofumi; Toyoda, Kenichi; Horita, Masahiro; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro

    2015-01-01

    We produced a thermostable TiO_2-(anatase)-coated multi-walled-carbon-nanotube (MWNT) nanocomposite for use in dye-sensitized solar cells (DSSCs) using biological supuramolecules as catalysts. We synthesized two different sizes of iron oxide nanoparticles (NPs) and arrayed the NPs on a silicon substrate utilizing two kinds of genetically modified cage-shaped proteins with silicon-binding peptide aptamers on their outer surfaces. Chemical vapor deposition (CVD) with the vapor–liquid-solid phase (VLS) method was applied to the substrate, and thermostable MWNTs with a diameter of 6 ± 1 nm were produced. Using a genetically modified cage-shaped protein with carbon-nanomaterials binding and Ti-mineralizing peptides as a catalyst, we were able to mineralize a titanium compound around the surface of the MWNT. The products were sintered, and thin TiO_2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO_2-coated MWNT nanocomposite to a DSSC photoelectrode improved current density by 11% and decreased electric resistance by 20% compared to MWNT-free reference DSSCs. These results indicate that a nanoscale TiO_2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO_2 photoelectrodes. (paper)

  16. Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

    Science.gov (United States)

    Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

    2017-02-01

    The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40 T , was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

  17. Improving the Efficiency of New Automatic Dishwashing Detergent Formulation by Addition of Thermostable Lipase, Protease and Amylase

    Directory of Open Access Journals (Sweden)

    Ashwini Naganthran

    2017-09-01

    Full Text Available The use of T1 lipase in automatic dishwashing detergent (ADD is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillus subtilis strain Rand, and Maltogenic amylase from Geobacillus sp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70 buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert‘s Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD “Finish” at 40 and 50 C.

  18. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  19. Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

    Science.gov (United States)

    Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

    2010-06-01

    D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

  20. Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

    Science.gov (United States)

    Bosshart, Andreas; Hee, Chee Seng; Bechtold, Matthias; Schirmer, Tilman; Panke, Sven

    2015-03-02

    Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Thermostable gel polymer electrolyte based on succinonitrile and ionic liquid for high-performance solid-state supercapacitors

    Science.gov (United States)

    Pandey, Gaind P.; Liu, Tao; Hancock, Cody; Li, Yonghui; Sun, Xiuzhi Susan; Li, Jun

    2016-10-01

    A flexible, free-standing, thermostable gel polymer electrolyte based on plastic crystalline succinonitrile (SN) and ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF4) entrapped in copolymer poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF-HFP) is prepared and optimized for application in solvent-free solid-state supercapacitors. The synthesized gel polymer electrolyte exhibits a high ionic conductivity over a wide temperature range (from ∼5 × 10-4 S cm-1 at -30 °C up to ∼1.5 × 10-2 S cm-1 at 80 °C) with good electrochemical stability window (-2.9 to 2.5 V). Thermal studies confirm that the SN containing gel polymer electrolyte remains stable in the same gel phase over a wide temperature range from -30 to 90 °C. The electric double layer capacitors (EDLCs) have been fabricated using activated carbon as active materials and new gel polymer electrolytes. Electrochemical performance of the EDLCs is assessed through cyclic voltammetry, galvanostatic charge-discharge cycling and impedance spectroscopy. The EDLC cells with the proper SN-containing gel polymer electrolyte has been found to give high specific capacitance 176 F g-1 at 0.18 A g-1 and 138 F g-1 at 8 A g-1. These solid-state EDLC cells show good cycling stability and the capability to retain ∼80% of the initial capacitance after 10,000 cycles.

  2. Thermostable hemicellulases of a bacterium, Geobacillus sp. DC3, isolated from the former Homestake gold mine in Lead, South Dakota.

    Science.gov (United States)

    Bergdale, Terran E; Hughes, Stephen R; Bang, Sookie S

    2014-04-01

    A thermophilic strain, Geobacillus sp. DC3, capable of producing hemicellulolytic enzymes was isolated from the 1.5-km depth of the former Homestake gold mine in Lead, South Dakota. The DC3 strain expressed a high level of extracellular endoxylanase at 39.5 U/mg protein with additional hemicellulases including β-xylosidase (0.209 U/mg) and arabinofuranosidase (0.230 U/mg), after the bacterium was grown in xylan for 24 h. Partially purified DC3 endoxylanase exhibited a molecular mass of approximately 43 kDa according to zymography with an optimal pH of 7 and optimal temperature of 70 °C. The kinetic constants, K m and V max, were 13.8 mg/mL and 77.5 μmol xylose/min·mg xylan, respectively. The endoxylanase was highly stable and maintained 70 % of its original activity after 16 h incubation at 70 °C. The thermostable properties and presence of three different hemicellulases of Geobacillus sp. DC3 strain support its potential application for industrial hydrolysis of renewable biomass such as lignocelluloses.

  3. CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

    Science.gov (United States)

    Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

    2013-07-01

    The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

  4. Thermostability and surface morphology of nano- and micro-filled NBR/CSM and CR/CSM rubber blends

    Directory of Open Access Journals (Sweden)

    M. MARINOVIC-CINCOVIC

    2004-02-01

    Full Text Available Acrylonitrile-butadiene rubber (NBR, polychloroprene rubber (CR, chlorosulphonated polyethylene rubber (CSM and their blends were cross-linked with sulphur, ethylene-thiourea, magnesium oxide or their combination. The effect of nano- and micro- particle sized of 35 pphr SiO2 on the thermostability and surface morphology of all the crosslinked systems was investigated. Identification of the structure of nano- and micro- particle sized SiO2 filled NBR/CSM and CR/CSM crosslinked systems was carried out by Fourier transform infrared spectroscopy (FTIR with an attenuated total reflectance (ATR extension. The thermal stability of the nano- and micro- particle sized SiO2 filled NBR/CSM and CR/CSM crosslinked systems were carried out by thermogravimetric analysis (TGA. The glass transition temperature (Tg of the samples was determined by differential scanning calorimetry (DSC. The morphology of the fracture surface of the crosslinked systems was carried out by scanning electron microscope (SEM. The results show when filled with nano-particle sized of SiO2 NBR/CSM and CR/CSM polymer matrix have a strong peak from SiO–C at 1079 cm-1. This suggests the an interaction between the SiO2, which should lead to an increased thermal stability, higher values of Tg, better dispersion the nano-SiO2 and more polish, without cracks than micro-filled NBR/CSM and CR/CSM crosslinked systems.

  5. Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

    Science.gov (United States)

    Gutarra, Melissa L E; Godoy, Mateus G; Maugeri, Francisco; Rodrigues, Maria Isabel; Freire, Denise M G; Castilho, Leda R

    2009-11-01

    The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

  6. Effect of chemical stabilizers on the thermostability and infectivity of a representative panel of freeze dried viruses.

    Directory of Open Access Journals (Sweden)

    Boris Pastorino

    Full Text Available As a partner of the European Virus Archive (EVA FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20°C, +4°C and +20°C for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID50 assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.

  7. Novel thermostable clostridial strains through protoplast fusion for enhanced biobutanol production at higher temperature—preliminary study

    Directory of Open Access Journals (Sweden)

    Muhammad Ferhan

    2016-01-01

    Full Text Available The objective of this study is to improve the thermal stability of clostridium strains for enhanced biobutanol production. Thermostable clostridia species were developed through protoplast fusion between mesophilic clostridial species (i.e., Clostridium beijerinckii and Clostridium acetobutylicum and thermophilic clostridial species (i.e., Clostridium thermocellum. Production of biobutanol was examined in the present preliminary study using the clostridium strains and their protoplast fusants using sugar mixture with composition identical to that of wheat straw acid hydrolysate. Maximum biobutanol production of 9.4 g/L was achieved by a fused strain at 45 °C with total sugar consumption of 66% compared to that at 35 °C (i.e., 8.4 g/L production and 64% total sugar consumption. Glucose and xylose uptake rates were generally higher compared to all other individual sugars in the feedstock. In general, average cell concentrations were in close proximity for all parenting and fused strains at 35 °C; i.e., in the range of 5.12 × 107 to 5.49 × 107 cells/mL. Average cell concentration of fusants between the mesophilic clostridial species and the thermophilic clostridial species slightly increased to ~ 5.62 × 107 cells/mL at a higher temperature of 45 °C. These results, in addition to the ones obtained for the butanol production, demonstrate enhanced thermal stability of both fusants at a higher temperature (45 °C.

  8. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  9. Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanine

    International Nuclear Information System (INIS)

    Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru; Kobayashi, Michihiko; Shimizu, Sakayu

    2003-01-01

    A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable β-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of β-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various β-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the β-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the β-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed β-cyano-L-alanine synthase. Heat stable β-cyano-L-alanine synthase can be applied to the synthesis of [4- 11 C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

  10. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from Lebanese cow’s milk

    Directory of Open Access Journals (Sweden)

    Imad AL Kassaa

    2016-12-01

    Full Text Available AL Kassaa, I., Safourim, N., Mostafa, N. and Hamze, M. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from lebanese cow’s milk. 2016. Lebanese Science Journal, 17(2: 166-176. Lactic acid bacteria (LAB are used in many fields such as fermentation agents, increasing nutritional value and improving organoleptic quality of food. Also they are used as probiotics and preservatives against pathogens and spoilage microbes by producing antimicrobial substances such as bacteriocins. Fifty cow’s milk samples were collected and 175 LAB isolates were isolated and identified by using biochemical method. Fifteen isolates showed an antimicrobial activity against Listeria monocytogenes ATCC® 19115™. One strain, BL4 which showed the strongest activity, was chosen to extract and characterize its antimicrobial substance in order to evaluate its potential use as a new food protective agent. This strain was identified as Enterococcus mundtii by pyrosequencing method. The active substance was extracted using solvent method. This Bacteriocin like Inhibitory Substances “BLIS” can support a high temperature (121 ˚C for a long time and resist pH variation. The BLIS BL4 can be considered as a peptide active against many food pathogen and food-spoilage microbes, such as Listeria monocytogenes and Penicillium spp. BLIS BL4 can be used in food application as bio-preservative to reduce food-spoilage and food-borne diseases in food products.

  11. Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

    Science.gov (United States)

    Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

    2003-04-01

    Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

  12. Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

    Science.gov (United States)

    Zhu, B C; Lo, J Y; Li, Y T; Li, S C; Jaynes, J M; Gildemeister, O S; Laine, R A; Ou, C Y

    1992-07-01

    A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

  13. Production and Characterization of Thermostable Bioplastic (Poly-beta-Hydroxybutyrate) from Bacillus Cereus NRRL-B-3711

    International Nuclear Information System (INIS)

    Asad-ur-Rehman, M.; Aslam, A.; Masood, R.; Aftab, M. N.; Ajmal, R.; Haq, I. U.

    2016-01-01

    The poly-beta-hydroxybutyrate (PHB) is a thermostable and biocompatible polyester produced by several bacteria under unbalanced nutritional conditions. Among Gram-positive bacteria, which are not well exploited for PHB production on industrial scale, Bacillus appears to be a prospective candidate due to its excellent biopolymer yield and less rigorous fermentation conditions. Batch culture fermentation was carried out for PHB production. The Bacillus cereus NRRL-B-3711 was the most efficient producer of PHB out of five Bacillus species. The optimized production was achieved with 2% glucose, 37 degree C, pH value of 8 and ammonium sulfate (2.5 g/L) as a nitrogen source. Carbon to nitrogen ratio of 10 significantly affects the PHB accumulation. The selected specie was able to accumulate PHB up to 56% (14.2) 0.07 g/L) on dry cell weight basis after optimization. This corresponds to a 1.87 folds increase in the production. The optical microscopy showed extremely flat surface of bioplastic thin film indicating the brittle failure of PHB under tensile loading. The FTIR analysis revealed C=O and -CH groups, with thermal properties e.g. Tg; 2 degree C, Tc; 54 degree C, Tm;162 degree C and percent crystallinity of 51.3 by differential scanning calorimeter (DSC), thus confirming the presence of PHB bioplastic and enhancing its industrial applications.The results surpassed those reported in the literature for PHB production. (author)

  14. Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate.

    Science.gov (United States)

    Kumar, Satyendra; Pahujani, Shweta; Ola, R P; Kanwar, S S; Gupta, Reena

    2006-06-01

    A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.

  15. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

    Directory of Open Access Journals (Sweden)

    Guo-Qing Zhang

    2013-01-01

    Full Text Available A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM.

  16. Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

    Energy Technology Data Exchange (ETDEWEB)

    Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

    2003-10-01

    A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

  17. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

    Science.gov (United States)

    Zhang, Guo-Qing; Wu, Ying-Ying; Ng, Tzi-Bun; Chen, Qing-Jun; Wang, He-Xiang

    2013-01-01

    A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM. PMID:23586045

  18. Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation.

    Science.gov (United States)

    Chatterjee, Joyee; Giri, Sudipta; Maity, Sujan; Sinha, Ankan; Ranjan, Ashish; Rajshekhar; Gupta, Suvroma

    2015-01-01

    Proteases are the most important group of enzymes utilized commercially in various arenas of industries, such as food, detergent, leather, dairy, pharmaceutical, diagnostics, and waste management, accounting for nearly 20% of the world enzyme market. Microorganisms of specially Bacillus genera serve as a vast repository of diverse set of industrially important enzymes and utilized for the large-scale enzyme production using a fermentation technology. Approximately 30%-40% of the cost of industrial enzymes originates from the cost of the growth medium. This study is attempted to produce protease from Bacillus subtilis (ATCC 6633) after optimization of various process parameters with the aid of solid-state fermentation using a cheap nutrient source such as wheat bran. B. subtilis (ATCC 6633) produces proteases of molecular weight 36 and 20 kDa, respectively, in the fermented medium as evident from SDS zymogram. Alkaline protease activity has been detected with optimum temperature at 50 °C and is insensitive to ethylenediaminetetraacetic acid. This thermostable alkaline protease exhibits dual pH optimum at 7 and 10 with moderate pH stability at alkaline pH range. It preserves its activity in the presence of detergent such as SDS, Tween 20, and Triton X-100 and may be considered as an effective additive to detergent formulation with some industrial importance. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  19. APPLICATION OF A PRIMAL-DUAL INTERIOR POINT ALGORITHM USING EXACT SECOND ORDER INFORMATION WITH A NOVEL NON-MONOTONE LINE SEARCH METHOD TO GENERALLY CONSTRAINED MINIMAX OPTIMISATION PROBLEMS

    Directory of Open Access Journals (Sweden)

    INTAN S. AHMAD

    2008-04-01

    Full Text Available This work presents the application of a primal-dual interior point method to minimax optimisation problems. The algorithm differs significantly from previous approaches as it involves a novel non-monotone line search procedure, which is based on the use of standard penalty methods as the merit function used for line search. The crucial novel concept is the discretisation of the penalty parameter used over a finite range of orders of magnitude and the provision of a memory list for each such order. An implementation within a logarithmic barrier algorithm for bounds handling is presented with capabilities for large scale application. Case studies presented demonstrate the capabilities of the proposed methodology, which relies on the reformulation of minimax models into standard nonlinear optimisation models. Some previously reported case studies from the open literature have been solved, and with significantly better optimal solutions identified. We believe that the nature of the non-monotone line search scheme allows the search procedure to escape from local minima, hence the encouraging results obtained.

  20. Engineering a disulfide bond in the lid hinge region of Rhizopus chinensis lipase: increased thermostability and altered acyl chain length specificity.

    Directory of Open Access Journals (Sweden)

    Xiao-Wei Yu

    Full Text Available The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for "interfacial activation" is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the "Disulfide by Design" algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t(1/2 value at 60°C and a 7°C increase of T(m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k(cat and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.

  1. Effect of pulsed electric field treatment on enzyme kinetics and thermostability of endogenous ascorbic acid oxidase in carrots (Daucus carota cv. Nantes).

    Science.gov (United States)

    Leong, Sze Ying; Oey, Indrawati

    2014-03-01

    The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

    Science.gov (United States)

    França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

    2016-10-15

    A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Constraint Network Analysis (CNA): a Python software package for efficiently linking biomacromolecular structure, flexibility, (thermo-)stability, and function.

    Science.gov (United States)

    Pfleger, Christopher; Rathi, Prakash Chandra; Klein, Doris L; Radestock, Sebastian; Gohlke, Holger

    2013-04-22

    For deriving maximal advantage from information on biomacromolecular flexibility and rigidity, results from rigidity analyses must be linked to biologically relevant characteristics of a structure. Here, we describe the Python-based software package Constraint Network Analysis (CNA) developed for this task. CNA functions as a front- and backend to the graph-based rigidity analysis software FIRST. CNA goes beyond the mere identification of flexible and rigid regions in a biomacromolecule in that it (I) provides a refined modeling of thermal unfolding simulations that also considers the temperature-dependence of hydrophobic tethers, (II) allows performing rigidity analyses on ensembles of network topologies, either generated from structural ensembles or by using the concept of fuzzy noncovalent constraints, and (III) computes a set of global and local indices for quantifying biomacromolecular stability. This leads to more robust results from rigidity analyses and extends the application domain of rigidity analyses in that phase transition points ("melting points") and unfolding nuclei ("structural weak spots") are determined automatically. Furthermore, CNA robustly handles small-molecule ligands in general. Such advancements are important for applying rigidity analysis to data-driven protein engineering and for estimating the influence of ligand molecules on biomacromolecular stability. CNA maintains the efficiency of FIRST such that the analysis of a single protein structure takes a few seconds for systems of several hundred residues on a single core. These features make CNA an interesting tool for linking biomacromolecular structure, flexibility, (thermo-)stability, and function. CNA is available from http://cpclab.uni-duesseldorf.de/software for nonprofit organizations.

  4. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Shalini RAI

    2014-03-01

    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  5. Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

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    P. Gururaj

    Full Text Available ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v inoculum, 2% (v/v castor oil (inducer, and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

  6. Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

    Science.gov (United States)

    Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

    2015-01-01

    Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

  7. A decoy set for the thermostable subdomain from chicken villin headpiece, comparison of different free energy estimators

    Directory of Open Access Journals (Sweden)

    Tosatto Silvio CE

    2005-12-01

    Full Text Available Abstract Background Estimators of free energies are routinely used to judge the quality of protein structural models. As these estimators still present inaccuracies, they are frequently evaluated by discriminating native or native-like conformations from large ensembles of so-called decoy structures. Results A decoy set is obtained from snapshots taken from 5 long (100 ns molecular dynamics (MD simulations of the thermostable subdomain from chicken villin headpiece. An evaluation of the energy of the decoys is given using: i a residue based contact potential supplemented by a term for the quality of dihedral angles; ii a recently introduced combination of four statistical scoring functions for model quality estimation (FRST; iii molecular mechanics with solvation energy estimated either according to the generalized Born surface area (GBSA or iv the Poisson-Boltzmann surface area (PBSA method. Conclusion The decoy set presented here has the following features which make it attractive for testing energy scoring functions: 1 it covers a broad range of RMSD values (from less than 2.0 Å to more than 12 Å; 2 it has been obtained from molecular dynamics trajectories, starting from different non-native-like conformations which have diverse behaviour, with secondary structure elements correctly or incorrectly formed, and in one case folding to a native-like structure. This allows not only for scoring of static structures, but also for studying, using free energy estimators, the kinetics of folding; 3 all structures have been obtained from accurate MD simulations in explicit solvent and after molecular mechanics (MM energy minimization using an implicit solvent method. The quality of the covalent structure therefore does not suffer from steric or covalent problems. The statistical and physical effective energy functions tested on the set behave differently when native simulation snapshots are included or not in the set and when averaging over the

  8. Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability.

    Science.gov (United States)

    Thomaeus, Ann; Naworyta, Agata; Mowbray, Sherry L; Widersten, Mikael

    2008-07-01

    A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro(186), Leu(266), His(269), and the His(153) imidazole. The hydroxyl of Tyr(149) is also an integrated component of the chain, which leads to the hydroxyl of Tyr(154). Available data suggest that Tyr(154) functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 A resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k (cat)/K (M) was similar or slightly increased compared with the wild-type reactions. k (cat) for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K (M). Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k (cat) but lowered thermostability.

  9. Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

    Directory of Open Access Journals (Sweden)

    Fatma Matpan Bekler

    2016-01-01

    Full Text Available A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purifi ed and characterized. Maximum enzyme production (1874.8 U/mL was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9 % were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE. The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 μmol and 0.929 μmol/min, espectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA and 1,10-phenanthroline (phen greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME and dithiothreitol (DTT to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB. Iodoacetamide (IAA and N-ethylmaleimide (NEM had a slight, whereas phenylmethylsulfonyl fluoride (PMSF had a strong inhibitory effect on the amylase activity.

  10. Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

    Science.gov (United States)

    Jørgensen, F; Hansen, O C; Stougaard, P

    2004-06-01

    The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

  11. Effects of Calcium Ions on the Thermostability and Spectroscopic Properties of the LH1-RC Complex from a New Thermophilic Purple Bacterium Allochromatium tepidum.

    Science.gov (United States)

    Kimura, Yukihiro; Lyu, Shuwen; Okoshi, Akira; Okazaki, Koudai; Nakamura, Natsuki; Ohashi, Akira; Ohno, Takashi; Kobayashi, Manami; Imanishi, Michie; Takaichi, Shinichi; Madigan, Michael T; Wang-Otomo, Zheng-Yu

    2017-05-18

    The light harvesting-reaction center (LH1-RC) complex from a new thermophilic purple sulfur bacterium Allochromatium (Alc.) tepidum was isolated and characterized by spectroscopic and thermodynamic analyses. The purified Alc. tepidum LH1-RC complex showed a high thermostability comparable to that of another thermophilic purple sulfur bacterium Thermochromatium tepidum, and spectroscopic characteristics similar to those of a mesophilic bacterium Alc. vinosum. Approximately 4-5 Ca 2+ per LH1-RC were detected by inductively coupled plasma atomic emission spectroscopy and isothermal titration calorimetry. Upon removal of Ca 2+ , the denaturing temperature of the Alc. tepidum LH1-RC complex dropped accompanied by a blue-shift of the LH1 Q y absorption band. The effect of Ca 2+ was also observed in the resonance Raman shift of the C3-acetyl νC═O band of bacteriochlorophyll-a, indicating changes in the hydrogen-bonding interactions between the pigment and LH1 polypeptides. Thermodynamic parameters for the Ca 2+ -binding to the Alc. tepidum LH1-RC complex indicated that this reaction is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. Our data support a hypothesis that Alc. tepidum may be a transitional organism between mesophilic and thermophilic purple bacteria and that Ca 2+ is one of the major keys to the thermostability of LH1-RC complexes in purple bacteria.

  12. Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

    International Nuclear Information System (INIS)

    Manikandan, K.; Bhardwaj, Amit; Ghosh, Amit; Reddy, V. S.; Ramakumar, S.

    2005-01-01

    A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution

  13. Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

    Energy Technology Data Exchange (ETDEWEB)

    Manikandan, K. [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bhardwaj, Amit [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ghosh, Amit [Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036 (India); Reddy, V. S. [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ramakumar, S., E-mail: ramak@physics.iisc.ernet.in [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012 (India)

    2005-08-01

    A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution.

  14. Study of the leaching of heavy metals from waste water sludge and incinerator's ash, using coupled thermostated columns and DTPA as complex agent

    International Nuclear Information System (INIS)

    Vite T, J.; Vite T, M.; Guerrero D, J.; Carreno de Leon, M.C.

    2000-01-01

    We studied the metallic composition from waste water sludge and incinerators ashes of an incinerator located in Toluca, Mexico, the qualitative studies were made using the Activation Analysis technique, and fluorescence X-ray techniques. The quantitative analysis of heavy metals in the wastes were made using Inductively coupled plasma atomic emission spectrometry (Icp-Aes). For leaching the samples, we used four coupled thermostated columns, each one had a p H of 2,5, 7 and 10. The flux of the air was of 1600 cc/min. The temperature was maintain constant in 60 Centigrade using a thermostated system. For this study we used 100 g of wastes mixed with mineral acid or sodium hydroxide to reach p H 2,5,7 and 10. We added a reducing and tensoactive agents and finally DTPA as complex agent. With this method, we obtain a better leaching efficiency using a complex agent. However the high DTPA cost, make this process expansive that is why we recommend to work with another classes of complex agents, that be cheaper to leach metals of different chemistry matrix. (Author)

  15. Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry.

    Science.gov (United States)

    Niu, Chengtuo; Zhu, Linjiang; Hill, Annie; Alex Speers, R; Li, Qi

    2017-01-01

    To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing. A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg -1 and 189 s -1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml -1 , which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively. The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

  16. Purification and properties of a thermostable pullulanase from Clostridium thermosulfurgenes EM1 which hydrolyses both. alpha. -1,6 and. alpha. -1,4-glycosidic linkages

    Energy Technology Data Exchange (ETDEWEB)

    Spreinat, A [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the {alpha}-1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an {alpha}-1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K{sub m} values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60deg-65deg C and a pH optimum at 5.5-6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. (orig.).

  17. Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2

    International Nuclear Information System (INIS)

    Shariff, Fairolniza Mohd; Rahman, Raja Noor Zaliha Raja Abd.; Ali, Mohd Shukuri Mohamad; Chor, Adam Leow Thean; Basri, Mahiran; Salleh, Abu Bakar

    2010-01-01

    Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P2 1 2 1 2 1 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V M ) of 2.85 Å 3 Da −1 and a solvent content of 57%

  18. Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup.

    Science.gov (United States)

    Jia, Dong-Xu; Zhou, Lin; Zheng, Yu-Guo

    2017-04-01

    Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn 2+ at 85°C for 48h. The K m and k cat /K m values for ToGI were 81.46mM and 21.77min -1 mM -1 , respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Production of extremely alkaliphilic, halotolerent, detergent, and thermostable mannanase by the free and immobilized cells of Bacillus halodurans PPKS-2. Purification and characterization.

    Science.gov (United States)

    Vijayalaxmi, S; Prakash, P; Jayalakshmi, S K; Mulimani, V H; Sreeramulu, K

    2013-09-01

    The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0-16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.

  20. Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07.

    Science.gov (United States)

    Gaur, Rajeeva; Tiwari, Soni

    2015-03-19

    The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml(-1)) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca(2+), mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml(-1) and 769.230 μg ml(-1) min(-1), respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for

  1. Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

    Science.gov (United States)

    Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

    2017-07-01

    The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the

  2. Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

    Science.gov (United States)

    Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

    2018-06-08

    Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Thermostable Shelf Life Study

    Science.gov (United States)

    Perchonok, M. H.; Antonini, D. K.

    2008-01-01

    The objective of this project is to determine the shelf life end-point of various food items by means of actual measurement or mathematical projection. The primary goal of the Advanced Food Technology Project in these long duration exploratory missions is to provide the crew with a palatable, nutritious and safe food system while minimizing volume, mass, and waste. The Mars missions could be as long as 2.5 years with the potential of the food being positioned prior to the crew arrival. Therefore, it is anticipated that foods that are used during the Mars missions will require a 5 year shelf life. Shelf life criteria are safety, nutrition, and acceptability. Any of these criteria can be the limiting factor in determining the food's shelf life. Due to the heat sterilization process used for the thermostabilized food items, safety will be preserved as long as the integrity of the package is maintained. Nutrition and acceptability will change over time. Since the food can be the sole source of nutrition to the crew, a significant loss in nutrition may determine when the shelf life endpoint has occurred. Shelf life can be defined when the food item is no longer acceptable. Acceptability can be defined in terms of appearance, flavor, texture, or aroma. Results from shelf life studies of the thermostabilized food items suggest that the shelf life of the foods range from 0 months to 8 years, depending on formulation.

  4. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

    Directory of Open Access Journals (Sweden)

    Sigoillot Jean-Claude

    2009-11-01

    Full Text Available Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS. Results A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78, commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5, was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity, locust bean gum galactomannan, carob galactomannan (low viscosity, and 1,4-β-D-mannan (from carob are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

  5. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

    Science.gov (United States)

    Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

    2009-11-13

    Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

  6. Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes.

    Science.gov (United States)

    Mohammad, Balsam T; Al Daghistani, Hala I; Jaouani, Atef; Abdel-Latif, Saleh; Kennes, Christian

    2017-01-01

    The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis . This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

  7. Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes

    Directory of Open Access Journals (Sweden)

    Balsam T. Mohammad

    2017-01-01

    Full Text Available The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis. This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

  8. A Thermostable Salmonella Phage Endolysin, Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

    DEFF Research Database (Denmark)

    Oliveira, Hugo; Thiagarajan, Viruthachalam; Walmagh, Maarten

    2014-01-01

    Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined...... with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min......, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid...

  9. Pigeon pea waste as a novel, inexpensive, substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99.

    Science.gov (United States)

    Johnvesly, B; Manjunath, B R; Naik, G R

    2002-03-01

    Thermoalkaliphilic Bacillus sp. JB-99 was grown in a 250 ml Erlenmeyer flask containing 50 ml medium containing (g/l) Pigeon pea waste 10; NaNO3, 5.0; K2HPO4, 5.0; MgSO4 x 2H2O, 0.2 and Na2CO3, 10.0. Incubations were carried out at 50 degrees C on a rotary incubator shaker for 15 h. A high level of extra cellular thermostable protease activity was observed after 24 h incubation. The optimum temperature and pH for activity were 70 degrees C and 11, respectively, so this enzyme showed stable activity at high temperature and under alkaline conditions.

  10. A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

    Directory of Open Access Journals (Sweden)

    Hugo Oliveira

    Full Text Available Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68 and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively. Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermostability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

  11. Characterization of a thermostable recombinant l-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 and its application for the production of l-fructose and l-rhamnulose.

    Science.gov (United States)

    Chen, Ziwei; Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2018-04-01

    l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized. The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co 2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co 2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg -1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L -1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L -1 l-mannose, respectively. The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  12. Allergy to fish collagen: Thermostability of collagen and IgE reactivity of patients' sera with extracts of 11 species of bony and cartilaginous fish.

    Science.gov (United States)

    Kobayashi, Yukihiro; Kuriyama, Takuma; Nakagawara, Ryoko; Aihara, Michiko; Hamada-Sato, Naoko

    2016-10-01

    Parvalbumin was identified as a major fish allergen, and has been well investigated. Collagen was identified as a second allergen; however, its allergenic properties remain uncharacterized. Although fish is an important staple in coastal countries, its thermostability is unknown. Therefore, we aimed to determine the thermostability of fish collagen as an allergen. Meat of seven bony and four cartilaginous fishes was heated at various temperatures and times, and extracts were analyzed using SDS-PAGE, IgE-ELISA, and SPTs. Collagen was dissolved from heated meat of Pacific mackerel into a crude extract. Collagen in the extracts was degraded at a high heating load-140 °C (10 min) or 100 °C (320 min). However, ELISA revealed the IgE reactivities of patients' sera with the extracts were unchanged even after heating the samples. Patients strongly reacted to extract proteins of other bony fish, which were detected by patients' IgE even after heating at 100 °C (320 min). In contrast, reactivities of the extracts of cartilaginous fish were lower than those of bony fish. SPTs in one patient revealed that all bony and cartilaginous fish extracts prepared from heated meat elicited allergic reactions. The IgE reactivity of patients' sera to fish collagen in extracts was retained even when fish meat was treated by a high heating load. As for the fish collagen, the IgE reactivities to cartilaginous fish were lower than that to bony fish. Reducing IgE reactivity to fish meat using heat is difficult, and other modalities will be required to produce hypoallergenic fish meat. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  13. Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Jung-Sue [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of); Rhee, Jin-Kyu [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Oh, Jong-Won [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2006-02-01

    Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method. EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4–C6 at a temperature of 303–363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est{sub pc}-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 Å resolution from an EstE1 crystal; the crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 73.71, c = 234.23 Å. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient V{sub M} is calculated to be 2.2 Å{sup 3} Da{sup −1} and the solvent content is 44.1%.

  14. Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

    Science.gov (United States)

    Ueno, Tomohiro; Niimi, Hideki; Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

    2015-01-01

    Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and

  15. 包覆赤磷制备工艺及其热安定性研究%Preparation technique of microcapsulated red phosphorusand its thermostability

    Institute of Scientific and Technical Information of China (English)

    刘杰; 关华; 宋东明

    2017-01-01

    为了降低赤磷的吸湿性,提高其热安定性,该文采用氢氧化铝-密胺树脂包覆赤磷(MRP).通过单因素实验优化了制备包覆赤磷的工艺,采用傅里叶变换红外(FTIR)光谱仪和扫描电子显微镜(SEM)对包覆层进行了表征,用热重-差热分析法(TG-DTA)分析了包覆前后赤磷样品的热安定性.结果表明,制备包覆赤磷的最佳工艺参数为,九水合硝酸铝的质量为0.10 g,甲醛和三聚氰胺总质量为0.40 g、摩尔比为3∶1,反应温度为80 ℃,反应时间为1 h.在此工艺条件下,包覆赤磷(MRP)的吸湿率降幅最大,达到96.88%,且包覆物质量分数小于5%,包覆材料在赤磷表面的包覆状况良好.相比于未包覆赤磷,包覆后赤磷的外推起始温度、峰值温度和外推终点温度分别提高42.8 ℃、51.7 ℃和48.4 ℃;自发火温度提高58.8 ℃,表明包覆赤磷的热安定性大幅提高.%In order to reduce the hygroscopicity of red phosphorus and improve its thermostability,the aluminium hydroxide/melamine-formaldehyde resin microcapsulated red phosphorus(MRP)is successfully prepared.The process of preparing the coated red phosphorus is optimized by the single factor test.The coating layer of the red phosphorus is characterized by the Fourier transform infrared(FTIR)spectrometer and the scanning electron microscopy(SEM).The thermostability of the red phosphorus(RP)and the MRP is investigated by the thermogravimetric analysis-differential thermal analysis(TG-DTA)in air atmosphere.Results show that the optimal process parameters for preparing the coated red phosphorus are,the mass of the aluminum nitrate nonahydrate 0.10 g,the total mass of the formaldehyde and melamine 0.40 g,the molar ratio of the formaldehyde and the melamine 3∶1,the reaction temperature 80 ℃,the reaction time 1 h.Under these optimal process parameters,the moisture absorption ratio of MRP reduces 96.88%(the greatest reduction),the mass fraction of coating materials is below 5

  16. Enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c(552) through introduction of an extra methylene group into its hydrophobic protein interior.

    Science.gov (United States)

    Tai, Hulin; Irie, Kiyofumi; Mikami, Shin-ichi; Yamamoto, Yasuhiko

    2011-04-19

    Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c(552) (HT) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729-25734] indicated that a void space, which is large enough to accommodate a methyl group, exists in the hydrophobic protein interior near the heme. We tried to reduce the void space through the replacement of a Val by Ile or Leu (Val/Ile or Val/Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The Val/Ile mutation was found to cause further enhancement of the thermostability of the oxidized HT, as reflected in the increase of the denaturation temperature (T(m)) value by ∼ 3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E(m)) of the Val/Ile mutant exhibited a negative shift of ∼ 50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E(m) value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650-13651]. In contrast, the Val/Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T(m) values of the oxidized and reduced proteins by ∼ 3 and ∼ 5 deg, respectively, and the E(m) value of the Val/Leu mutant happened to be similar to that of the Val/Ile one. The E

  17. Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Pichapak Sriyapai

    2015-06-01

    Full Text Available A thermostable esterase gene (hydS14 was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG and catalytic triad (Ser88-Asp208-His235 of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases, has three conserved regions, and contains the novel motif (GY(FSLG, which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14 was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6, displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1. In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

  18. A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

    Science.gov (United States)

    Kim, Hye-Jung; Ryu, Se-Ah; Kim, Pil; Oh, Deok-Kun

    2003-01-01

    To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.

  19. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges.

    Science.gov (United States)

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, psalt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (psalt-bridges and smaller volume nonpolar residues (Gly, Ala and Val) and lesser occurrence of bulky polar residues in the thermophilic proteins. A more stoichiometric relationship amongst these factors minimized the hindrance due to side chain burial and increased compactness and secondary structural stability in thermophilic proteins.

  20. Characterization of an organic solvent-tolerant thermostable glucoamylase from a halophilic isolate, Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production.

    Science.gov (United States)

    Yu, Hui-Ying; Li, Xin

    2014-01-01

    A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin-layer chromatography revealed that glucose was the sole end-product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0-100°C), pH (7.0-12.0), and NaCl concentration (0-20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers.

  1. Development of a D-amino acids electrochemical sensor based on immobilization of thermostable D-Proline dehydrogenase within agar gel membrane

    International Nuclear Information System (INIS)

    Tani, Yuji; Tanaka, Katsuhito; Yabutani, Tomoki; Mishima, Yuji; Sakuraba, Haruhiko; Ohshima, Toshihisa; Motonaka, Junko

    2008-01-01

    A novel biosensor for determination of D-amino acids (DAAs) in biological samples by using an electrode based on immobilization of a thermostable D-Proline dehydrogenase (D-Pro DH) within an agar gel membrane was developed. The electrode was simply prepared by spin-coating the agar solution with the D-Pro DH on a glassy carbon (GC) electrode. An electrocatalytic oxidation current of 2,6-dichloroindophenol (DCIP) was observed at -100 mV vs. Ag/AgCl with the addition of 5 and 20 mmol L -1 D-proline. The current response and its relative standard deviation were 0.15 μA and 7.6% (n = 3), respectively, when it was measured in a pH 8.0 phosphate buffer solution containing 10 mmol L -1 D-proline and 0.5 mmol L -1 DCIP at 50 deg. C. The current response of D-proline increased with increase of the temperature of the sample solution up to 70 deg. C. The electrocatalytic response at the D-Pro DH/agar immobilized electrode subsequently maintained for 80 days. Finally, the D-Pro DH/agar immobilized electrode was applied to determination of DAAs in a human urine sample. The determined value of DAAs in the human urine and its R.S.D. were 1.39 ± 0.12 mmol L -1 and 8.9% (n = 3), respectively

  2. Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry.

    Science.gov (United States)

    Niu, Canfang; Yang, Peilong; Luo, Huiying; Huang, Huoqing; Wang, Yaru; Yao, Bin

    2017-08-30

    Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

  3. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    Science.gov (United States)

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  4. Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation.

    Science.gov (United States)

    Liang, Min; Chen, Min; Liu, Xinying; Zhai, Yafei; Liu, Xian-wei; Zhang, Houcheng; Xiao, Min; Wang, Peng

    2012-02-01

    The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.

  5. Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans

    International Nuclear Information System (INIS)

    Doan, Thi-Ngoc-Thanh; Prabhu, Ponnandy; Kim, Jin-Kwang; Ahn, Yeh-Jin; Natarajan, Sampath; Kang, Lin-Woo; Park, Geon Tae; Lim, Sang-Boem; Lee, Jung-Kul

    2010-01-01

    l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution. l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å 3 Da −1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method

  6. Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77

    Directory of Open Access Journals (Sweden)

    Wajdi Thebti

    2016-01-01

    Full Text Available A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1.

  7. Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure.

    Science.gov (United States)

    Ishak, Siti Nor Hasmah; Aris, Sayangku Nor Ariati Mohamad; Halim, Khairul Bariyyah Abd; Ali, Mohd Shukuri Mohamad; Leow, Thean Chor; Kamarudin, Nor Hafizah Ahmad; Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd

    2017-09-25

    Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.

  8. thermo-stable alpha-amylase(S) from irradiated microbial origin utilizing agricultural and environmental wastes under solid state fermentation conditions

    International Nuclear Information System (INIS)

    Moustafa, O.E.A.

    2002-01-01

    an investigation concerning the production of thermo-stable α-amylases by thermophilic bacterial and fungal isolates has been undertaken. nine thermophilic bacteria and five teen fungi were isolated from different localities viz. phyllosphere of water hyacinth, different desert plants leaves, fermented dough, oven dust, garbage , and soil. their amylolytic activities were tested by dinitrosalicylic acid color reagent (Dns) method when grown on some environmental pollutants (garbage and water hyacinth) as well as industrial wastes (Bagasse, biscuit, corn flex and dough residues ) as the sole carbon source at 65 o C for bacterial and at 50 o C for fungal isolates . isolates No. B 1 ,B 2 ,B 5 ,B 6 ,B 7 ,B 8 ,B 9 , and F 4 ,F 6 ,F 8 ,F 1 2,F 1 3 and F 1 5, exhibited the highest α -amylase production when grown on water hyacinth, while B 4 ,F 3 ,F 1 1 and F 1 3, on dough ; (B 3 ,F 9 and F 1 0 ) on bagasse and ( F 1 ,F 2 ,F 5 ,F 7 ,F 1 1 and F 1 4) on garbage. Out of the nine identified bacterial isolated, only two isolates viz; actinobacillus actinomycetemcomitans, B1 and strepto bacillus moniliformis, B 7 , exhibited the ability to produce high percentages of α amylases at 55 o C (while still able to produce the enzyme within 45-70 o C)

  9. Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

    Science.gov (United States)

    Yildirim, Vildan; Baltaci, Mustafa Ozkan; Ozgencli, Ilknur; Sisecioglu, Melda; Adiguzel, Ahmet; Adiguzel, Gulsah

    2017-12-01

    An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K M and V max values were calculated as 0.197 mg/mL and 7.29 μmol.mL - 1 .min - 1 , respectively.

  10. Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

    Directory of Open Access Journals (Sweden)

    Nadia Andrea Andreani

    2016-12-01

    Full Text Available Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

  11. A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

    Science.gov (United States)

    Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

    2017-12-11

    The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

  12. Evaluating the effects of buffer conditions and extremolytes on thermostability of granulocyte colony-stimulating factor using high-throughput screening combined with design of experiments.

    Science.gov (United States)

    Ablinger, Elisabeth; Hellweger, Monika; Leitgeb, Stefan; Zimmer, Andreas

    2012-10-15

    In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis. pH was identified as most critical factor regarding thermal stability. The most stabilizing buffer, sodium glutamate, and sodium acetate were determined for further investigations. Second we tested the effect of 6 naturally occurring extremolytes (trehalose, sucrose, ectoine, hydroxyectoine, sorbitol, mannitol) on the thermal stability of G-CSF, using a central composite circumscribed design. At low pH (3.8) and low buffer concentration (5 mM) all extremolytes led to a significant increase in thermal stability except the addition of ectoine which resulted in a strong destabilization of G-CSF. Increasing pH and buffer concentration led to an increase in thermal stability with all investigated extremolytes. The described systematic approach allowed to create a ranking of stabilizing extremolytes at different buffer conditions. Copyright © 2012. Published by Elsevier B.V.

  13. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

    Directory of Open Access Journals (Sweden)

    Steven M. Swift

    2015-06-01

    Full Text Available Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

  14. Effect of enzymatic (thermostable α-amylase) treatment on the physicochemical and antioxidant properties of extruded rice incorporated with soybean flour.

    Science.gov (United States)

    Xu, Enbo; Wu, Zhengzong; Pan, Xiaowei; Long, Jie; Wang, Fang; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

    2016-04-15

    In order to determine the effect of enzymatic extrusion on the physicochemical and antioxidant properties of rice/soybean mixture, different mass ratios (100/0, 95/5, 85/15, 70/30, 50/50 and 25/75%, w/w) were treated with thermostable α-amylase. The reduced special mechanical energy and the enhanced product temperature were closely and regularly linked with the increase of soybean content. The bulk density and water solubility index increased, and the water absorption index and viscosities decreased remarkably after enzymatic extrusion, however, the modification caused by α-amylase were dramatically eliminated with the increase of soybean content to ∼50%. Moreover, the addition of enzyme exhibited an improvement of the total phenolic/flavonoid content (TPC/TFC) and antioxidant capacities compared to traditional extrusion. The TPC/TFC retention of extrudate (ratios of 85/15 and 70/30%) attained over 90%, but dramatically decreased (72.91 and 67.81%, respectively) with soybean added to 75%, probably due to the great reduction of starch substrate for enzymatic hydrolysis. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  15. Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity.

    Science.gov (United States)

    Shrinivas, Dengeti; Kumar, Raghwendra; Naik, G R

    2012-01-01

    The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.

  16. A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

    Science.gov (United States)

    Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

    2010-11-01

    A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

  17. An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5.

    Science.gov (United States)

    Rachadech, W; Navacharoen, A; Ruangsit, W; Pongtharangkul, T; Vangnai, A S

    2010-01-01

    Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.

  18. Default logic and specification of nonmonotonic reasoning

    NARCIS (Netherlands)

    Engelfriet, J.; Marek, V.W.; Treur, J.; Truszczynski, M.

    2001-01-01

    In this paper constructions leading to the formation of belief sets by agents are studied. The focus is on the situation when possible belief sets are built incrementally in stages. An infinite sequence of theories that represents such a process is called a reasoning trace. A set of reasoning traces

  19. Semantic matchmaking with nonmonotonic description logics

    CERN Document Server

    Grimm, S

    2009-01-01

    Semantic web has grown into a mature field of research. Its methods find innovative applications on and off the World Wide Web. Its underlying technologies have significant impact on adjacent fields of research and on industrial applications. This new book series reports on the state-of-the-art in foundations, methods, and applications of semantic web and its underlying technologies. It is a central forum for the communication of recent developments and comprises research monographs, textbooks and edited volumes on all topics related to the semantic web. In this first volume several non-monoto

  20. Nonmonotonic Reasoning as a Temporal Activity

    OpenAIRE

    Schwartz, Daniel G.

    2014-01-01

    A {\\it dynamic reasoning system} (DRS) is an adaptation of a conventional formal logical system that explicitly portrays reasoning as a temporal activity, with each extralogical input to the system and each inference rule application being viewed as occurring at a distinct time step. Every DRS incorporates some well-defined logic together with a controller that serves to guide the reasoning process in response to user inputs. Logics are generic, whereas controllers are application-specific. E...

  1. Non-monotonic compositional dependence of isothermal bulk modulus of the (Mg1–xMnxCr2O4 spinel solid solutions, and its origin and implication

    Directory of Open Access Journals (Sweden)

    Xi Liu

    2016-12-01

    Full Text Available The compressibility of the spinel solid solutions, (Mg1−xMnxCr2O4 with x = 0.00 (0, 0.20 (0, 0.44 (2, 0.61 (2, 0.77 (2 and 1.00 (0, has been investigated by using a diamond-anvil cell coupled with synchrotron X-ray radiation up to ∼10 GPa (ambient T. The second-order Birch–Murnaghan equation of state was used to fit the PV data, yielding the following values for the isothermal bulk moduli (KT, 198.2 (36, 187.8 (87, 176.1 (32, 168.7 (52, 192.9 (61 and 199.2 (61 GPa, for the spinel solid solutions with x = 0.00 (0, 0.20 (0, 0.44 (2, 0.61 (2, 0.77 (2 and 1.00 (0, respectively (KT′ fixed as 4. The KT value of the MgCr2O4 spinel is in good agreement with existing experimental determinations and theoretical calculations. The correlation between the KT and x is not monotonic, with the KT values similar at both ends of the binary MgCr2O4MnCr2O4, but decreasing towards the middle. This non-monotonic correlation can be described by two equations, KT = −49.2 (11x + 198.0 (4 (x ≤ ∼0.6 and KT = 92 (41x + 115 (30 (x ≥ ∼0.6, and can be explained by the evolution of the average bond lengths of the tetrahedra and octahedra of the spinel solid solutions. Additionally, the relationship between the thermal expansion coefficient and composition is correspondingly reinterpreted, the continuous deformation of the oxygen array is demonstrated, and the evolution of the component polyhedra is discussed for this series of spinel solid solutions. Our results suggest that the correlation between the KT and composition of a solid solution series may be complicated, and great care should be paid while estimating the KT of some intermediate compositions from the KT of the end-members.

  2. Lactate dehydrogenase of Mugil sp. (Mugilidae, Perciformes. Lack of electrokinetic, thermostability and kinetic differences among individuals with different number of scales

    Directory of Open Access Journals (Sweden)

    Marcelo dos Santos

    2000-03-01

    Full Text Available The scale number in lateral sets (SNS of Mugil sp. (Mugilidae, Perciformes collected in the lagoon-estuarine region of Cananéia, State of São Paulo ranges from 33 to 39. Electrokinetic, kinetic and thermostability properties of lactate dehydrogenase (LDH were tested to determine if individuals with different SNS correspond to different species or populations of mullet. As in many other teleosts, LDH-A*, LDH-B*, and LDH-C* loci were detected. Through a two-fold serial dilution method applied to 10 different tissues of Mugil sp., a bidirectionally divergent expression of these loci was suggested. No association among LDH electrophoretic pattern, thermal inactivation, kinetic responses and different SNS was observed. The apparent Km (pyr values obtained here were similar to Km values obtained by other authors for muscle and heart LDH or their purified isoforms. The effect of NaCl on Km and Vmax values of Mugil sp. (35 and 39 SNS individuals indicates that this salt behaves as a competitive inhibitor, since it decreases enzyme-substrate affinity. Thus, electrokinetic and thermostability behavior, Km and Vmax values and the effect of NaCl do not permit us to consider these mullets, with SNS ranging from 33 to 39, as belonging to different populations or species.O número de escamas em séries laterais (SNS de exemplares de Mugil sp. (Mugilidae, Perciformes coletados na região estuarino-lagunar de Cananéia, Estado de São Paulo, varia de 33 a 39. A fim de tentar determinar se exemplares com diferentes SNS corresponderiam a diferentes espécies ou populações de tainhas, foram analisadas as propriedades eletrocinéticas, cinéticas e de termoestabilidade da sua lactato desidrogenase (LDH. A exemplo de muitos teleósteos, a LDH de Mugil sp. mostrou-se codificada por 3 locos gênicos: LDH-A*, LDH-B* e LDH-C*. Método de diluições seriadas aplicado a 10 diferentes tecidos dessa espécie sugeriu um padrão bidirecionalmente divergente de express

  3. Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.

    Science.gov (United States)

    He, Dong; Luo, Wen; Wang, Zhiyuan; Lv, Pengmei; Yuan, Zhenhong; Huang, Shaowei; Xv, Jingliang

    2017-07-01

    Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T 50 60 -value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Directory of Open Access Journals (Sweden)

    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  5. Application of a novel alkali-tolerant thermostable DyP-type peroxidase from Saccharomonospora viridis DSM 43017 in biobleaching of eucalyptus kraft pulp.

    Directory of Open Access Journals (Sweden)

    Wangning Yu

    Full Text Available Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50-80°C. It also showed broad pH adaptability (>35% activity at pH 4.0-9.0 and alkali-tolerance (>80% activity after incubation at pH 5-10 for 1 h at 37°C, and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0. SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.

  6. Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments.

    Science.gov (United States)

    Hesp, J R; Poolman, T M; Budge, C; Batten, L; Alexander, F; McLuckie, G; O'Brien, S; Wells, P; Raven, N D H; Sutton, J M

    2010-02-01

    Adenylate kinase (tAK), a thermostable enzyme, was assessed as a possible means of providing a quantitative measure of cleaning efficacy suitable for validating the performance of an automated washer disinfector (AWD) during routine use. Two indicator formulations were developed using either a commercially available washer disinfector soil or a protein-based soil. Each indicator consisted of 100 microg (in test soil) of tAK dried on to a steel or plastic surface. These indicators were placed in each basket of a washer disinfector and processed alongside soiled surgical instruments during a standard day's operation. After processing, remaining tAK activity was detected using a rapid enzyme assay (2 min detection time) in a handheld hygiene monitor. The amount of tAK remaining on each indictor after a full AWD cycle was found to range from 0.1 to 0.4 ng, which represented a mean log(10) removal of 5.8+/-0.3. There was no statistical difference in the residual tAK activity between individual runs or the position of the indicator in the machine. The tAK indicator was also used to analyse the protein removal within each component of the wash cycle. These results demonstrated that all phases of the wash process contributed to the removal of the protein load, with the main wash alone being responsible for 3.6-4.0 log(10) reductions in protein activity. We propose that a quantitative cleaning index using such rapid readout indicator devices would provide a valuable addition to the methodologies for validating cleaning processes.

  7. Characterization of a Highly Thermostable and Organic Solvent-Tolerant Copper-Containing Polyphenol Oxidase with Dye-Decolorizing Ability from Kurthia huakuii LAM0618T.

    Directory of Open Access Journals (Sweden)

    Xiang Guo

    Full Text Available Laccases are green biocatalysts that possess attractive advantages for the treatment of resistant environmental pollutants and dye effluents. A putative laccase-like gene, laclK, encoding a protein of 29.3 kDa and belonging to the Cu-oxidase_4 superfamily, was cloned and overexpressed in Escherichia coli. The purified recombinant protein LaclK (LaclK was able to oxidize typical laccase substrates such as 2,6-dimethoxyphenol and l-dopamine. The characteristic adsorption maximums of typical laccases at 330 nm and 610 nm were not detected for LaclK. Cu2+ was essential for substrate oxidation, but the ratio of copper atoms/molecule of LaclK was determined to only be 1:1. Notably, the optimal temperature of LaclK was 85°C with 2,6-dimethoxyphenol as substrates, and the half-life approximately 3 days at 80°C. Furthermore, 10% (v/v organic solvents (methanol, ethanol, isopropyl alcohol, butyl alcohol, Triton x-100 or dimethyl sulfoxide could promote enzymatic activity. LaclK exhibited wide-spectrum decolorization ability towards triphenylmethane dyes, azo dyes and aromatic dyes, decolorizing 92% and 94% of Victoria Blue B (25 μM and Ethyl Violet (25 μM, respectively, at a concentration of 60 U/L after 1 h of incubation at 60°C. Overall, we characterized a novel thermostable and organic solvent-tolerant copper-containing polyphenol oxidase possessing dye-decolorizing ability. These unusual properties make LaclK an alternative for industrial applications, particularly processes that require high-temperature conditions.

  8. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

    OpenAIRE

    Sigoillot Jean-Claude; Kim-Anh To; Haltrich Dietmar; Berrin Jean-Guy; Thi-Thu Dang; Bien-Cuong Do; Yamabhai Montarop

    2009-01-01

    Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pret...

  9. Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

    Science.gov (United States)

    Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

    2017-02-01

    , a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity. Copyright © 2017 American Society for Microbiology.

  10. Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization.

    Directory of Open Access Journals (Sweden)

    Huiping Liu

    Full Text Available Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid (ABTS, syringaldazine (SGZ, guaiacol, and 2,6-dimethoxyphenol (2,6-DMP as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0-11.0 and thermostable at 40°C-90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.

  11. Application of alkaline thermo-stable lipase(s) enzyme produced from irradiated microbial isolate in the field of detergent technology

    International Nuclear Information System (INIS)

    Ahmed, O.E.A.M.S

    2010-01-01

    (s) from Bacillus brevis B 2 was studied. It was found that:1-The optimum incubation temperature for maximum lipase(s) formation is 70 degree C after 24h at ph 11.5.2- Maximum lipase(s) biosynthesis attained at concentration of 1% for each of fish-wastes and yeast extract with inoculum size equal 2 ml/100 ml culture medium incubating under shaking condition.3-Using fish-wastes-yeast extract medium fortified with disaccharide (sucrose or maltose or lactose) was the best nutritional medium for lipase formation. On contrary none of the tested nitrogen sources affect the induction of the enzyme.4-The addition of some surfactant to the nutritional medium revealed that Tween-20 slightly increase lipase(s) synthesis while the presence of zinc sulfate (100 ppm) and folic acid (500 ppm) enhanced the formation of lipase(s) by B. brevis B 2 . 5-Exposure of the experimental organism to 1 KGy gamma-irradiation resulted in slight increase in the level of enzyme. Thermoalkalo-stable lipase(s) from B. brevis B 2 was purified 21.8-fold using ammonium sulphate precipitation (100% saturation) followed by fractionation on Sephadex G-200 column chromatography. The properties of the enzyme were studied.Maximal enzyme activity occurred at 80 degree C. In addition, exposure of the enzyme to 70-80 degree C for 1h resulted remaining activities equal 98-90% respectively, indicating that it was thermo-stable. Optimum ph for maximal enzyme activity was ph 9.5 and exposure of the enzyme to alkaline condition (ph 9 - 10.5) for 1h resulted remaining activity from 80-90%, indicating the alkalo-stable nature of the enzyme. - The relationship between enzyme activity and enzyme concentration indicated that the extent of catalytic action was a function of the concentration of the enzyme. Maximal enzyme activity obtained after 60 minute and the reaction rate increased as a result of increasing substrate concentration, up to 1.2 mg of p-nitrophenyl palmitate, with some saturation at the highest concentration

  12. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters

    Directory of Open Access Journals (Sweden)

    Hüseyin Ilgü

    2018-03-01

    Full Text Available The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg, agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5, an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  13. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters.

    Science.gov (United States)

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Colas, Claire; Ucurum, Zöhre; Schlessinger, Avner; Fotiadis, Dimitrios

    2018-03-20

    The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures ( T m s) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting T m s indicated stabilization of AdiC variants upon ligand binding, in which T m s and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  14. Polymerization of calcium caseinates solutions induced by gamma irradiation

    International Nuclear Information System (INIS)

    Lacroix, M.; Jobin, M.; Mezgheni, E.; Srour, M.; Boileau, S.

    1998-01-01

    Solutions of calcium caseinate (5%) combined with propylene glycol (PG) or triethylene glycol(TEG) (0, 2.5% and 5%) and used for the development of edible films and coatings, were irradiated at doses between 0 to 128 kGy. Solutions were chromatographed through toyopearl HW 55F resin to observe the effect of irradiation on cross-link reactions. In unirradiated calcium caseinate solutions, two peaks could be observed (fractions 30 and 37) while samples irradiated at 64 kGy and 128 kGy showed one shifted peak at fraction 32 and 29 respectively. No effect of the plasticizers was observed. According to proteins standards of knowed molecular weights, the molecular weight of calcium caseinate increased approximately 10 times when irradiated at 128 kGy and 5 times when irradiated at 64 kGy. The physico-chemical properties of bio-films prepared with the irradiated solutions, demonstrated that tensile strength at break increased with increase of irradiation dose. A maximum dose was obtained at 16 kGy

  15. Aggregation in charged nanoparticles solutions induced by different interactions

    Energy Technology Data Exchange (ETDEWEB)

    Abbas, S.; Kumar, Sugam; Aswal, V. K., E-mail: vkaswal@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Kohlbrecher, J. [Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen (Switzerland)

    2016-05-23

    Small-angle neutron scattering (SANS) has been used to study the aggregation of anionic silica nanoparticles as induced through different interactions. The nanoparticle aggregation is induced by addition of salt (NaCl), cationic protein (lysozyme) and non-ionic surfactant (C12E10) employing different kind of interactions. The results show that the interaction in presence of salt can be explained using DLVO theory whereas non-DLVO forces play important role for interaction of nanoparticles with protein and surfactant. The presence of salt screens the repulsion between charged nanoparticles giving rise to a net attraction in the DLVO potential. On the other hand, strong electrostatic attraction between nanoparticle and oppositely charged protein leads to protein-mediated nanoparticle aggregation. In case of non-ionic surfactant, the relatively long-range attractive depletion interaction is found to be responsible for the particle aggregation. Interestingly, the completely different interactions lead to similar kind of aggregate morphology. The nanoparticle aggregates formed are found to have mass fractal nature having a fractal dimension (~2.5) consistent with diffusion limited type of fractal morphology in all three cases.

  16. A system for nonmonotonic rules on the web

    NARCIS (Netherlands)

    Antoniou, G.; Bikakis, A.; Wagner, G.R.

    2004-01-01

    Defeasible reasoning is a rule-based approach for efficient reasoning with incomplete and inconsistent information. Such reasoning is, among others, useful for ontology integration, where conflicting information arises naturally; and for the modeling of business rules and policies, where rules with

  17. Nonmonotonic energy harvesting efficiency in biased exciton chains

    NARCIS (Netherlands)

    Vlaming, S.M.; Malyshev, V.A.; Knoester, J.

    2007-01-01

    We theoretically study the efficiency of energy harvesting in linear exciton chains with an energy bias, where the initial excitation is taking place at the high-energy end of the chain and the energy is harvested (trapped) at the other end. The efficiency is characterized by means of the average

  18. Expert system for failures detection and non-monotonic reasoning

    International Nuclear Information System (INIS)

    Assis, Abilio de; Schirru, Roberto

    1997-01-01

    This paper presents the development of a shell denominated TIGER that has the purpose to serve as environment to the development of expert systems in diagnosis of faults in industrial complex plants. A model of knowledge representation and an inference engine based on non monotonic reasoning has been developed in order to provide flexibility in the representation of complex plants as well as performance to satisfy restrictions of real time. The TIGER is able to provide both the occurred fault and a hierarchical view of the several reasons that caused the fault to happen. As a validation of the developed shell a monitoring system of the critical safety functions of Angra-1 has been developed. 7 refs., 7 figs., 2 tabs

  19. Infinitary default logic for specification of nonmonotonic reasoning

    NARCIS (Netherlands)

    Engelfriet, Joeri; Marek, V. Wiktor; Treur, Jan; Truszczynski, Miroslaw

    1996-01-01

    In this paper we study constructions leading to the formation of belief sets by agents. We focus on the situation when possible belief sets axe built incrementally in stages. We call an infinite sequence of theories that represents such a process a reasoning trace. A set of reasoning traces

  20. Nonmonotonic quantum-to-classical transition in multiparticle interference

    DEFF Research Database (Denmark)

    Ra, Young-Sik; Tichy, Malte; Lim, Hyang-Tag

    2013-01-01

    Quantum-mechanical wave–particle duality implies that probability distributions for granular detection events exhibit wave-like interference. On the single-particle level, this leads to self-interference—e.g., on transit across a double slit—for photons as well as for large, massive particles...... that interference fades away monotonically with increasing distinguishability—in accord with available experimental evidence on the single- and on the many-particle level. Here, we demonstrate experimentally and theoretically that such monotonicity of the quantum-to-classical transition is the exception rather than...

  1. The Non-Monotonic Effect of Financing Constraints on Investment

    DEFF Research Database (Denmark)

    Hirth, Stefan; Viswanatha, Marc

    We analyze investment timing in a discrete-time framework with two possible investment dates, which is an extension of the model by Lyandres (2007). We derive an investment threshold that is U-shaped in the firm's liquid funds, a result similar to the infinite-horizon models by Boyle and Guthrie...... (2003) and Hirth and Uhrig-Homburg (2007). However, due to the tractability of our model, we can more clearly explain the relevant trade-offs leading to the U-shape: The firm balances financing costs and abandonment risk for present and future investment, respectively....

  2. Derivation of Monotone Decision Models from Non-Monotone Data

    NARCIS (Netherlands)

    Daniëls, H.A.M.; Velikova, M.V.

    2003-01-01

    The objective of data mining is the extraction of knowledge from databases. In practice, one often encounters difficulties with models that are constructed purely by search, without incorporation of knowledge about the domain of application.In economic decision making such as credit loan approval or

  3. Empirical Modelling of Nonmonotonous Behaviour of Shear Viscosity

    Czech Academy of Sciences Publication Activity Database

    David, Jiří; Filip, Petr; Kharlamov, Alexander

    2013-01-01

    Roč. 2013, August (2013) ISSN 1687-6822 R&D Projects: GA ČR GA103/09/2066 Institutional support: RVO:67985874 Keywords : shear viscosity * Galindo-rosales * Carreau-yasuda Subject RIV: BK - Fluid Dynamics Impact factor: 0.500, year: 2012 http://www.hindawi.com/journals/amse/2013/658187/

  4. Study of the leaching of heavy metals from waste water sludge and incinerator's ash, using coupled thermostated columns and DTPA as complex agent; Estudio de la extraccion de metales pesados de lodos y cenizas de aguas residuales usando columnas termostatizadas acopladas y DTPA como agente complejante

    Energy Technology Data Exchange (ETDEWEB)

    Vite T, J.; Vite T, M.; Guerrero D, J.; Carreno de Leon, M.C. [Departamento de Estudios del Ambiente, Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)

    2000-07-01

    We studied the metallic composition from waste water sludge and incinerators ashes of an incinerator located in Toluca, Mexico, the qualitative studies were made using the Activation Analysis technique, and fluorescence X-ray techniques. The quantitative analysis of heavy metals in the wastes were made using Inductively coupled plasma atomic emission spectrometry (Icp-Aes). For leaching the samples, we used four coupled thermostated columns, each one had a p H of 2,5, 7 and 10. The flux of the air was of 1600 cc/min. The temperature was maintain constant in 60 Centigrade using a thermostated system. For this study we used 100 g of wastes mixed with mineral acid or sodium hydroxide to reach p H 2,5,7 and 10. We added a reducing and tensoactive agents and finally DTPA as complex agent. With this method, we obtain a better leaching efficiency using a complex agent. However the high DTPA cost, make this process expansive that is why we recommend to work with another classes of complex agents, that be cheaper to leach metals of different chemistry matrix. (Author)

  5. Production and characterization of thermostable xylanase from ...

    African Journals Online (AJOL)

    ajl2

    2013-02-20

    Feb 20, 2013 ... Penicillium, .... production in liquid medium ranged from 0.11 to 0.21. U/ml (data not shown). ... Lane 2 represents the negative control (Bacillus sp.). Lane 3 is .... circulans D1 in submerged fermentation using response surface.

  6. Biochemical characterization of a thermoactive and thermostable ...

    African Journals Online (AJOL)

    faouzi

    h on a rotary shaker set at 200 rpm at a temperature of 30°C. Growth was followed ... The medium culture, obtained after 24 h with an inoculum size of 2. × 10⁷ cells/ml, was .... (1997) showed that SHL was not inactivated by anionic detergents ...

  7. Characterization and engineering of thermostable glycoside hydrolases

    NARCIS (Netherlands)

    Lieshout, van J.F.T.

    2007-01-01

    Glycosidehydrolasesform a class of enzymes that play an important role in sugar-converting processes. They are applied as biocatalyst in both the hydrolysis of natural polymers to mono- andoligo-saccharides, and the reverse hydrolysis or

  8. Optimization and Characterization of Thermostable Endo and ...

    African Journals Online (AJOL)

    http://dx.doi.org/10.4314/star.v3i4.16 · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL · AJOL's Partners · Terms and Conditions of Use · Contact AJOL · News. OTHER RESOURCES... for Researchers · for Journals · for Authors · for Policy Makers ...

  9. Production and characterization of thermostable xylanase from ...

    African Journals Online (AJOL)

    ajl2

    2013-02-20

    Feb 20, 2013 ... produced from Trichoderma (Huitron et al., 2008). Only a ... around their colonies against a red background, were selected and .... Resistance to antibiotic ..... Xylanases of marine fungi potential use for biobleaching of paper.

  10. 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.

  11. Purification and characterization of a thermostable glucoamylase ...

    African Journals Online (AJOL)

    Glucoamylase (GA) from Aspergillus flavus HBF34 strain was partially purified 120 folds using starch affinity chromatography. Two isoenzymes (GA1 and GA2) were identified by polyacrylamide gel electrophoresis (PAGE) zymography. Sodium dodecyl sulfate (SDS)-PAGE analysis revealed that one of the enzymes consist ...

  12. Purification and characterization of a thermostable glucoamylase ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-06-07

    Jun 7, 2010 ... was 6.0 and the optimum temperature was 60°C. GA was found to be stable at temperatures up to .... on starch plates were inoculated to test for amylase secretion, ..... starch-digesting glucoamylase production of Rhizopus sp.

  13. Engineering thermostable xylanase enzyme mutant from Bacillus ...

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... waste treatment, fuel and chemical production, paper and pulp industries; but these applications ... approaches have been taken: screening organisms from various ... and site-directed mutagenesis was applied on this.

  14. Internal electric fields of electrolytic solutions induced by space-charge polarization

    Science.gov (United States)

    Sawada, Atsushi

    2006-10-01

    The dielectric dispersion of electrolytic solutions prepared using chlorobenzene as a solvent and tetrabutylammonium tetraphenylborate as a solute is analyzed in terms of space-charge polarization in order to derive the ionic constants, and the Stokes radius obtained is discussed in comparison with the values that have been measured by conductometry. A homogeneous internal electric field is assumed for simplicity in the analysis of the space-charge polarization. The justification of the approximation by the homogeneous field is discussed from two points of view: one is the accuracy of the Stokes radius value observed and the other is the effect of bound charges on electrodes in which they level the highly inhomogeneous field, which has been believed in the past. In order to investigate the actual electric field, numerical calculations based on the Poisson equation are carried out by considering the influence of the bound charges. The variation of the number of bound charges with time is clarified by determining the relaxation function of the dielectric constant attributed to the space-charge polarization. Finally, a technique based on a two-field approximation, where homogeneous and hyperbolic fields are independently applied in relevant frequency ranges, is introduced to analyze the space-charge polarization of the electrolytic solutions, and further improvement of the accuracy in the determination of the Stokes radius is achieved.

  15. Adsorption of Antibiotics on Graphene and Biochar in Aqueous Solutions Induced by π-π Interactions

    Science.gov (United States)

    Peng, Bingquan; Chen, Liang; Que, Chenjing; Yang, Ke; Deng, Fei; Deng, Xiaoyong; Shi, Guosheng; Xu, Gang; Wu, Minghong

    2016-08-01

    The use of carbon based materials on the removal of antibiotics with high concentrations has been well studied, however the effect of this removal method is not clear on the actual concentration of environments, such as the hospital wastewater, sewage treatment plants and aquaculture wastewater. In this study, experimental studies on the adsorption of 7 antibiotics in environmental concentration of aqueous solutions by carbon based materials have been observed. Three kinds of carbon materials have shown very fast adsorption to antibiotics by liquid chromatography-tandem mass spectrometry (LC-MS-MS) detection, and the highest removal efficiency of antibiotics could reach to 100% within the range of detection limit. Surprisedly, the adsorption rate of graphene with small specific surface area was stronger than other two biochar, and adsorption rate of the two biochar which have approximate specific surface and different carbonization degree, was significantly different. The key point to the present observation were the π-π interactions between aromatic rings on adsorbed substance and carbon based materials by confocal laser scanning microscope observation. Moreover, adsorption energy markedly increased with increasing number of the π rings by using the density functional theory (DFT), showing the particular importance of π-π interactions in the adsorption process.

  16. The synthesis of nucleotide in the aqueous solution induced by low energy ions

    International Nuclear Information System (INIS)

    Shi Huaibin; Shao Chunlin; Wang Xiangqin; Yu Zengliang

    2000-08-01

    A new apparatus was designed to induce reactions in aqueous solution by introducing low energy ions into the aqueous solution, this apparatus overcome the defaults of the old ones which demanded vacuum and made it possible to study the action among solutions, it also expanded the ion implantation biology. The role of low energy ions was introduced into the study of the origin of life, primitive earth conditions were imitated to study prior-life synthesis of nucleotide by introducing low energy ions into aqueous solution, low energy N + was implanted into adenine supersaturation solution including D-ribose and NH 4 H 2 PO 4 , it was confirmed that 5'-AMP was gained by HPLC analysis of the products. In comparison with other methods in this field, this one is simpler and nearer to the primitive earth conditions, thus it provided a new try for the studying of the origin of life

  17. Stability properties of the Euler-Korteweg system with nonmonotone pressures

    KAUST Repository

    Giesselmann, Jan; Tzavaras, Athanasios

    2016-01-01

    We establish a relative energy framework for the Euler-Korteweg system with non-convex energy. This allows us to prove weak-strong uniqueness and to show convergence to a Cahn-Hilliard system in the large friction limit. We also use relative energy

  18. Stability properties of the Euler-Korteweg system with nonmonotone pressures

    KAUST Repository

    Giesselmann, Jan

    2016-12-21

    We establish a relative energy framework for the Euler-Korteweg system with non-convex energy. This allows us to prove weak-strong uniqueness and to show convergence to a Cahn-Hilliard system in the large friction limit. We also use relative energy to show that solutions of Euler-Korteweg with convex energy converge to solutions of the Euler system in the vanishing capillarity limit, as long as the latter admits sufficiently regular strong solutions.

  19. Renormalization in charged colloids: non-monotonic behaviour with the surface charge

    International Nuclear Information System (INIS)

    Haro-Perez, C; Quesada-Perez, M; Callejas-Fernandez, J; Schurtenberger, P; Hidalgo-Alvarez, R

    2006-01-01

    The static structure factor S(q) is measured for a set of deionized latex dispersions with different numbers of ionizable surface groups per particle and similar diameters. For a given volume fraction, the height of the main peak of S(q), which is a direct measure of the spatial ordering of latex particles, does not increase monotonically with the number of ionizable groups. This behaviour cannot be described using the classical renormalization scheme based on the cell model. We analyse our experimental data using a renormalization model based on the jellium approximation, which predicts the weakening of the spatial order for moderate and large particle charges. (letter to the editor)

  20. Subtractive, divisive and non-monotonic gain control in feedforward nets linearized by noise and delays

    OpenAIRE

    Mejias, Jorge F.; Payeur, Alexandre; Selin, Erik; Maler, Leonard; Longtin, André

    2014-01-01

    The control of input-to-output mappings, or gain control, is one of the main strategies used by neural networks for the processing and gating of information. Using a spiking neural network model, we studied the gain control induced by a form of inhibitory feedforward circuitry—also known as “open-loop feedback”—, which has been experimentally observed in a cerebellum-like structure in weakly electric fish. We found, both analytically and numerically, that this network displays three different...

  1. Non-monotonic permeability variation during colloidal transport: Governing equations and analytical model

    Science.gov (United States)

    Chequer, L.; Russell, T.; Behr, A.; Genolet, L.; Kowollik, P.; Badalyan, A.; Zeinijahromi, A.; Bedrikovetsky, P.

    2018-02-01

    Permeability decline associated with the migration of natural reservoir fines impairs the well index of injection and production wells in aquifers and oilfields. In this study, we perform laboratory corefloods using aqueous solutions with different salinities in engineered rocks with different kaolinite content, yielding fines migration and permeability alteration. Unusual permeability growth has been observed at high salinities in rocks with low kaolinite concentrations. This has been attributed to permeability increase during particle detachment and re-attachment of already mobilised fines by electrostatic attraction to the rock in stagnant zones of the porous space. We refine the traditional model for fines migration by adding mathematical expressions for the particle re-attachment rate, particle detachment with delay relative to salinity decrease, and the attached-concentration-dependency of permeability. A one-dimensional flow problem that accounts for those three effects allows for an exact analytical solution. The modified model captures the observed effect of permeability increase at high water salinities in rocks with low kaolinite concentrations. The developed model matches the coreflooding data with high accuracy, and the obtained model coefficients vary within their usual intervals.

  2. Option Valuation with Volatility Components, Fat Tails, and Non-Monotonic Pricing Kernels

    DEFF Research Database (Denmark)

    Babaoglu, Kadir; Christoffersen, Peter; Heston, Steven L.

    We nest multiple volatility components, fat tails and a U-shaped pricing kernel in a single option model and compare their contribution to describing returns and option data. All three features lead to statistically significant model improvements. A U-shaped pricing kernel is economically most im...

  3. Stability of dynamical systems on the role of monotonic and non-monotonic Lyapunov functions

    CERN Document Server

    Michel, Anthony N; Liu, Derong

    2015-01-01

    The second edition of this textbook provides a single source for the analysis of system models represented by continuous-time and discrete-time, finite-dimensional and infinite-dimensional, and continuous and discontinuous dynamical systems.  For these system models, it presents results which comprise the classical Lyapunov stability theory involving monotonic Lyapunov functions, as well as corresponding contemporary stability results involving non-monotonicLyapunov functions.Specific examples from several diverse areas are given to demonstrate the applicability of the developed theory to many important classes of systems, including digital control systems, nonlinear regulator systems, pulse-width-modulated feedback control systems, and artificial neural networks.   The authors cover the following four general topics:   -          Representation and modeling of dynamical systems of the types described above -          Presentation of Lyapunov and Lagrange stability theory for dynamical sy...

  4. Non-Monotonicity of Fertility in Human Capital Accumulation and Economic Growth

    OpenAIRE

    Spyridon Boikos; Alberto Bucci; Thanasis Stengos

    2012-01-01

    This paper investigates the relationship between per-capita human capital investment and the birth rate. Since the consequences of higher fertility (birth rate) on per-capita human capital accumulation (the so-called dilution effect) are not the same (in sign and magnitude) across different groups of countries with different birth rates, we analyze the growth impact of a non-linear dilution-effect. The main predictions of the model (concerning the relationship between population and economic ...

  5. Nonmonotonic anisotropy in charge conduction induced by antiferrodistortive transition in metallic SrTiO3

    Science.gov (United States)

    Tao, Qian; Loret, Bastien; Xu, Bin; Yang, Xiaojun; Rischau, Carl Willem; Lin, Xiao; Fauqué, Benoît; Verstraete, Matthieu J.; Behnia, Kamran

    2016-07-01

    Cubic SrTiO3 becomes tetragonal below 105 K. The antiferrodistortive (AFD) distortion leads to clockwise and counterclockwise rotation of adjacent TiO6 octahedra. This insulator becomes a metal upon the introduction of extremely low concentration of n -type dopants. However, signatures of the structural phase transition in charge conduction have remained elusive. Employing the Montgomery technique, we succeed in resolving the anisotropy of charge conductivity induced by the AFD transition, in the presence of different types of dopants. We find that the slight lattice distortion (liquids, the anisotropy has opposite signs for elastic and inelastic scattering. Increasing the concentration of dopants leads to a drastic shift in the temperature of the AFD transition either upward or downward. The latter result puts strong constraints on any hypothetical role played by the AFD soft mode in the formation of Cooper pairs and the emergence of superconductivity in SrTiO3.

  6. Peaks and Valleys : Experimental Asset Markets With Non-Monotonic Fundamentals

    NARCIS (Netherlands)

    Noussair, C.N.; Powell, O.R.

    2008-01-01

    We report the results of an experiment designed to measure how well asset market prices track fundamentals when the latter experience peaks and troughs. We observe greater price efficiency in markets in which fundamentals rise to a peak and then decline, than in markets in which fundamentals decline

  7. An ultra-low-voltage electronic implementation of inertial neuron model with nonmonotonous Liao's activation function.

    Science.gov (United States)

    Kant, Nasir Ali; Dar, Mohamad Rafiq; Khanday, Farooq Ahmad

    2015-01-01

    The output of every neuron in neural network is specified by the employed activation function (AF) and therefore forms the heart of neural networks. As far as the design of artificial neural networks (ANNs) is concerned, hardware approach is preferred over software one because it promises the full utilization of the application potential of ANNs. Therefore, besides some arithmetic blocks, designing AF in hardware is the most important for designing ANN. While attempting to design the AF in hardware, the designs should be compatible with the modern Very Large Scale Integration (VLSI) design techniques. In this regard, the implemented designs should: only be in Metal Oxide Semiconductor (MOS) technology in order to be compatible with the digital designs, provide electronic tunability feature, and be able to operate at ultra-low voltage. Companding is one of the promising circuit design techniques for achieving these goals. In this paper, 0.5 V design of Liao's AF using sinh-domain technique is introduced. Furthermore, the function is tested by implementing inertial neuron model. The performance of the AF and inertial neuron model have been evaluated through simulation results, using the PSPICE software with the MOS transistor models provided by the 0.18-μm Taiwan Semiconductor Manufacturer Complementary Metal Oxide Semiconductor (TSM CMOS) process.

  8. Nonmonotonic and spatial-temporal dynamic slope effects on soil erosion during rainfall-runoff processes

    Science.gov (United States)

    Wu, Songbai; Yu, Minghui; Chen, Li

    2017-02-01

    The slope effect on flow erosivity and soil erosion still remains a controversial issue. This theoretical framework explained and quantified the direct slope effect by coupling the modified Green-Ampt equation accounting for slope effect on infiltration, 1-D kinematic wave overland flow routing model, and WEPP soil erosion model. The flow velocity, runoff rate, shear stress, interrill, and rill erosion were calculated on 0°-60° isotropic slopes with equal horizontal projective length. The results show that, for short-duration rainfall events, the flow erosivity and erosion amounts exhibit a bell-shaped trend which first increase with slope gradient, and then decrease after a critical slope angle. The critical slope angles increase significantly or even vanish with increasing rainfall duration but are nearly independent of the slope projective length. The soil critical shear stress, rainfall intensity, and temporal patterns have great influences on the slope effect trend, while the other soil erosion parameters, soil type, hydraulic conductivity, and antecedent soil moisture have minor impacts. Neglecting the slope effect on infiltration would generate smaller erosion and reduce critical slope angles. The relative slope effect on soil erosion in physically based model WEPP was compared to those in the empirical models USLE and RUSLE. The trends of relative slope effect were found quite different, but the difference may diminish with increasing rainfall duration. Finally, relatively smaller critical slope angles could be obtained with the equal slope length and the range of variation provides a possible explanation for the different critical slope angles reported in previous studies.

  9. Nonmonotonic temperature dependence of critical current in diffusive d-wave junctions

    NARCIS (Netherlands)

    Yokoyama, T.; Tanaka, Y.; Golubov, Alexandre Avraamovitch; Asano, Y.

    2006-01-01

    We study the Josephson effect in D/I/DN/I/D junctions, where I, DN, and D denote an insulator, a diffusive normal metal, and a d-wave superconductor, respectively. The Josephson current is calculated based on the quasiclassical Green's function theory with a general boundary condition for

  10. Qualitative Stability of a Class of Non-Monotone Variational Inclusions. Application in Electronics

    Czech Academy of Sciences Publication Activity Database

    Adly, S.; Outrata, Jiří

    2013-01-01

    Roč. 20, č. 1 (2013), s. 43-66 ISSN 0944-6532 R&D Projects: GA AV ČR IAA100750802; GA ČR(CZ) GAP201/12/0671 Institutional support: RVO:67985556 Keywords : Hemivariational inequalities * Nonsmooth and variational analysis * Aubin property * Isolated calmness * Non-regular electrical circuits Subject RIV: BA - General Mathematics Impact factor: 0.592, year: 2013 http://library.utia.cas.cz/separaty/2013/MTR/outrata-0392738.pdf

  11. Degenerate Power in Multilevel Mediation: The Non-Monotonic Relationship between Power & Effect Size

    Science.gov (United States)

    Kelcey, Ben

    2015-01-01

    Like studies focused on the detection of a main or total effect, a principal consideration in the design of studies examining mediation is the power with which mediation effects can be detected if they exist (e.g., Raudenbush, 1997). However, unlike studies concerning the detection of main effects, literature has not established power formulas for…

  12. Semiparametric approach for non-monotone missing covariates in a parametric regression model

    KAUST Repository

    Sinha, Samiran; Saha, Krishna K.; Wang, Suojin

    2014-01-01

    mechanism helps to nullify (or reduce) the problems due to non-identifiability that result from the non-ignorable missingness mechanism. The asymptotic properties of the proposed estimator are derived. Finite sample performance is assessed through simulation

  13. Why Friedman's Non-monotonic Reasoning Defies Hempel's Covering Law Model

    NARCIS (Netherlands)

    M.C.W. Janssen (Maarten); Y-H. Tan (Yao-Hua)

    1991-01-01

    textabstractIn this paper we will show that Hempel's covering law model can't deal very well with explanations that are based on incomplete knowledge. In particular the symmetry thesis, which is an important aspect of the covering law model, turns out to be problematic for these explanations. We

  14. Why Friedman's non-monotonic reasoning defies Hempel's covering law model

    NARCIS (Netherlands)

    M.C.W. Janssen (Maarten); Y.H. Tan (Yao Hua)

    1991-01-01

    textabstractIn this paper we will show that Hempel's covering law model can't deal very well with explanations that are based on incomplete knowledge. In particular the symmetry thesis, which is an important aspect of the covering law model, turns out to be problematic for these explanations. We

  15. Monotonic and Nonmonotonic Lag Effects in Paired-Associate and Recognition Memory Paradigms

    Science.gov (United States)

    Glenberg, Arthur M.

    1976-01-01

    Three experiments are reported investigating the relationship between response recall and the spacing of repetitions as a function of the retention interval. The results of the experiments support the theory which emphasizes the nature of the cues available for retrieval. (Author/RM)

  16. Instability of an infiltration-driven dissolution-precipitation front with a nonmonotonic porosity profile

    Science.gov (United States)

    Kondratiuk, Paweł; Dutka, Filip; Szymczak, Piotr

    2016-04-01

    Infiltration of a rock by an external fluid very often drives it out of chemical equilibrium. As a result, alteration of the rock mineral composition occurs. It does not however proceed uniformly in the entire rock volume. Instead, one or more reaction fronts are formed, which are zones of increased chemical activity, separating the altered (product) rock from the yet unaltered (primary) one. The reaction fronts propagate with velocities which are usually much smaller than those of the infiltrating fluid. One of the simplest examples of such alteration is the dissolution of some of the minerals building the primary rock. For instance, calcium carbonate minerals in the rock matrix can be dissolved by infiltrating acidic fluids. In such a case the product rock has higher porosity and permeability than the primary one. Due to positive feedbacks between the reactant transport, fluid flow, and porosity generation, the reaction fronts in porosity-generating replacement systems are inherently unstable. An arbitrarily small protrusion of the front gets magnified and develops into a highly porous finger-like or funnel-like structure. This feature of dissolution fronts, dubbed the "reactive-infiltration instability" [1], is responsible for the formation of a number of geological patterns, such as solution pipes or various karst forms. It is also of practical importance, since spontaneous front breakup and development of localized highly porous flow paths (a.k.a. "wormholes") is favourable by petroleum engineers, who apply acidization to oil-bearing reservoirs in order to increase their permeability. However, more complex chemical reactions might occur during infiltration of a rock by a fluid. In principle, the products of dissolution might react with other species present either in the fluid or in the rock and reprecipitate [2]. The dissolution and precipitation fronts develop and and begin to propagate with equal velocities, forming a single dissolution-precipitation front. The porosity profile is not monotonic as in the case of pure dissolution, but it typically has a minimum in the vicinity of the front. Additionally, the porosity difference between the initial rock far-downstream and the well-developed secondary rock far-upstream can be either negative or positive, which either destabilizes of stabilized the front. We propose a theoretical model of a simple infiltration-driven dissolution-precipitation system and find the morphology of the resulting planar reaction front. By performing linear stability analysis of the stationary planar solutions we show that the front can be unstable for a wide range of control parameters, even if the porosity of the secondary rock is lower than the porosity of the primary rock. Next, by numerical simulations of the full nonlinear model we present the long-term evolution of the system. [1] D. Chadam et al., IMA J. Appl. Math. 36, 207-221, 1986. [2] A. Putnis, Rev. Mineral. Geochemistry, 70(1), 87-124, 2009.

  17. Production of Thermostab Streptomyc ction of Thermostable Alkaline ...

    African Journals Online (AJOL)

    and temperature of 500C. Therefore ... Actinomycetes species and determining the cultural conditions se yields. ... ials for protease production on skimmed milk agar, only Norcar ... produced crude enzyme demonstrated optimal activity at pH 8.

  18. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  19. A thermostable Cas9 with increased lifetime in human plasma

    OpenAIRE

    Harrington, LB; Paez-Espino, D; Staahl, BT; Chen, JS; Ma, E; Kyrpides, NC; Doudna, JA

    2017-01-01

    © 2017 The Author(s). CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas...

  20. Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

    Science.gov (United States)

    Ribeiro, Liliane Fraga Costa; Vaz, Marcelo Gomes Marçal Vieira; Chaves-Alves, Virgínia Maria; Vanetti, Maria Cristina Dantas; Vanetti, Maria Cristina Dantas; Kasuya, Maria Catarina Megumi; Passos, Flávia Maria Lopes; do Nascimento, Antônio Galvão

    2012-01-01

    Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65°C for 21 hours, losing only 15–25% activity. Fungus incubation at 50°C for 12 hours and at 65°C for 24 hours increased the amount of xylose produced. PMID:24031818

  1. Characterization of a thermostable Bacillus subtilis β-amylase

    African Journals Online (AJOL)

    ... 70 0C respectively, and the thermal stability curve gave a maximum activity of 9.75 U at 70oC for 60 min of incubation. Bacillus subtilis â-amylase is valuable for maltose production, which can be hydrolyzed further by other groups of amylase for the production of high cassava glucose syrup used as sweeteners in the food ...

  2. Thermostability of reconstituted newcastle disease virus strains at 36 ...

    African Journals Online (AJOL)

    Haemagglutination (HA) test was employed to determine the stability of HA titers of reconstituted form of Hitchner – B1 (B1), LaSota (L) and Komarov (K) strains of Newcastle Disease Vaccine (NDV) at 360c. The temperature treatment method was through incubation (in water bath) of the reconstituted vaccines at selected ...

  3. Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710

    Directory of Open Access Journals (Sweden)

    Saleh A. Mohamed

    2015-06-01

    Full Text Available Two inulinases (Inu2 and Inu3 were purified from Rhizopus oligosporus NRRL 2710 by chromatography on DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of Inu2 and Inu3 were determined to be 76 and 30 kDa, respectively. Inu2 and Inu3 had the same pH optimum at 5.0, temperature optimum at 50 and 60 °C, and thermal stability up to 60 and 70 °C for 1 h, respectively. Inu2 and Inu3 had low km values (0.93 and 0.70 mM, respectively indicating the high affinity toward inulin. Mg2+, Ca2+, Zn2+ and EDTA did not significantly influence the enzyme activity. Ni2+, Cu2+, Fe2+ and Co2+ showed a partial inhibitory effect, and Hg2+ had a strong inhibitory effect. p-Chloromercuribenzoate had a partial inhibitory effect on Inu2. From these findings, R. oligosporus inulinases can be beneficial enzymes for industrial enzymatic production of high fructose syrup.

  4. Purification and biochemical properties of a new thermostable ...

    African Journals Online (AJOL)

    The enzyme was stable for a long time-period up to 50°C and for 1 h at 60°C. Although the xylanase had a lower carboxymethylcellulase activity, it lacked activity towards substituted xylan, xylobiose, inulin, starch, polygalacturonic acid or pNP glycosides. Kinetic parameters indicated higher efficiency in the hydrolysis of ...

  5. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  6. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

    Science.gov (United States)

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  7. A thermostable messenger RNA based vaccine against rabies.

    Science.gov (United States)

    Stitz, Lothar; Vogel, Annette; Schnee, Margit; Voss, Daniel; Rauch, Susanne; Mutzke, Thorsten; Ketterer, Thomas; Kramps, Thomas; Petsch, Benjamin

    2017-12-01

    Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

  8. Enhancing RGI lyase thermostability by targeted single point mutations

    DEFF Research Database (Denmark)

    Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten

    2013-01-01

    Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering...

  9. Research of thermostability of phthalates of oxyalkylated alcohols

    Directory of Open Access Journals (Sweden)

    MAZITOVA Aliya Karamovna

    2018-04-01

    Full Text Available The production of polyvinyl chloride (PVC requires implication of a large number of plasticizers. Ester compounds are the most suitable for plasticization. Currently the existing range of industrial plasticizers does not satisfy a need of the polymer industry and it is of great importance to develop new plasticizers that meet modern requirements. Therefore, the elaboration of new ester compounds with effective plasticizing properties, is relevant and has not only scientific value, but also practical one. One of the basic requirements for ester plasticizers is their resistance to thermal oxidation. Accordingly, we considered the effect of oxygen at 150оC (the temperature of conversion of PVC-plastic compounds on a wide range of oxyalated alcohol phthalates developed earlier. The first group consisted of aromatic alcohols, the second group – phthalates of oxyalkylated aliphatic alcohols. The first group showed high thermal stability, the latter should be stabilized by the addition of antioxidants. It is known that aromatic hydrocarbon derivatives are widely used as tabilizers of thermooxidative disrupture. The main function of the mentioned compounds is to suppress the catalytic action of air oxygen in energy impacts on polymers and plasticizers. Consequently, we investigated the effect of Agidol-1 on increasing the stability of phthalate plasticizers to thermaloxidative degradation.

  10. Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia.

    Science.gov (United States)

    Nascimento, Kyria Santiago; Nascimento, Francisco Lucas Faustino do; Silva, Mayara Torquato Lima; Nobre, Camila Bezerra; Moreira, Cleane Gomes; Brizeno, Luiz André Cavalcante; da Ponte, Edson Lopes; Assreuy, Ana Maria Sampaio; Cavada, Benildo Sousa

    2016-06-01

    Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose-mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d-mannose and d-sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein-carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Directed evolution of a thermostable l-aminoacylase biocatalyst

    DEFF Research Database (Denmark)

    Parker, Brenda M.; Taylor, Ian N.; Woodley, John

    2011-01-01

    Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l......-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution....... The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of kcat values for N...

  12. Characterization of Thermostable Cellulases Produced by Bacillus and Geobacillus Strains

    Science.gov (United States)

    Bacterial community composition of thermophilic (60 deg C) mixed cellulose-enrichment cultures was examined by constructing a 16S rDNA clone library which demonstrated major lineages affiliated to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria. A tot...

  13. Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization

    DEFF Research Database (Denmark)

    Hameed, Uzma; Price, Ian; Ikram-Ul-Haq

    2017-01-01

    and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb(1+), K(1+) and Ca(2+) ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13...... of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases....

  14. Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37 Produção e glucoamilase por Aspergillus flavus A1.1 e Thermomyces lanuginosus A13.37

    Directory of Open Access Journals (Sweden)

    Eleni Gomes

    2005-03-01

    Full Text Available Thirteen thermophilic fungal strains were isolated from agricultural soil, tubers and compost samples in tropical Brazil. Two strains were selected based on of their ability to produce considerable glucoamylase activity while growing in liquid medium at 45ºC with starch as the only carbon source. They were identified as Aspergillus flavus A1.1 and Thermomyces lanuginosus A 13.37 Tsiklinsky. The experiment to evaluate the effect of carbon source, temperature and initial pH of the medium on enzyme production was developed in a full factorial design (2x2x3. Enzyme productivity was influenced by the type of starch used as carbon source. Cassava starch showed to be a better substrate than corn starch for glucoamylase production by A. flavus but for T. lanuginosus the difference was not significant. Enzyme activities were determined using as substrates 0.3% soluble starch, 0.3% maltose or 0.3% of starch plus 0.1% maltose. The enzymes from A. flavus A1.1 hydrolyzed soluble starch preferentially but also exhibited a significant maltase activity. Moreover higher quantities of glucose were released when the substrate used was a mixture of starch and maltose, suggesting that this fungus produced two types of enzyme. In the case T. lanuginosus A 13.37, the substrate specificity test indicated that the enzyme released also hydrolyzed starch more efficiently than maltose, but there was no increase in the liberation of glucose when a mixture of starch and maltose was used as substrate, suggesting that only one type of enzyme was secreted. Glucoamylases produced from A. flavus A1.1 and T. lanuginous A.13-37 have high optimum temperature (65ºC and 70ºC and good thermostability in the absence of substrate (maintaining 50% of activity for 5 and 8 hours, respectively, at 60ºC and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for industrial starch processing.Entre 13 linhagens de fungos filamentosos

  15. Synthesis of 5'-CMP and 5'-dCMP in aqueous solution induced by low energy ions implantation

    International Nuclear Information System (INIS)

    Shi Huaibin; Shao Chunlin; Wang Xiangqin; Yu Zengliang

    2001-01-01

    Low energy N + ions produced by N 2 are accelerated and then introduced into aqueous solution to induce chemical reactions. This process avoids the need of a vacuum chamber and makes it possible to investigate the actions of low energy ions in aqueous solution. In order to explore prebiotic synthesis of nucleotide via reaction between low energy ions and aqueous solution under the primitive earth conditions, low energy N + is implanted into aqueous solution containing cytosine, D-ribose, D-2-deoxyribose and NH 4 H 2 PO 4 . It is confirmed that 5'-CMP and 5'-dCMP are produced by HPLC and 1 H-NMR analyses. The relation between yields of 5'-CMP and 5'-dCMP and irradiation time has been obtained

  16. Non-monotonic variations of the nucleation free energy in a glass-forming ultra-soft particles fluid.

    Science.gov (United States)

    Desgranges, Caroline; Delhommelle, Jerome

    2018-06-18

    Using molecular dynamics simulation, we study the impact of the degree of supercooling on the crystal nucleation of ultra-soft particles, modeled with the Gaussian core potential. Focusing on systems with a high number density, our simulations reveal dramatically different behaviors as the degree of supercooling is varied. In the moderate supercooling regime, crystal nucleation proceeds as expected from classical nucleation theory, with a decrease in the free energy of nucleation, as well as in the size of the critical nucleus, as supercooling is increased. On the other hand, in the large supercooling regime, we observe an unusual reversal of behavior with an increase in the free energy of nucleation and in the critical size, as supercooling is increased. This unexpected result is analyzed in terms of the interplay between the glass transition and the crystal nucleation process. Specifically, medium range order crystal-like domains, with structural features different from that of the crystal nucleus, are found to form throughout the system when the supercooling is very large. These, in turn, play a pivotal role in the increase in the free energy of nucleation, as well as in the critical size, as the temperature gets closer to the glass transition.

  17. Nonmonotonous electron mobility due to structurally induced resonant coupling of subband states in an asymmetric double quantum well

    Directory of Open Access Journals (Sweden)

    R. K. Nayak

    2015-11-01

    Full Text Available We show that sharp nonmonotic variation of low temperature electron mobility μ can be achieved in GaAs/AlxGa1-xAs barrier delta-doped double quantum well structure due to quantum mechanical transfer of subband electron wave functions within the wells. We vary the potential profile of the coupled structure as a function of the doping concentration in order to bring the subbands into resonance such that the subband energy levels anticross and the eigen states of the coupled structure equally share both the wells thereby giving rise to a dip in mobility. When the wells are of equal widths, the dip in mobility occurs under symmetric doping of the side barriers. In case of unequal well widths, the resonance can be obtained by suitable asymmetric variation of the doping concentrations. The dip in mobility becomes sharp and also the wavy nature of mobility takes a rectangular shape by increasing the barrier width. We show that the dip in mobility at resonance is governed by the interface roughness scattering through step like changes in the subband mobilities. It is also gratifying to show that the drop in mobility at the onset of occupation of second subband is substantially supressed through the quantum mechanical transfer of subband wave functions between the wells. Our results can be utilized for performance enhancement of coupled quantum well devices.

  18. Visual search in ecological and non-ecological displays: evidence for a non-monotonic effect of complexity on performance.

    Directory of Open Access Journals (Sweden)

    Philippe Chassy

    Full Text Available Considerable research has been carried out on visual search, with single or multiple targets. However, most studies have used artificial stimuli with low ecological validity. In addition, little is known about the effects of target complexity and expertise in visual search. Here, we investigate visual search in three conditions of complexity (detecting a king, detecting a check, and detecting a checkmate with chess players of two levels of expertise (novices and club players. Results show that the influence of target complexity depends on level of structure of the visual display. Different functional relationships were found between artificial (random chess positions and ecologically valid (game positions stimuli: With artificial, but not with ecologically valid stimuli, a "pop out" effect was present when a target was visually more complex than distractors but could be captured by a memory chunk. This suggests that caution should be exercised when generalising from experiments using artificial stimuli with low ecological validity to real-life stimuli.

  19. Origin of the non-monotonic variance of Tc in the 1111 iron based superconductors with isovalent doping

    Science.gov (United States)

    Usui, Hidetomo; Suzuki, Katsuhiro; Kuroki, Kazuhiko

    2015-01-01

    Motivated by recent experimental investigations of the isovalent doping iron-based superconductors LaFe(AsxP1-x)O1-yFy and NdFe(AsxP1-x)O1-yFy, we theoretically study the correlation between the local lattice structure, the Fermi surface, the spin fluctuation-mediated superconductivity, and the composition ratio. In the phosphides, the dXZ and dYZ orbitals barely hybridize around the Γ point to give rise to two intersecting ellipse shape Fermi surfaces. As the arsenic content increases and the Fe-As-Fe bond angle is reduced, the hybridization increases, so that the two bands are mixed to result in concentric inner and outer Fermi surfaces, and the orbital character gradually changes to dxz and dyz, where x–y axes are rotated by 45 degrees from X–Y. This makes the orbital matching between the electron and hole Fermi surfaces better and enhances the spin fluctuation within the dxz/yz orbitals. On the other hand, the hybridization splits the two bands, resulting in a more dispersive inner band. Hence, there is a trade-off between the density of states and the orbital matching, thereby locally maximizing the dxz/yz spin fluctuation and superconductivity in the intermediate regime of As/P ratio. The consistency with the experiment strongly indicate the importance of the spin fluctuation played in this series of superconductors. PMID:26073071

  20. Periodic solutions for a non-monotone family of delayed differential equations with applications to Nicholson systems

    Science.gov (United States)

    Faria, Teresa

    2017-07-01

    For a family of n-dimensional periodic delay differential equations which encompasses a broad set of models used in structured population dynamics, the existence of a positive periodic solution is obtained under very mild conditions. The proof uses the Schauder fixed point theorem and relies on the permanence of the system. A general criterion for the existence of a positive periodic solution for Nicholson's blowflies periodic systems (with both distributed and discrete time-varying delays) is derived as a simple application of our main result, generalizing the few existing results concerning multi-dimensional Nicholson models. In the case of a Nicholson system with discrete delays all multiples of the period, the global attractivity of the positive periodic solution is further analyzed, improving results in recent literature.

  1. Analysis of a Dynamic Viscoelastic Contact Problem with Normal Compliance, Normal Damped Response, and Nonmonotone Slip Rate Dependent Friction

    Directory of Open Access Journals (Sweden)

    Mikaël Barboteu

    2016-01-01

    Full Text Available We consider a mathematical model which describes the dynamic evolution of a viscoelastic body in frictional contact with an obstacle. The contact is modelled with a combination of a normal compliance and a normal damped response law associated with a slip rate-dependent version of Coulomb’s law of dry friction. We derive a variational formulation and an existence and uniqueness result of the weak solution of the problem is presented. Next, we introduce a fully discrete approximation of the variational problem based on a finite element method and on an implicit time integration scheme. We study this fully discrete approximation schemes and bound the errors of the approximate solutions. Under regularity assumptions imposed on the exact solution, optimal order error estimates are derived for the fully discrete solution. Finally, after recalling the solution of the frictional contact problem, some numerical simulations are provided in order to illustrate both the behavior of the solution related to the frictional contact conditions and the theoretical error estimate result.

  2. Molecular characterisation of the thermostability and catalytic properties of enzymes from hyperthermophiles

    NARCIS (Netherlands)

    Lebbink, J.H.G.

    1999-01-01

    Hyperthermophilic organisms are able to survive and reproduce optimally between 80°C and 113°C. Most of them belong to the domain of the Archaea, although several hyperthermophilic Bacteria have been described. One of the major questions regarding hyperthermophiles concerns the molecular

  3. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

    Science.gov (United States)

    Cline, J; Braman, J C; Hogrefe, H H

    1996-09-15

    The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

  4. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

    OpenAIRE

    Cline, J; Braman, J C; Hogrefe, H H

    1996-01-01

    The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at p...

  5. 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.

  6. Characterization of novel thermostable bacterial Laccase-like multi-copper oxidases

    DEFF Research Database (Denmark)

    Brander, Søren; Mikkelsen, Jørn Dalgaard

    decolorization, bleaching of paper pulp, delignification of biomass and remediation of polluted water. In order to widen the applicability of LMCOs, it is important to establish the properties and substrate specificities of naturally occurring LMCOs. This is especially true for LMCOs from bacteria, whose role...

  7. Characterizing a thermostable Cas9 for bacterial genome editing and silencing

    NARCIS (Netherlands)

    Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke F.; Vrouwe, Valentijn; Finger Bou, Max; Naduthodi, Mihris I.S.; Gussak, Alex; Brinkman, Rudolf B.L.; Kranenburg, Van Richard; Oost, Van Der John

    2017-01-01

    CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including

  8. Fabrication of electrospun almond gum/PVA nanofibers as a thermostable delivery system for vanillin.

    Science.gov (United States)

    Rezaei, Atefe; Tavanai, Hossein; Nasirpour, Ali

    2016-10-01

    In this study, the fabrication of vanillin incorporated almond gum/polyvinyl alcohol (PVA) nanofibers through electrospinning has been investigated. Electrospinning of only almond gum was proved impossible. It was found that the aqueous solution of almond gum/PVA (80:20, concentration=7% (w/w)) containing 3% (w/w) vanillin could have successfully electrospun to uniform nanofibers with diameters as low as 77nm. According to the thermal analysis, incorporated vanillin in almond gum/PVA nanofibers showed higher thermal stability than free vanillin, making this composite especially suitable for high temperature applications. XRD and FTIR analyses proved the presence of vanillin in the almond gum/PVA nanofibers. It was also found that vanillin was dispersed as big crystallites in the matrix of almond gum/PVA nanofibers. FTIR analysis showed almond gum and PVA had chemical cross-linking by etheric bonds between COH groups of almond gum and OH groups of PVA. Also, in the nanofibers, there were no major interaction between vanillin and either almond gum or PVA. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study

    Science.gov (United States)

    Pathak, Arup K.; Bandyopadhyay, Tusar

    2017-04-01

    Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

  10. The effect of carbon sources on the expression level of thermostable ...

    African Journals Online (AJOL)

    Jane

    2011-10-12

    Oct 12, 2011 ... 1 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. ... production of detergents, animal skin-based industry and food processing ... and minimal microbial contamination are achievable.

  11. Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114

    Directory of Open Access Journals (Sweden)

    Amanda Reges de Sena

    2014-01-01

    Full Text Available Tannase (EC 3.1.1.20 is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L. Skeels leaves, and identified as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL. Response surface methodology was employed to evaluate the effect of variables (pH and temperature, and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. Km was found to be 7.18·10–4 mol/L and vmax=250.00 U/mL. The tannase activity was the highest in the presence of Ca2+ at a concentration of 5·10–3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3 % of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

  12. A thermostable metal-tolerant laccase with bioremediation potential from a marine-derived fungus

    Digital Repository Service at National Institute of Oceanography (India)

    DeSouza-Ticlo, D.; Sharma, D.; Raghukumar, C.

    their optimum pH and temperature). From these results, K cat and specificity constant of Lac IId with ABTS were derived (Das et al., 2001). Substrate specificity and inhibitors of Lac IId. Oxygen uptake during reaction of Lac IId with substrates, non... from Q 10 values between 60 o and 70 o C at this pH was 2.5 kJ mol -1 whereas from the Arrhenius plot, it was 8.15 kJ mol -1 . The K m , V max and K cat of Lac IId were compared using ABTS and syringaldazine; maximum specificity constant (K...

  13. Modeling the thermostability of surface functionalisation by oxygen, hydroxyl, and water on nanodiamonds.

    Science.gov (United States)

    Lai, Lin; Barnard, Amanda S

    2011-06-01

    Understanding nanodiamond functionalisation is of great importance for biological and medical applications. Here we examine the stabilities of oxygen, hydroxyl, and water functionalisation of the nanodiamonds using the self-consistent charge density functional tight-binding simulations. We find that the oxygen and hydroxyl termination are thermodynamically favourable and form strong C–O covalent bonds on the nanodiamond surface in an O2 and H2 gas reservoir, which confirms previous experiments. Yet, the thermodynamic stabilities of oxygen and hydroxyl functionalisation decrease dramatically in a water vapour reservoir. In contrast, H2O molecules are found to be physically adsorbed on the nanodiamond surface, and forced chemical adsorption results in decomposition of H2O. Moreover, the functionalisation efficiency is found to be facet dependent. The oxygen functionalisation prefers the {100} facets as opposed to alternative facets in an O2 and H2 gas reservoir. The hydroxyl functionalisation favors the {111} surfaces in an O2 and H2 reservoir and the {100} facets in a water vapour reservoir, respectively. This facet selectivity is found to be largely dependent upon the environmental temperature, chemical reservoir, and morphology of the nanodiamonds.

  14. Evaluation of flocculating performance of a thermostable bioflocculant produced by marine Bacillus sp.

    Science.gov (United States)

    Okaiyeto, Kunle; Nwodo, Uchechukwu U; Mabinya, Leonard V; Okoli, Arinze S; Okoh, Anthony I

    2016-01-01

    This study assessed the bioflocculant (named MBF-W7) production potential of a bacterial isolate obtained from Algoa Bay, Eastern Cape Province of South Africa. The 16S ribosomal deoxyribonucleic acids gene sequence analysis showed 98% sequence similarity to Bacillus licheniformis strain W7. Optimum culture conditions for MBF-W7 production include 5% (v/v) inoculum size, maltose and NH4NO3 as carbon and nitrogen sources of choice, medium pH of 6 as the initial pH of the growth medium. Under these optimal conditions, maximum flocculating activity of 94.9% was attained after 72 h of cultivation. Chemical composition analyses showed that the purified MBF-W7 was a glycoprotein which was predominantly composed of polysaccharides 73.7% (w/w) and protein 6.2% (w/w). Fourier transform infrared spectroscopy revealed the presence of hydroxyl, carboxyl and amino groups as the main functional groups identified in the bioflocculant molecules. Thermogravimetric analyses showed the thermal decomposition profile of MBF-W7. Scanning electron microscopy imaging revealed that bridging played an important role in flocculation. MBF-W7 exhibited excellent flocculating activity for kaolin clay suspension at 0.2 mg/ml over a wide pH range of 3-11; with the maximal flocculation rate of 85.8% observed at pH 3 in the presence of Mn(2+). It maintained and retained high flocculating activity of over 70% after heating at 100°C for 60 min. MBF-W7 showed good turbidity removal potential (86.9%) and chemical oxygen demand reduction efficiency (75.3%) in Tyume River. The high flocculating rate of MBF-W7 makes it an attractive candidate to replace chemical flocculants utilized in water treatment.

  15. Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Eichler, Jerry; Ivanova, Natalia; Axen, Seth D.; Kerfeld, Cheryl A.; Chen, Feng; Kyrpides, Nikos; Hugenholtz, Philip; Cheng, Jan-Fang; Sale, Kenneth L.; Simmons, Blake; Rubin, Eddy

    2011-02-17

    Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20percent w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.

  16. A Novel Highly Thermostable Multifunctional Beta-Glycosidase from Crenarchaeon Acidilobus saccharovorans

    Directory of Open Access Journals (Sweden)

    Vadim M. Gumerov

    2015-01-01

    Full Text Available We expressed a putative β-galactosidase Asac_1390 from hyperthermophilic crenarchaeon Acidilobus saccharovorans in Escherichia coli and purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1, pNP-β-D-glucopyranoside (246 U mg−1, pNP-β-D-xylopyranoside (72 U mg−1, and pNP-β-D-mannopyranoside (28 U mg−1. Thus the enzyme was actually a multifunctional β-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

  17. Influence of ligand binding on structure and thermostability of human alpha(1)-acid glycoprotein

    Czech Academy of Sciences Publication Activity Database

    Kopecký, V. Jr.; Ettrich, Rüdiger; Pazderka, T.; Hofbauerová, Kateřina; Řeha, David; Baumruk, V.

    2016-01-01

    Roč. 29, č. 2 (2016), s. 70-79 ISSN 0952-3499 Institutional support: RVO:61388971 Keywords : orosomucoid * binding site * Raman spectroscopy Subject RIV: CE - Biochemistry Impact factor: 2.175, year: 2016

  18. Cloning and characterization of a thermostable 2- deoxy-D-ribose-5 ...

    African Journals Online (AJOL)

    Analysis of the presumptive 2-deoxy-D-ribose 5-phosphate aldolase gene from Aciduliprofundum boonei revealed an open reading frame (ORF) encoding 222 amino acids, which was subcloned and then expressed in Escherichia coli. The recombinant DERA protein was purified to apparent homogeneity. The enzyme ...

  19. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

    Science.gov (United States)

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-10-15

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Production of a Thermostable Α-Amylase and its Assay using ...

    African Journals Online (AJOL)

    Screening for amylolytic properties from obtained isolates was carried out on starch agar plates while production and characterization of α-amylase was carried out using submerged fermentation. The isolated organism was identified as Bacillus licheniformis. Physiological studies on the isolate showed that temperature of ...

  1. Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

    Science.gov (United States)

    de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

    1995-05-01

    Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

  2. Contact materials for thermostable resistors on the base of Ni-Re alloy

    International Nuclear Information System (INIS)

    Yusipov, H.Yu.; Glasman, L.I.; Arskaya, E.P.; Lazarev, Eh.M.; Korotkov, N.A.

    1979-01-01

    Given are the electron diffraction analysis results and the operational characteristics of the contact materials, used in the heat-resistant thin-filmed resistors (TFR), made on the basis of the Ni-Re system alloy. The results are compared with the pure nickel. Operational tests of the thin-filmed resistors, having (NR10-VP) alloy contacts, showed that the departure of the resistors nominals is almost twice as small as that for the resistors, having pure nickel contacts. The use of this alloy permits to increase the thermal stability and durability of the TFRs, if they are used under extreme conditions

  3. Optical bistability of a thin film of resonant atoms in a phase-sensitive thermostate

    International Nuclear Information System (INIS)

    Basharov, A.M.

    1995-01-01

    It is shown theoretically that when a thin film of two-level atoms interacting with a resonant coherent electromagnetic wave is additionally illuminated with a squeezed field, a bistable transmission/reflection regime for coherent waves is obtained. This regime depends strongly on the phase difference between the coherent and the squeezed fields. New regimes, including a bistable regime, for the interaction of a coherent field with a film of resonant atoms are predicted based on this phenomenon. 14 refs., 5 figs

  4. Thermostability of the coating, antigen and immunostimulator in an adjuvanted oral capsule vaccine formulation.

    Science.gov (United States)

    Longet, Stephanie; Aversa, Vincenzo; O'Donnell, Daire; Tobias, Joshua; Rosa, Monica; Holmgren, Jan; Coulter, Ivan S; Lavelle, Ed C

    2017-12-20

    Oral vaccines present an attractive alternative to injectable vaccines for enteric diseases due to ease of delivery and the induction of intestinal immunity at the site of infection. However, susceptibility to gastrointestinal proteolysis, limited transepithelial uptake and a lack of clinically acceptable adjuvants present significant challenges. A further challenge to mass vaccination in developing countries is the very expensive requirement to maintain the cold chain. We recently described the effectiveness of a Single Multiple Pill ® (SmPill ® ) adjuvanted capsule approach to enhance the effectiveness of a candidate enterotoxigenic Escherichia coli (ETEC) oral vaccine. Here it was demonstrated that this delivery system maintains the antigenicity of ETEC colonisation factor antigen I (CFA/I) and the immunostimulatory activity of the orally active α-Galactosylceramide (α-GalCer) adjuvant after storage of SmPill ® minispheres under room temperature and extreme storage conditions for several months. In addition, the internal structure of the cores of SmPill ® minispheres and antigen release features at intestinal pH were found to be preserved under all these conditions. However, changes in the surface morphology of SmPill ® minispheres leading to the antigen release at gastric pH were observed after a few weeks of storage under extreme conditions. Those modifications were prevented by the introduction of an Opadry ® White film coating layer between the core of SmPill ® minispheres and the enteric coating. Under these conditions, protection against antigen release at gastric pH was maintained even under high temperature and humidity conditions. These results support the potential of the SmPill ® minisphere approach to maintain the stability of an adjuvanted whole cell killed oral vaccine formulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Generalized Langevin dynamics of a nanoparticle using a finite element approach: Thermostating with correlated noise

    Science.gov (United States)

    Uma, B.; Swaminathan, T. N.; Ayyaswamy, P. S.; Eckmann, D. M.; Radhakrishnan, R.

    2011-09-01

    A direct numerical simulation (DNS) procedure is employed to study the thermal motion of a nanoparticle in an incompressible Newtonian stationary fluid medium with the generalized Langevin approach. We consider both the Markovian (white noise) and non-Markovian (Ornstein-Uhlenbeck noise and Mittag-Leffler noise) processes. Initial locations of the particle are at various distances from the bounding wall to delineate wall effects. At thermal equilibrium, the numerical results are validated by comparing the calculated translational and rotational temperatures of the particle with those obtained from the equipartition theorem. The nature of the hydrodynamic interactions is verified by comparing the velocity autocorrelation functions and mean square displacements with analytical results. Numerical predictions of wall interactions with the particle in terms of mean square displacements are compared with analytical results. In the non-Markovian Langevin approach, an appropriate choice of colored noise is required to satisfy the power-law decay in the velocity autocorrelation function at long times. The results obtained by using non-Markovian Mittag-Leffler noise simultaneously satisfy the equipartition theorem and the long-time behavior of the hydrodynamic correlations for a range of memory correlation times. The Ornstein-Uhlenbeck process does not provide the appropriate hydrodynamic correlations. Comparing our DNS results to the solution of an one-dimensional generalized Langevin equation, it is observed that where the thermostat adheres to the equipartition theorem, the characteristic memory time in the noise is consistent with the inherent time scale of the memory kernel. The performance of the thermostat with respect to equilibrium and dynamic properties for various noise schemes is discussed.

  6. The use of thermostable bacterial hemicellulases improves the conversion of lignocellulosic biomass to valuable molecules.

    Science.gov (United States)

    Rakotoarivonina, Harivony; Revol, Pierre-Vincent; Aubry, Nathalie; Rémond, Caroline

    2016-09-01

    The hydrolysis of xylans, one of the main classes of carbohydrates that constitute lignocellulosic biomass, requires the synergistic action of several enzymes. The development of efficient enzymatic strategies for hydrolysis remains a challenge in the pursuit of viable biorefineries, particularly with respect to the valorisation of pentoses. The approach developed in this work is based on obtaining and characterising hemicellulasic cocktails from Thermobacillus xylanilyticus after culturing this bacterium on the hemicellulose-rich substrates wheat bran and wheat straw, which differ in their chemistries. The two obtained cocktails (WSC and WBC, for cocktails obtained from wheat straw and wheat bran, respectively) were resistant to a broad range of temperature and pH conditions. At 60 °C, both cocktails efficiently liberated pentoses and phenolic acids from wheat bran (liberating more than 60, 30 and 40 % of the total xylose, arabinose and ferulic acid in wheat bran, respectively). They acted to a lesser extent on the more recalcitrant wheat straw, with hydrolytic yields of more than 30 % of the total arabinose and xylose content and 22 % of the ferulic acid content. Hydrolysis is associated with a high rate of sugar monomerisation. When associated with cellulases, high quantities of glucose were also obtained. On wheat bran, total glucose yields were improved by 70 % compared to the action of cellulases alone. This improvement was obtained by cellulase complementation either with WSC or with WBC. On wheat straw, similar levels of total glucose were obtained for cellulases alone or complemented with WSC or WBC. Interestingly, the complementation of cellulases with WSC or WBC induced an increase in the monomeric glucose yield of more than 20 % compared to cellulases alone.

  7. Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost

    Directory of Open Access Journals (Sweden)

    ÉVILIN G. DE MARCO

    2017-10-01

    Full Text Available ABSTRACT During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1. For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1 for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

  8. Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant

    NARCIS (Netherlands)

    Rahman, D. Y.; Sarian, F. D.; van Wijk, A.; Martinez-Garcia, M.; van der Maarel, M. J. E. C.

    The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae

  9. Improvement of thermostability and activity of firefly luciferase through [TMG][Ac] ionic liquid mediator.

    Science.gov (United States)

    Ebrahimi, Mehdi; Hosseinkhani, Saman; Heydari, Akbar; Khavari-Nejad, Ramazan Ali; Akbari, Jafar

    2012-10-01

    Firefly luciferase catalyzes production of light from luciferin in the presence of Mg(2+)-ATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K(m) for ATP.

  10. A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

    Science.gov (United States)

    Wang, Xiaoyu; Luo, Huiying; Yu, Wangning; Ma, Rui; You, Shuai; Liu, Weina; Hou, Lingyu; Zheng, Fei; Xie, Xiangming; Yao, Bin

    2016-05-15

    A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Antimicrobial activity and high thermostability of a novel blissecreted by enterococcus mundtiiisolated from Lebanese cow's milk

    International Nuclear Information System (INIS)

    Al Kassaa, I.; Safourim, N.; Mostafa, N.; Hamze, M.

    2016-01-01

    Lactic acid bacteria (LAB) are used in many fields such as fermentation agents, increasing nutritional value and improving organoleptic quality of food. Also they are use das probiotics and preservatives against pathogensand spoilage microbes by producing antimicrobial substances such as bacteriocins. Fifty cow's milk samples were collected and 175 LAB isolates were isolated and identified by using biochemical method. Fifteen isolates showedan antimicrobial activity against Listeria monocytogenesATCC® 19115™.One strain, BL4 which showed the strongest activity, was chosen to extract and characterize its antimicrobial substance in order to evaluate its potential use as a new food protective agent. This strain was identified as Enterococcus mundtiibypyrosequencing method.The active substance was extracted using solvent method.This Bacteriocin likeInhibitory Substances “BLIS”can support a high temperature (121 °C) for a long time and resist pH variation.The BLIS BL4 can be considered as a peptide active against many food pathogen and food-spoilage microbes, such as Listeriamonocytogenes and Penicillium spp. BLISBL4can be used in food application as bio-preservative to reduce food-spoilage and food-borne diseases in food product. (author

  12. Purification, crystallization and preliminary crystallographic analysis of a thermostable endonuclease IV from Thermotoga maritima

    International Nuclear Information System (INIS)

    Hughes, Ronny C.; Tomanicek, Stephen J.; Ng, Joseph D.; Coates, Leighton

    2009-01-01

    The overexpression, purification and crystallization of endonuclease IV from T. maritima are reported. The crystals belonged to the hexagonal space group P6 1 and diffracted to 2.36 Å resolution. The DNA-repair enzyme endonuclease IV from the thermophilic bacterium Thermotoga maritima MSB8 (reference sequence NC-000853) has been expressed in Escherichia coli and crystallized for X-ray analysis. T. maritima endonuclease IV is a 287-amino-acid protein with 32% sequence identity to E. coli endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting-drop vapor-diffusion method. The protein crystallized in space group P6 1 , with one biological molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.39 Å 3 Da −1 and 47% solvent content. The unit-cell parameters of the crystals were a = b = 123.2, c = 35.6 Å. Microseeding and further optimization yielded crystals with an X-ray diffraction limit of 2.36 Å. A single 70° data set was collected and processed, resulting in an overall R merge and a completeness of 9.5% and 99.3%, respectively

  13. TtMCO: A highly thermostable laccase-like multicopper oxidase from the thermophilic Thermobaculum terrenum

    DEFF Research Database (Denmark)

    Brander, Søren; Mikkelsen, Jørn Dalgaard; Kepp, Kasper Planeta

    2015-01-01

    This paper reports the identification, heterologous expression in Escherichia coli and characterization of TtMCO from the thermophilic bacterium Thermobaculum terrenum, the first laccase-like multi-copper oxidase (LMCO) from the distinct Phylum Chloroflexi. TtMCO has only 39% identity to its...... closest characterized homologue, CotA from Bacillus subtilis, but sequence and spectrophotometry confirmed copper coordination similar to that of LMCOs. TtMCO is extremely thermophilic with a half-time of inactivation of 2.24 days at 70 degrees C and 350 min at 80°C and pH 7, consistent...

  14. 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.

  15. 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.).

  16. Production of a thermostable B-glucosidase by a mesophilic fungus aspergillus niger NIAB 280

    International Nuclear Information System (INIS)

    Iqbal, M.J.; Rajoka, M.I.; Malik, K.A.

    1991-01-01

    Aspergillus niger NIAB 280 produced extracellular B- glucosidease when grown on different ligno cellulosic substrates. Wheat bran was found to be the best inducer of this enzyme. Maximum enzyme production was found at initial pH 5. When the organisms was grown on easily available carbohydrates, the enzyme was induced only to a basic level essential for microbial growth. The optimum pH for enzyme activity was found to be 3 whereas optimum temperature was 55 degree C. The enzyme retained 100% activity when exposed to a temperature of 50 degree C for 5 days. When this enzyme was added to FPase from T. reesei and celluclast (a commercial cellulase from Novazyme), the %age saccharification as well as glucose production from lignocellulosic biomass was doubled. (author)

  17. Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Brillowska-Dabrowska, Anna; Ahring, Birgitte Kiær

    2013-01-01

    Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis......) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg......, pH 8.0 and at low Mg2+ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3'-5' exonuclease activity and at Mg2+ concentration > 2.0 mM 3' nuclease activity was observed. Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long...

  18. Effect of mutations on the thermostability of Aspergillus aculeatus β-1, 4-galactanase

    DEFF Research Database (Denmark)

    Terpenholt, Søs; De Maria, Leonardo; Olsson, Mats H.M.

    2015-01-01

    New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool......, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions......R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability. Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also...

  19. Investigation of the Thermostability of Bovine Submaxillary Mucin (BSM) and its Impact on Lubrication

    DEFF Research Database (Denmark)

    Madsen, Jan Busk; Pakkanen, Kirsi I.; Lee, Seunghwan

    2013-01-01

    Bovine Submaxillary Mucin (BSM) generates thin film layers via spontaneous adsorption onto hydrophobic surfaces such as Poly(dimethylsiloxane) (PDMS) and High Density Polyethylene (HDPE). A characteristic feature of mucin is its tribological- or lubricating properties. Circular dichroismspectrosc......Bovine Submaxillary Mucin (BSM) generates thin film layers via spontaneous adsorption onto hydrophobic surfaces such as Poly(dimethylsiloxane) (PDMS) and High Density Polyethylene (HDPE). A characteristic feature of mucin is its tribological- or lubricating properties. Circular...

  20. Characterizing a thermostable Cas9 for bacterial genome editing and silencing

    DEFF Research Database (Denmark)

    Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke Fenna

    2017-01-01

    CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including...