WorldWideScience

Sample records for nonmonotonic solute-induced thermostability

  1. A modal nonmonotonic logic

    Institute of Scientific and Technical Information of China (English)

    林作铨

    1996-01-01

    A modal nonmonotonic logic is presented based on an experiential modal semantics on typicality and exception.The syntactic and semantics of modal nonmonotonic logic are provided,and the completeness theorem and the theorems relating it to major nonmonotonic logics are proved.It directly formalizes the intuition of nonmonotonic reasoning.Among other things,it provides us a first-order extension of default logic and autoepistemic logic,and simultaneously has the capability of circumscription to infer universal statement.It has important applications in logic programming and deductive data base.As a result,it provides a uniform basis for various nonmonotonic logics,from which the correspondent relationship among major nonmonotonic logics can coincide.

  2. Nonmonotonic diffusion in crowded environments

    Science.gov (United States)

    Putzel, Gregory Garbès; Tagliazucchi, Mario; Szleifer, Igal

    2015-01-01

    We study the diffusive motion of particles among fixed spherical crowders. The diffusers interact with the crowders through a combination of a hard-core repulsion and a short-range attraction. The long-time effective diffusion coefficient of the diffusers is found to depend non-monotonically on the strength of their attraction to the crowders. That is, for a given concentration of crowders, a weak attraction to the crowders enhances diffusion. We show that this counterintuitive fact can be understood in terms of the mesoscopic excess chemical potential landscape experienced by the diffuser. The roughness of this excess chemical potential landscape quantitatively captures the nonmonotonic dependence of the diffusion rate on the strength of crowder-diffuser attraction; thus it is a purely static predictor of dynamic behavior. The mesoscopic view given here provides a unified explanation for enhanced diffusion effects that have been found in various systems of technological and biological interest. PMID:25302920

  3. 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....

  4. Causation, explanation and nonmonotonic temporal reasoning

    NARCIS (Netherlands)

    Grünwald, P.D.

    1997-01-01

    We introduce a new approach to reasoning about action and change using nonmonotonic logic. The approach is arrived at by applying Pearl's theory of causal networks to logical formalizations of temporal reasoning domains. It handles complicated reasoning domains involving concurrent actions, actions

  5. 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. 

  6. 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

    2012-05-01

    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.

  7. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    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.

  8. Complexity of Non-Monotonic Logics

    CERN Document Server

    Thomas, Michael

    2010-01-01

    Over the past few decades, non-monotonic reasoning has developed to be one of the most important topics in computational logic and artificial intelligence. Different ways to introduce non-monotonic aspects to classical logic have been considered, e.g., extension with default rules, extension with modal belief operators, or modification of the semantics. In this survey we consider a logical formalism from each of the above possibilities, namely Reiter's default logic, Moore's autoepistemic logic and McCarthy's circumscription. Additionally, we consider abduction, where one is not interested in inferences from a given knowledge base but in computing possible explanations for an observation with respect to a given knowledge base. Complexity results for different reasoning tasks for propositional variants of these logics have been studied already in the nineties. In recent years, however, a renewed interest in complexity issues can be observed. One current focal approach is to consider parameterized problems and ...

  9. Thermostability of montmorillonitic clays

    Directory of Open Access Journals (Sweden)

    Petr Jelínek

    2014-05-01

    Full Text Available Bentonite is one of the most widespread used clays connected with various applications. In the case of foundry technology, bentonite is primarily used as a binder for mold manufacture. Thermal stability of bentonites is a natural property of clay minerals and it depends on the genesis, source and chemical composition of the clay. This property is also closely connected to bentonite structure. According to DTA analysis if only one peak of dehydroxylation is observed (about 600 ºC, the cis- isomerism of bentonite is expected, while two peaks of de-hydroxylation (about 550 and 850 ºC are expected in the trans- one. In this overview, the bentonite structure, the water – bentonite interaction and the swelling behavior of bentonite in connection with the general technological properties of bentonite molding mixture are summarized. Further, various types of methods for determination of bentonite thermostability are discussed, including instrumental analytical methods as well as methods that employ evaluation of various technological properties of bentonite binders and/or bentonite molding mixtures.

  10. 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.

  11. 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.

  12. Thermostability of montmorillonitic clays

    Institute of Scientific and Technical Information of China (English)

    Petr Jelnek; Stanisaw M.Dobosz; Jaroslav Beo; Katarzyna Major-Gabry

    2014-01-01

    Bentonite is one of the most widespread used clays connected with various applications. In the case of foundry technology, bentonite is primarily used as a binder for mold manufacture. Thermal stability of bentonites is a natural property of clay minerals and it depends on the genesis, source and chemical composition of the clay. This property is also closely connected to bentonite structure. According to DTA analysis if only one peak of dehydroxylation is observed (about 600 ºC), thecis- isomerism of bentonite is expected, while two peaks of de-hydroxylation (about 550 and 850 ºC) are expected in thetrans- one. In this overview, the bentonite structure, the water - bentonite interaction and the sweling behavior of bentonite in connection with the general technological properties of bentonite molding mixture are summarized. Further, various types of methods for determination of bentonite thermostability are discussed, including instrumental analytical methods as wel as methods that employ evaluation of various technological properties of bentonite binders and/or bentonite molding mixtures.

  13. Fluorescence and Thermostability of Nanometer Porphyrin Trimer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A nanometer porphyrin trimer was firstly synthesized with 1,3-dibromopropane as a bridge-linked agent and the fluorescence property and thermostability were studied. The results show that the fluorescence property and thermostability of the trimer are different from those of monoporphyrin. The effects of the molecule structure on the optical property and the thermostability were also studied in detail.

  14. A CLASS OF NONMONOTONE CONJUGATE GRADIENT METHODS FOR NONCONVEX FUNCTIONS

    Institute of Scientific and Technical Information of China (English)

    LiuYun; WeiZengxin

    2002-01-01

    This paper discusses the global convergence of a class of nonmonotone conjugate gradient methods (NM methods) for nonconvex object functions. This class of methods includes the nonmonotone counterpart of modified Polak-Ribiere method and modified Hestenes-Stiefel method as special cases.

  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 Synaptic Excitation and Imbalanced Inhibition Underlying Cortical Intensity Tuning

    OpenAIRE

    Wu, Guangying K.; Li, Pingyang; Tao, Huizhong W.; Zhang, Li I.

    2006-01-01

    Intensity-tuned neurons, characterized by their nonmonotonic response-level function, may play important roles in the encoding of sound intensity-related information. The synaptic mechanisms underlying intensity-tuning remain yet unclear. Here, in vivo whole-cell recordings in rat auditory cortex revealed that intensity-tuned neurons, mostly clustered in a posterior zone, receive imbalanced tone-evoked excitatory and inhibitory synaptic inputs. Excitatory inputs exhibit nonmonotonic intensity...

  17. Layered neural networks with non-monotonic transfer functions

    Science.gov (United States)

    Katayama, Katsuki; Sakata, Yasuo; Horiguchi, Tsuyoshi

    2003-01-01

    We investigate storage capacity and generalization ability for two types of fully connected layered neural networks with non-monotonic transfer functions; random patterns are embedded into the networks by a Hebbian learning rule. One of them is a layered network in which a non-monotonic transfer function of even layers is different from that of odd layers. The other is a layered network with intra-layer connections, in which the non-monotonic transfer function of inter-layer is different from that of intra-layer, and inter-layered neurons and intra-layered neurons are updated alternately. We derive recursion relations for order parameters for those layered networks by the signal-to-noise ratio method. We clarify that the storage capacity and the generalization ability for those layered networks are enhanced in comparison with those with a conventional monotonic transfer function when non-monotonicity of the transfer functions is selected optimally. We also point out that some chaotic behavior appears in the order parameters for the layered networks when non-monotonicity of the transfer functions increases.

  18. Nonmonotonic Aging and Memory Retention in Disordered Mechanical Systems

    Science.gov (United States)

    Lahini, Yoav; Gottesman, Omer; Amir, Ariel; Rubinstein, Shmuel M.

    2017-02-01

    We observe nonmonotonic aging and memory effects, two hallmarks of glassy dynamics, in two disordered mechanical systems: crumpled thin sheets and elastic foams. Under fixed compression, both systems exhibit monotonic nonexponential relaxation. However, when after a certain waiting time the compression is partially reduced, both systems exhibit a nonmonotonic response: the normal force first increases over many minutes or even hours until reaching a peak value, and only then is relaxation resumed. The peak time scales linearly with the waiting time, indicating that these systems retain long-lasting memory of previous conditions. Our results and the measured scaling relations are in good agreement with a theoretical model recently used to describe observations of monotonic aging in several glassy systems, suggesting that the nonmonotonic behavior may be generic and that athermal systems can show genuine glassy behavior.

  19. Lugol's solution-induced painless thyroiditis

    Science.gov (United States)

    Yang, Ji Wei

    2017-01-01

    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. Learning points: Lugol’s solution is used for thyroidectomy preparation in patients with Graves’ disease and for the control of severe thyrotoxicosis and thyroid storm. Iodine excess can cause both hypothyroidism and thyrotoxicosis. Thyroid glands with an underlying pathology are particularly susceptible to the

  20. Adaptive nonmonotone line search method for unconstrained optimization

    Institute of Scientific and Technical Information of China (English)

    Qunyan ZHOU; Wenyu SUN

    2008-01-01

    In this paper, an adaptive nonmonotone line search method for unconstrained minimization problems is proposed. At every iteration, the new algorithm selects only one of the two directions: a Newton-type direc-tion and a negative curvature direction, to perform the line search. The nonmonotone technique is included in the backtracking line search when the Newton-type direction is the search direction. Furthermore, if the negative curvature direction is the search direction, we increase the steplength un-der certain conditions. The global convergence to a stationary point with second-order optimality conditions is established. Some numerical results which show the efficiency of the new algorithm are reported.

  1. Modeling non-monotone risk aversion using SAHARA utility functions

    NARCIS (Netherlands)

    A. Chen; A. Pelsser; M. Vellekoop

    2011-01-01

    We develop a new class of utility functions, SAHARA utility, with the distinguishing feature that it allows absolute risk aversion to be non-monotone and implements the assumption that agents may become less risk averse for very low values of wealth. The class contains the well-known exponential and

  2. A NONMONOTONE CONJUGATE GRADIENT ALGORITHM FOR UNCONSTRAINED OPTIMIZATION

    Institute of Scientific and Technical Information of China (English)

    DAI Yuhong

    2002-01-01

    Conjugate gradient methods are very important methods for unconstrained optimization, especially for large scale problems. In this paper, we propose a new conjugate gradient method, in which the technique of nonmonotone line search is used. Under mild assumptions, we prove the global convergence of the method. Some numerical results are also presented.

  3. Reasoning Biases, Non-Monotonic Logics, and Belief Revision

    NARCIS (Netherlands)

    Dutilh Novaes, Catarina; Veluwenkamp, Herman

    2017-01-01

    A range of formal models of human reasoning have been proposed in a number of fields such as philosophy, logic, artificial intelligence, computer science, psychology, cognitive science etc.: various logics (epistemic logics; non-monotonic logics), probabilistic systems (most notably, but not exclusi

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

    CSIR Research Space (South Africa)

    Casini, G

    2013-06-01

    Full Text Available and modeling of defeasible information and non-monotonic reasoning services. Here we formalize a possible way of introducing non-monotonic reasoning into ORM2 schemas, enriching the language with special set of new constraints....

  5. EQUILIBRIUM ALGORITHMS WITH NONMONOTONE LINE SEARCH TECHNIQUE FOR SOLVING THE TRAFFIC ASSIGNMENT PROBLEMS

    Institute of Scientific and Technical Information of China (English)

    ZHAO Hui; GAO Ziyou

    2005-01-01

    This paper presents a unified framework of the nonmonotone convex combination algorithms (such as Frank-Wolfe Algorithm) for solving the traffic assignment problems. Global convergence results are established under mild conditions. The line search procedure used in our algorithm includes the nonmonotone Armijo rule, the nonmonotone Goldstein rule and the nonmonotone Wolfe rule as special cases. So, the new algorithm can be viewed as a generalization of the regular convex combination algorithm.

  6. Measurement of non-monotonic Casimir forces between silicon nanostructures

    Science.gov (United States)

    Tang, L.; Wang, M.; Ng, C. Y.; Nikolic, M.; Chan, C. T.; Rodriguez, A. W.; Chan, H. B.

    2017-01-01

    Casimir forces are of fundamental interest because they originate from quantum fluctuations of the electromagnetic field. Apart from controlling this force via the optical properties of materials, a number of novel geometries have been proposed to generate repulsive and/or non-monotonic Casimir forces between bodies separated by vacuum gaps. Experimental realization of these geometries, however, is hindered by the difficulties in alignment when the bodies are brought into close proximity. Here, using an on-chip platform with integrated force sensors and actuators, we circumvent the alignment problem and measure the Casimir force between two surfaces with nanoscale protrusions. We demonstrate that the force depends non-monotonically on the displacement. At some displacements, the Casimir force leads to an effective stiffening of the nanomechanical spring. Our findings pave the way for exploiting the Casimir force in nanomechanical systems using structures of complex and non-conventional shapes.

  7. AN ADAPTIVE NONMONOTONIC TRUST REGION METHOD WITH CURVILINEAR SEARCHES

    Institute of Scientific and Technical Information of China (English)

    Qun-yan Zhou; Wen-yu Sun

    2006-01-01

    In this paper, an algorithm for unconstrained optimization that employs both trust region techniques and curvilinear searches is proposed. At every iteration, we solve the trust region subproblem whose radius is generated adaptively only once. Nonmonotonic backtracking curvilinear searches are performed when the solution of the subproblem is unacceptable. The global convergence and fast local convergence rate of the proposed algorithms are established under some reasonable conditions. The results of numerical experiments are reported to show the effectiveness of the proposed algorithms.

  8. Modeling argumentation based semantics using non-monotonic reasoning

    OpenAIRE

    2005-01-01

    Argumentation theory is an alternative style of formalizing non-monotonic reasoning. It seems, argumentation theory is a suitable framework for practical and uncertain reasoning, where arguments support conclusions. Dung's approach is an unifying framework which has played an influential role on argumentation research and Artificial Intelligence. Even though the success of the argumentation theory, it seems that argumentation theory is so far from being efficiently implemented like the logic ...

  9. Non-monotonic effect of confinement on the glass transition

    Science.gov (United States)

    Varnik, Fathollah; Franosch, Thomas

    2016-04-01

    The relaxation dynamics of glass forming liquids and their structure are influenced in the vicinity of confining walls. This effect has mostly been observed to be a monotonic function of the slit width. Recently, a qualitatively new behaviour has been uncovered by Mittal and coworkers, who reported that the single particle dynamics in a hard-sphere fluid confined in a planar slit varies in a non-monotonic way as the slit width is decreased from five to roughly two particle diametres (Mittal et al 2008 Phys. Rev. Lett. 100 145901). In view of the great potential of this effect for applications in those fields of science and industry, where liquids occur under strong confinement (e.g. nano-technology), the number of researchers studying various aspects and consequences of this non-monotonic behaviour has been rapidly growing. This review aims at providing an overview of the research activity in this newly emerging field. We first briefly discuss how competing mechanisms such as packing effects and short-range attraction may lead to a non-monotonic glass transition scenario in the bulk. We then analyse confinement effects on the dynamics of fluids using a thermodynamic route which relates the single particle dynamics to the excess entropy. Moreover, relating the diffusive dynamics to the Widom’s insertion probability, the oscillations of the local dynamics with density at moderate densities are fairly well described. At high densities belonging to the supercooled regime, however, this approach breaks down signaling the onset of strongly collective effects. Indeed, confinement introduces a new length scale which in the limit of high densities and small pore sizes competes with the short-range local order of the fluid. This gives rise to a non-monotonic dependence of the packing structure on confinement, with a corresponding effect on the dynamics of structural relaxation. This non-monotonic effect occurs also in the case of a cone-plate type channel, where the degree

  10. 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

  11. 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.

  12. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    Bioethanol from lignocellulosic biomass is among the green alternatives to fossil fuels, but as the processing techniques are today, gasolin is still heaper than bioethanol. Lignocellulose, which remains the primary resource for ioethanol production in Denmark, is complex when it comes to release...... 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...

  13. NONMONOTONE PRECONDITIONAL CURVILINEAR PATH ALGORITHMS FOR UNCONSTRAINED OPTIMIZATION

    Institute of Scientific and Technical Information of China (English)

    朱德通

    2003-01-01

    This paper presents nonmonotonic quasi-Newton algorithms via two pre-conditional curvilinear paths, the preconditional modified gradient path and the precon-ditional optimal path, for unconstrained optimization problem. We employ the stableBunch-Parlett factorization method to form two curvilinear paths very easily. Thenonmonotone criterion is used to speed up the convergence progress in the contoursof objective function with large curvature. Theoretical analyses are given which provethat the proposed algorithms are globally convergent and have a local superlinear con-vergence rate under some reasonable conditions. The results of numerical experimentsare reported to show the effectiveness of the proposed algorithms.

  14. 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). While Lyandres could only show non-monotonicity of investment in market frictions, we derive an investment threshold that is U-shaped in the firm's ......'s liquid funds, a result similar to the infinite-horizon model by Boyle and Guthrie (2003). However, due to the tractability of our model, we can more clearly explain the relevant trade-offs leading to the U-shape....

  15. A NONMONOTONE TRUST REGION ALGORITHM FOR NONLINEAR OPTIMIZATION SUBJECT TO GENERAL CONSTRAINTS

    Institute of Scientific and Technical Information of China (English)

    Hongchao Zhang

    2003-01-01

    In this paper we present a nonmonotone trust region algorithm for general nonlinear constrained optimization problems. The main idea of this paper is to combine Yuan's technique[1] with a nonmonotone method similar to Ke and Han [2]. This new algorithm may not only keep the robust properties of the algorithm given by Yuan, but also have some advantages led by the nonmonotone technique. Under very mild conditions, global convergence for the algorithm is given. Numerical experiments demonstrate the efficiency of the algorithm.

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

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... 2School of Biological and Chemical Engineering, Zhejiang University ... factorial central composite design (CCD) was chosen to explain the combined ... obtained by the statistical analysis showed that corn starch at 19.8 g/l, ... the optimization process. .... would inhibited the thermostable protease synthesis.

  17. 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...... the rule whenever more than two particles interfere. As the distinguishability of the particles is continuously increased, different numbers of particles effectively interfere, which leads to interference signals that are, in general, nonmonotonic functions of the distinguishability of the particles...

  18. Chaos in neural networks with a nonmonotonic transfer function

    CERN Document Server

    Caroppo, D; Nardulli, Giuseppe; Stramaglia, S

    1999-01-01

    Time evolution of diluted neural networks with a nonmonotonic transfer function is analitically described by flow equations for macroscopic variables. The macroscopic dynamics shows a rich variety of behaviours: fixed-point, periodicity and chaos. We examine in detail the structure of the strange attractor and in particular we study the main features of the stable and unstable manifolds, the hyperbolicity of the attractor and the existence of homoclinic intersections. We also discuss the problem of the robustness of the chaos and we prove that in the present model chaotic behaviour is fragile (chaotic regions are densely intercalated with periodicity windows), according to a recently discussed conjecture. Finally we perform an analysis of the microscopic behaviour and in particular we examine the occurrence of damage spreading by studying the time evolution of two almost identical initial configurations. We show that for any choice of the parameters the two initial states remain microscopically distinct.

  19. Parallel Variable Distribution Algorithm for Constrained Optimization with Nonmonotone Technique

    Directory of Open Access Journals (Sweden)

    Congying Han

    2013-01-01

    Full Text Available A modified parallel variable distribution (PVD algorithm for solving large-scale constrained optimization problems is developed, which modifies quadratic subproblem QPl at each iteration instead of the QPl0 of the SQP-type PVD algorithm proposed by C. A. Sagastizábal and M. V. Solodov in 2002. The algorithm can circumvent the difficulties associated with the possible inconsistency of QPl0 subproblem of the original SQP method. Moreover, we introduce a nonmonotone technique instead of the penalty function to carry out the line search procedure with more flexibly. Under appropriate conditions, the global convergence of the method is established. In the final part, parallel numerical experiments are implemented on CUDA based on GPU (Graphics Processing unit.

  20. The global convergence of the non-quasi-Newton methods with non-monotone line search

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The non-quasi-Newton methods for unconstrained optimization was investigated. Non-monotone line search procedure is introduced, which is combined with the non-quasi-Newton family. Under the uniform convexity assumption on objective function, the global convergence of the non-quasi-Newton family was proved.Numerical experiments showed that the non-monotone line search was more effective.

  1. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    Bioethanol from lignocellulosic biomass is among the green alternatives to fossil fuels, but as the processing techniques are today, gasolin is still heaper than bioethanol. Lignocellulose, which remains the primary resource for ioethanol production in Denmark, is complex when it comes to release...... 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...

  2. 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...... condition for producing non-monotonic deposition profiles. The described physics by the additional equation may be different in different experimental settings....

  3. Non-monotonicity of trace distance under tensor products

    Energy Technology Data Exchange (ETDEWEB)

    Maziero, Jonas, E-mail: jonas.maziero@ufsm.br [Universidade Federal de Santa Maria (UFSM), RS (Brazil). Departamento de Fisica

    2015-10-15

    The trace distance (TD) possesses several of the good properties required for a faithful distance measure in the quantum state space. Despite its importance and ubiquitous use in quantum information science, one of its questionable features, its possible non-monotonicity under taking tensor products of its arguments (NMuTP), has been hitherto unexplored. In this article, we advance analytical and numerical investigations of this issue considering different classes of states living in a discrete and finite dimensional Hilbert space. Our results reveal that although this property of TD does not show up for pure states and for some particular classes of mixed states, it is present in a non-negligible fraction of the regarded density operators. Hence, even though the percentage of quartets of states leading to the NMuTP drawback of TD and its strength decrease as the system's dimension grows, this property of TD must be taken into account before using it as a figure of merit for distinguishing mixed quantum states. (author)

  4. Molecular basis for the thermostability of Newcastle disease virus

    Science.gov (United States)

    Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against Newcastle disease, due to their decreased dependence on cold chain for transport and storage. However, the genetic basis underlying the NDV thermostability is poorly understood. In this stud...

  5. Engineering proteins for thermostability through rigidifying flexible sites.

    Science.gov (United States)

    Yu, Haoran; Huang, He

    2014-01-01

    Engineering proteins for thermostability is an exciting and challenging field since it is critical for broadening the industrial use of recombinant proteins. Thermostability of proteins arises from the simultaneous effect of several forces such as hydrophobic interactions, disulfide bonds, salt bridges and hydrogen bonds. All of these interactions lead to decreased flexibility of polypeptide chain. Structural studies of mesophilic and thermophilic proteins showed that the latter need more rigid structures to compensate for increased thermal fluctuations. Hence flexibility can be an indicator to pinpoint weak spots for enhancing thermostability of enzymes. A strategy has been proven effective in enhancing proteins' thermostability with two steps: predict flexible sites of proteins firstly and then rigidify these sites. We refer to this approach as rigidify flexible sites (RFS) and give an overview of such a method through summarizing the methods to predict flexibility of a protein, the methods to rigidify residues with high flexibility and successful cases regarding enhancing thermostability of proteins using RFS.

  6. 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

    Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and ...... of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases....

  7. Infeasible-interior-point algorithm for a class of nonmonotone complementarity problems and its computational complexity

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This paper presents an infeasible-interior-point algorithm for aclass of nonmonotone complementarity problems, and analyses its convergence and computational complexity. The results indicate that the proposed algorithm is a polynomial-time one.

  8. Periodic Solutions of Periodic Delay Predator-Prey System with Nonmonotonic Functional Response

    Institute of Scientific and Technical Information of China (English)

    宋永利; 韩茂安

    2003-01-01

    By using the continuation theorem of coincidence degree theory, sufficient conditions are obtained for theexistence of positive periodic solutions of a delayed predator-prey system with nonmonotonic functional response ina periodic environment.

  9. 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.

  10. Thermostable Subunit Vaccines for Pulmonary Delivery

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    -administrable, can be distributed independently of functioning freezers and refrigerators, and can be designed to induce mucosal and/or cell-mediated immunity, which is attractive for a number of diseases requiring stimulation of local mucosal immunity for protection. However, the design and delivery of thermostable...... dry powder-based vaccines represents a technological challenge: It calls for careful formulation and dosage form design, combined with cheap and efficient delivery devices, which must be engineered via a thorough understanding of the physiological barrier and the requirements for induction of mucosal...... 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....

  11. 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.

  12. 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.

  13. More data, less information? Potential for nonmonotonic information growth using GEE.

    Science.gov (United States)

    Shoben, Abigail B; Rudser, Kyle D; Emerson, Scott S

    2017-01-01

    Statistical intuition suggests that increasing the total number of observations available for analysis should increase the precision with which parameters can be estimated. Such monotonic growth of statistical information is of particular importance when data are analyzed sequentially, such as in confirmatory clinical trials. However, monotonic information growth is not always guaranteed, even when using a valid, but inefficient estimator. In this article, we demonstrate the theoretical possibility of nonmonotonic information growth when using generalized estimating equations (GEE) to estimate a slope and provide intuition for why this possibility exists. We use theoretical and simulation-based results to characterize situations that may result in nonmonotonic information growth. Nonmonotonic information growth is most likely to occur when (1) accrual is fast relative to follow-up on each individual, (2) correlation among measurements from the same individual is high, and (3) measurements are becoming more variable further from randomization. In situations that may lead to nonmonotonic information growth, study designers should plan interim analyses to avoid situations most likely to result in nonmonotonic information growth.

  14. Convergence of a smoothing algorithm for symmetric cone complementarity problems with a nonmonotone line search

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In this paper, we propose a smoothing algorithm for solving the monotone symmetric cone complementarity problems (SCCP for short) with a nonmonotone line search. We show that the nonmonotone algorithm is globally convergent under an assumption that the solution set of the problem concerned is nonempty. Such an assumption is weaker than those given in most existing algorithms for solving optimization problems over symmetric cones. We also prove that the solution obtained by the algorithm is a maximally complementary solution to the monotone SCCP under some assumptions.

  15. Non-monotonic dynamics and crosstalk in signaling pathways and their implications for pharmacology

    Science.gov (United States)

    van Wijk, Roeland; Tans, Sander J.; Wolde, Pieter Rein Ten; Mashaghi, Alireza

    2015-06-01

    Currently, drug discovery approaches commonly assume a monotonic dose-response relationship. However, the assumption of monotonicity is increasingly being challenged. Here we show that for two simple interacting linear signaling pathways that carry two different signals with different physiological responses, a non-monotonic input-output relation can arise with simple network topologies including coherent and incoherent feed-forward loops. We show that non-monotonicity of the response functions has severe implications for pharmacological treatment. Fundamental constraints are imposed on the effectiveness and toxicity of any drug independent of its chemical nature and selectivity due to the specific network structure.

  16. VARIED STEPLENGTH NONMONOTONE PATTERN SEARCH METHOD%变步长非单调模式搜索法

    Institute of Scientific and Technical Information of China (English)

    杨春; 倪勤

    2005-01-01

    A varied steplength nonmonotone pattern search method is proposed in this paper. The varied steplength search strategy is designed in this method such that the pattern direction is more approximated to efficient descent direction. The interpolation and nonmonotone technique are used for improving local search and global convergence. The theoretical and numerical results show that this method is an efficient direct search method.

  17. 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.

  18. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Manoj Kumar, PhD

    2011-04-26

    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.

  20. Global Convergence of the Broyden's Class of Quasi-Newton Methods with Nonmonotone Linesearch

    Institute of Scientific and Technical Information of China (English)

    Da-chuan Xu

    2003-01-01

    In this paper, the Broyden class of quasi-Newton methods for unconstrained optimization is investigated. Non-monotone linesearch procedure is introduced, which is combined with the Broyden's class. Under the convexity assumption on objective function, the global convergence of the Broyden's class is proved.

  1. Bifurcations of a predator-prey model with non-monotonic response function

    NARCIS (Netherlands)

    Broer, H.W.; Naudot, Vincent; Roussarie, Robert; Saleh, Khairul

    2005-01-01

    A 2-dimensional predator-prey model with five parameters is investigated, adapted from the Volterra-Lotka system by a non-monotonic response function. A description of the various domains of structural stability and their bifurcations is given. The bifurcation structure is reduced to four organising

  2. Computation of non-monotonic Lyapunov functions for continuous-time systems

    Science.gov (United States)

    Li, Huijuan; Liu, AnPing

    2017-09-01

    In this paper, we propose two methods to compute non-monotonic Lyapunov functions for continuous-time systems which are asymptotically stable. The first method is to solve a linear optimization problem on a compact and bounded set. The proposed linear programming based algorithm delivers a CPA1

  3. Nonmonotonous concentration behaviour of T sub c in Van-Hove scenario

    CERN Document Server

    Kozlov, A N

    2002-01-01

    Dependence of T sub c on doping in the Hubbard model with a low- repulsion in the range of carrier concentration, where the Cooper amplitude depends on the system vicinity to spin density wave transition, is considered. It is shown that in the Van Hove scenario in the range of optimal doping nonmonotonous dependence of T sub c on carrier concentration is observed

  4. Spreading Speed for a Periodic Reaction-diffusion Model with Nonmonotone Birth Function

    Institute of Scientific and Technical Information of China (English)

    HUANG Ye-hui; WENG Pei-xuan

    2012-01-01

    A reaction-diffusion model for a single spccies with age structure and nonlocal reaction for periodic time t is derived.Some results about the model with monotone birth function are firstly introduced,and then by constructing two auxiliary equations and squeezing method,the spreading speed for the system with nonmonotone birth function is obtained.

  5. An unconstrained optimization method using nonmonotone second order Goldstein's line search

    Institute of Scientific and Technical Information of China (English)

    Wen-yu; SUN; Qun-yan; ZHOU

    2007-01-01

    In this paper, an unconstrained optimization method using the nonmonotone second order Goldstein's line search is proposed. By using the negative curvature information from the Hessian,the sequence generated is shown to converge to a stationary point with the second order optimality conditions. Numerical tests on a set of standard test problems confirm the efficiency of our new method.

  6. 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....

  7. 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

  8. A strategy to model nonmonotonic dose-response curve and estimate IC50.

    Science.gov (United States)

    Zhang, Hui; Holden-Wiltse, Jeanne; Wang, Jiong; Liang, Hua

    2013-01-01

    The half-maximal inhibitory concentration IC[Formula: see text] is an important pharmacodynamic index of drug effectiveness. To estimate this value, the dose response relationship needs to be established, which is generally achieved by fitting monotonic sigmoidal models. However, recent studies on Human Immunodeficiency Virus (HIV) mutants developing resistance to antiviral drugs show that the dose response curve may not be monotonic. Traditional models can fail for nonmonotonic data and ignore observations that may be of biologic significance. Therefore, we propose a nonparametric model to describe the dose response relationship and fit the curve using local polynomial regression. The nonparametric approach is shown to be promising especially for estimating the IC[Formula: see text] of some HIV inhibitory drugs, in which there is a dose-dependent stimulation of response for mutant strains. This model strategy may be applicable to general pharmacologic, toxicologic, or other biomedical data that exhibits a nonmonotonic dose response relationship for which traditional parametric models fail.

  9. Structural Basis for the Thermostability of Sulfur Oxygenase Reductases

    Institute of Scientific and Technical Information of China (English)

    尤晓颜; 孟珍; 陈栋炜; 郭旭; Josef Zeyer; 刘双江; 姜成英

    2012-01-01

    The thermostability of three sulfur oxygenase reductases (SORs) was investigated from thermoacidophilic achaea Acidianus tengchongensis (SORAT) and Sulfolobus tokodaii (SORsT) as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 (SORsB). The optimal temperatures for catalyzing sulfur oxidation were 80 ℃ (SORAT), 85 ℃ (SORsT), and 70 ℃ (SORsB), respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min (SORAT), 58 min (SORsT), and 37 min (SORsB). In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA (from Acidianus ambivalens) as a template. The results suggest that thermostability was dependent on: (a) high number of the charged amino acid glutamic acid and the flexible amino acid proline, (b) low number of the therraolabile amino acid glutamine, (c) increased number of ion pairs, (d) decreased ratio of hydrophobie accessible solvent surface area (ASA) to charged ASA, and (e) increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not signifieantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.

  10. THERMOSTABILITY OF RESPIRATORY TERMINAL OXIDASES IN THE LIPID ENVIRONMENT

    NARCIS (Netherlands)

    Elferink, Marieke G.L.; Bosmal, Tjibbe; Lolkema, Juke S.; Gleiszner, Michael; Driessen, Arnold J.M.; Konings, Wil N.

    1995-01-01

    The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-e oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the a

  11. Changing the thermostability of Bacillus licheniformis a-amylase

    OpenAIRE

    De Cordt, S.; Saraiva, J.; Hendrickx, M.; Maesmans, G.; Tobback, P

    1993-01-01

    We applied "solvent engineering" (i.e. variation of environmental conditions) to and/or irnrnobilized Baci//us licheniforrnis a-amylase covalently onto porous glass beads. In this way, important alterations in its thermostability characteristics (kinactrE Ainactw) ere achieved.

  12. Mixtures of thermostable enzymes show high performance in biomass saccharification.

    Science.gov (United States)

    Kallioinen, Anne; Puranen, Terhi; Siika-aho, Matti

    2014-07-01

    Optimal enzyme mixtures of six Trichoderma reesei enzymes and five thermostable enzyme components were developed for the hydrolysis of hydrothermally pretreated wheat straw, alkaline oxidised sugar cane bagasse and steam-exploded bagasse by statistically designed experiments. Preliminary studies to narrow down the optimization parameters showed that a cellobiohydrolase/endoglucanase (CBH/EG) ratio of 4:1 or higher of thermostable enzymes gave the maximal CBH-EG synergy in the hydrolysis of hydrothermally pretreated wheat straw. The composition of optimal enzyme mixtures depended clearly on the substrate and on the enzyme system studied. The optimal enzyme mixture of thermostable enzymes was dominated by Cel7A and required a relatively high amount of xylanase, whereas with T. reesei enzymes, the high proportion of Cel7B appeared to provide the required xylanase activity. The main effect of the pretreatment method was that the required proportion of xylanase was higher and the proportion of Cel7A lower in the optimized mixture for hydrolysis of alkaline oxidised bagasse than steam-exploded bagasse. In prolonged hydrolyses, less Cel7A was generally required in the optimal mixture. Five-component mixtures of thermostable enzymes showed comparable hydrolysis yields to those of commercial enzyme mixtures.

  13. A Strategy to Model Nonmonotonic Dose-Response Curve and Estimate IC50

    OpenAIRE

    Hui Zhang; Jeanne Holden-Wiltse; Jiong Wang; Hua Liang

    2013-01-01

    The half-maximal inhibitory concentration IC[Formula: see text] is an important pharmacodynamic index of drug effectiveness. To estimate this value, the dose response relationship needs to be established, which is generally achieved by fitting monotonic sigmoidal models. However, recent studies on Human Immunodeficiency Virus (HIV) mutants developing resistance to antiviral drugs show that the dose response curve may not be monotonic. Traditional models can fail for nonmonotonic data and igno...

  14. Non-monotonic size dependence of diffusion and levitation effect: a mode-coupling theory analysis.

    Science.gov (United States)

    Nandi, Manoj Kumar; Banerjee, Atreyee; Bhattacharyya, Sarika Maitra

    2013-03-28

    We present a study of diffusion of small tagged particles in a solvent, using mode coupling theory (MCT) analysis and computer simulations. The study is carried out for various interaction potentials. For the first time, using MCT, it is shown that only for strongly attractive interaction potential with allowing interpenetration between the solute-solvent pair the diffusion exhibits a non-monotonic solute size dependence which has earlier been reported in simulation studies [P. K. Ghorai and S. Yashonath, J. Phys. Chem. B 109, 5824-5835 (2005)]. For weak attractive and repulsive potential the solute size dependence of diffusion shows monotonic behaviour. It is also found that for systems where the interaction potential does not allow solute-solvent interpenetration, the solute cannot explore the neck of the solvent cage. Thus these systems even with strong attractive interaction will never show any non-monotonic size dependence of diffusion. This non-monotonic size dependence of diffusion has earlier been connected to levitation effect [S. Yashonath and P. Santikary, J. Phys. Chem. 98, 6368 (1994)]. We also show that although levitation is a dynamic phenomena, the effect of levitation can be obtained in the static radial distribution function.

  15. The Global Convergence of Self-Scaling BFGS Algorithm with Nonmonotone Line Search for Unconstrained Nonconvex Optimization Problems

    Institute of Scientific and Technical Information of China (English)

    Hong Xia YIN; Dong Lei DU

    2007-01-01

    The self-scaling quasi-Newton method solves an unconstrained optimization problem by scaling the Hessian approximation matrix before it is updated at each iteration to avoid the possible large eigenvalues in the Hessian approximation matrices of the objective function. It has been proved in the literature that this method has the global and superlinear convergence when the objective function is convex (or even uniformly convex). We propose to solve unconstrained nonconvex optimization problems by a self-scaling BFGS algorithm with nonmonotone linear search. Nonmonotone line search has been recognized in numerical practices as a competitive approach for solving large-scale nonlinear problems. We consider two different nonmonotone line search forms and study the global convergence of these nonmonotone self-scale BFGS algorithms. We prove that, under some weaker condition than that in the literature, both forms of the self-scaling BFGS algorithm are globally convergent for unconstrained nonconvex optimization problems.

  16. Permanence of a general discrete-time two-species-interaction model with non-monotonic per capita growth rates

    CERN Document Server

    Kang, Yun

    2011-01-01

    Combined with all density-dependent factors, the per capita growth rate of a species may be non-monotonic. One important consequence is that species may suffer from weak Allee effects or strong Allee effects. In this paper, we study the permanence of a discrete-time two-species-interaction model with non-monotonic per capita growth rates for the first time. By using the average Lyapunov functions and extending the ecological concept of the relative nonlinearity, we find a simple sufficient condition for guaranteeing the permanence of systems that can model complicated two-species interactions. The extended relative nonlinearity allows us to fully characterize the effects of nonlinearities in the per capita growth functions with non-monotonicity. These results are illustrated with specific two species competition and predator-prey models of generic forms with non-monotone per capita growth rates.

  17. A Review on Chemical Effects in Aqueous Solution induced by Plasma with Glow Discharge

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Chemical effects in different aqueous solutions induced by plasma with glow dis charge electrolysis (GDE) and contact glow discharge electrolysis (CGDE) are described in this paper. The experimental and discharge characteristics are also reviewed. These are followed by a discussion of their mechanisms of both anodic and cathodic CGDE..

  18. 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.

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

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    In the past century, vaccines have contributed to a significant improvement in global public health by preventing a number of infectious diseases. Despite this, the vaccine field is still facing challenges related to incomplete vaccine coverage and persistent difficult vaccine targets......, 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...

  20. Structure prediction and functional analyses of a thermostable lipase obtained from Shewanella putrefaciens.

    Science.gov (United States)

    Khan, Faez Iqbal; Nizami, Bilal; Anwer, Razique; Gu, Ke-Ren; Bisetty, Krishna; Hassan, Md Imtaiyaz; Wei, Dong-Qing

    2017-08-01

    Previous experimental studies on thermostable lipase from Shewanella putrefaciens suggested the maximum activity at higher temperatures, but with little information on its conformational profile. In this study, the three-dimensional structure of lipase was predicted and a 60 ns molecular dynamics (MD) simulation was carried out at temperatures ranging from 300 to 400 K to better understand its thermostable nature at the molecular level. MD simulations were performed in order to predict the optimal activity of thermostable lipase. The results suggested strong conformational temperature dependence. The thermostable lipase maintained its bio-active conformation at 350 K during the 60 ns MD simulations.

  1. Short Time Uniqueness Results for Solutions of Nonlocal and Non-monotone Geometric Equations

    CERN Document Server

    Barles, Guy; Mitake, Hiroyoshi

    2010-01-01

    We describe a method to show short time uniqueness results for viscosity solutions of general nonlocal and non-monotone second-order geometric equations arising in front propagation problems. Our method is based on some lower gradient bounds for the solution. These estimates are crucial to obtain regularity properties of the front, which allow to deal with nonlocal terms in the equations. Applications to short time uniqueness results for the initial value problems for dislocation type equations, asymptotic equations of a FitzHugh-Nagumo type system and equations depending on the Lebesgue measure of the fronts are presented.

  2. Nonmonotonic Recursive Polynomial Expansions for Linear Scaling Calculation of the Density Matrix.

    Science.gov (United States)

    Rubensson, Emanuel H

    2011-05-10

    As it stands, density matrix purification is a powerful tool for linear scaling electronic structure calculations. The convergence is rapid and depends only weakly on the band gap. However, as will be shown in this letter, there is room for improvements. The key is to allow for nonmonotonicity in the recursive polynomial expansion. On the basis of this idea, new purification schemes are proposed that require only half the number of matrix-matrix multiplications compared to previous schemes. The speedup is essentially independent of the location of the chemical potential and increases with decreasing band gap.

  3. Non-monotonicity in the quantum-classical transition: Chaos induced by quantum effects

    CERN Document Server

    Kapulkin, A; Kapulkin, Arie; Pattanayak, Arjendu K.

    2007-01-01

    The transition from classical to quantum behavior for chaotic systems is understood to be accompanied by the suppression of chaotic effects as the relative size of $\\hbar$ is increased. We show evidence to the contrary in the behavior of the quantum trajectory dynamics of a dissipative quantum chaotic system, the double-well Duffing oscillator. The classical limit in the case considered has regular behavior, but as the effective $\\hbar$ is increased we see chaotic behavior. This chaos then disappears deeper into the quantum regime, which means that the quantum-classical transition in this case is non-monotonic in $\\hbar$.

  4. Interface failure modes explain non-monotonic size-dependent mechanical properties in bioinspired nanolaminates

    Science.gov (United States)

    Song, Z. Q.; Ni, Y.; Peng, L. M.; Liang, H. Y.; He, L. H.

    2016-03-01

    Bioinspired discontinuous nanolaminate design becomes an efficient way to mitigate the strength-ductility tradeoff in brittle materials via arresting the crack at the interface followed by controllable interface failure. The analytical solution and numerical simulation based on the nonlinear shear-lag model indicates that propagation of the interface failure can be unstable or stable when the interfacial shear stress between laminae is uniform or highly localized, respectively. A dimensionless key parameter defined by the ratio of two characteristic lengths governs the transition between the two interface-failure modes, which can explain the non-monotonic size-dependent mechanical properties observed in various laminate composites.

  5. A New Nonmonotonic Trust Region Algorithm for A Class of Unconstraied Nonsmooth Optimization

    Institute of Scientific and Technical Information of China (English)

    欧宜贵; 侯定丕

    2002-01-01

    This paper preasents a new trust region algorithm for solving a class of composite nonsmooth optimizations.It is distinguished by the fact that this method does not enforce strict monotonicity of the objective function values at successive itereates and that this method extends the existing results for this type of nonlinear optimization with smooth ,or piecewis somooth,or convex objective functions or their composition It is pyoved that this algorithm is globally convergent under certain conditions.Finally,some numerical results for several optimization problems are reported which show that the nonmonotonic trust region method is competitive with the usual trust region method.

  6. CONVERGENCE PROPERTIES OF PROJECTEDGRADIENT METHODS WITH NONMONOTONIC BACK TRACKING TECHNIQUE FOR CONVEX CONSTRAINED OPTIMIZATION

    Institute of Scientific and Technical Information of China (English)

    ZHU Detong

    2000-01-01

    This paper proposes projected gradient algorithms in association with using both trust region and line search techniques for convex constrained optimization problems.The mixed strategy is adopted which switches to back tracking steps when a trial projected gradient step produced by the trust region subproblem is unacceptable. A nonmonotone criterion is used to speed up the convergence progress in some curves with large curvature.A theoretical analysis is given which proves that the proposed algorithms are globally convergent and have local superlinear convergence rate under some reasonable conditions.The results of numerical experiments are reported to show the effectiveness of the proposed algorithms.

  7. CURVILINEAR PATHS AND TRUST REGION METHODS WITH NONMONOTONIC BACK TRACKING TECHNIQUE FOR UNCONSTRAINED OPTIMIZATION

    Institute of Scientific and Technical Information of China (English)

    De-tong Zhu

    2001-01-01

    In this paper we modify type approximate trust region methods via two curvilinear paths for unconstrained optimization. A mixed strategy using both trust region and line search techniques is adopted which switches to back tracking steps when a trial step produced by the trust region subproblem is unacceptable. We give a series of properties of both optimal path and modified gradient path. The global convergence and fast local convergence rate of the proposed algorithms are established under some reasonable conditions. A nonmonotonic criterion is used to speed up the convergence progress in some ill-conditioned cases.

  8. Global stability and attractivity of a network-based SIS epidemic model with nonmonotone incidence rate

    Science.gov (United States)

    Wei, Xiaodan; Liu, Lijun; Zhou, Wenshu

    2017-03-01

    In this paper, we study the global stability and attractivity of the endemic equilibrium for a network-based SIS epidemic model with nonmonotone incidence rate. The model was introduced in Li (2015). We prove that the endemic equilibrium is globally asymptotically stable if α (a parameter of this model) is sufficiently large, and is globally attractive if the transmission rate λ satisfies λ/λc ∈(1 , 2 ] , where λc is the epidemic threshold. Some numerical experiments are also presented to illustrate the theoretical results.

  9. A simple class of singular, two species Vlasov equilibria sustaining nonmonotonic potential distributions

    Energy Technology Data Exchange (ETDEWEB)

    Nocera, L.; Palumbo, L. J. [CNR-IPCF, Theoretical Plasma Physics, Via Moruzzi 1, I-56124 Pisa (Italy)

    2013-01-15

    We present new elementary, exact weak singular solutions of the steady state, two species, electrostatic, one dimensional Vlasov-Poisson equations. The distribution of the hot, finite mass, mobile ions is assumed to be log singular at the position of the electric potential's minimum. We show that the electron energy distributions on opposite sides of this minimum are not equal. This leads to a jump discontinuity of the electron distribution across its separatrix. A simple relation exists between the difference of these two electron distributions and that of the ions. The velocity Fourier transform of the electron singular distribution is smooth and appears as a simple Neumann series. Elementary, finite amplitude profiles of the electric potential result from Poisson equation, which are smoothly, but nonmonotonically and asymmetrically distributed in space. Two such profiles are given explicitly as appropriate for a nonmonotonic double layer and for a plasma bounded by a surface. The distributions of both electrons and ions supporting such potential meet smooth and kinetically stable boundary conditions at one plasma boundary. For sufficiently small potential to electron temperature ratios, the nonthermal, discontinuous electron distribution resulting at the other plasma boundary is also stable against Landau damped perturbations of the electron distribution.

  10. Canonical single field slow-roll inflation with a non-monotonic tensor

    CERN Document Server

    German, Gabriel; Hidalgo, Juan Carlos; Sussman, Roberto A

    2015-01-01

    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 $\\epsilon$ and its derivatives $\\epsilon^{\\prime }$ and $\\epsilon^{\\prime\\prime }$, thereby extracting general conditions on the tensor $r$ and the running $n_{sk}$. Of particular interest is a non-monotonic $\\epsilon$ with a maximum where universality conditions are found among the observables. In models with a monotonically increasing $\\epsilon$ the running is expected to be always negative for positive $\\epsilon^{\\prime\\prime }$. To accommodate a large tensor that meets the limiting values allowed by the Planck data, we study a non-monotonic $\\epsilon$ decreasing during most part of inflation. Since at $\\phi_{H}$, at which the perturbations are produced, some $50$ $-$ $60$ $e$-folds before the end of inflation, $\\epsilon$ is increasing, we thus require that $\\epsilon$ develops a maximum for $\\phi > \\phi_{H}$ after which $\\epsilon$ decreas...

  11. Quantitative structure-activity relationships of selected phenols with non-monotonic dose-response curves

    Institute of Scientific and Technical Information of China (English)

    GAO ChangAn; ZHANG AiQian; LIN Yuan; YIN DaQiang; WANG LianSheng

    2009-01-01

    Particular non-monotonic dose-response curves of many endocrine disrupting chemicals (EDCs) suggest the existence of diverse toxicity mechanisms at different dose levels. As a result, the biologi-cal activities of EDCs cannot be simply exhibited by unique EC/LD<,50. values, and the quantitative structure-activity relationship (QSAR) analysis for non-monotonic dose-response relationship be-comes an unknown field in the environmental science. In this paper, nine phenols with inverted U-shaped dose-response curves in lymphocyte proliferation test of Carassius auratus were selected. The binding interactions between the phenols and several typical EDCs-related receptors were then explored in a molecular simulation study. The estrogen receptor (ER), androgen receptor (AR), thyroid hormone receptor (TR), bacterial O2 sensing FixL protein (FixL), aryl hydrocarbon receptor (AhR), and the peroxisome proliferator-activated receptor (PPAR) were the target receptors in the study. Linear regression QSAR models for the low and high exposure levels of the compounds were developed separately. The results indicated that the lymphocyte proliferation in the low-dose range might involve ER-mediated process, while the proliferation inhibition in the high dose range was dominated by the acute toxicity of phenols due to receptor occupancy and cell damage.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, R.; Steinmann, V.; Mangan, N. M.; Brandt, R. E.; Poindexter, J. R.; Jaramillo, R.; Mailoa, J. P.; Hartman, K.; Polizzotti, A.; Buonassisi, T. [Massachusetts Institute of Technology, Cambridge, Cambridge, Massachusetts 02139 (United States); Yang, C.; Gordon, R. G. [Harvard University, Cambridge, Massachusetts 02138 (United States)

    2015-05-18

    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.

  13. Health inequality and non-monotonicity of the health related social welfare function.

    Science.gov (United States)

    Dutta, Indranil

    2007-03-01

    In a recent paper in this journal Abasolo and Tsuchiya [Abasolo, I., Tsuchiya, A., 2004. Exploring social welfare functions and violation of monotonicity: an example from inequalities in health. Journal of Health Economics 23, 313-329] have strongly argued for the use of a non-monotonic health related social welfare function. This note discusses both the limitations of the measure proposed by Abasolo and Tsuchiya [Abasolo, I., Tsuchiya, A., 2004. Exploring social welfare functions and violation of monotonicity: an example from inequalities in health. Journal of Health Economics 23, 313-329] and the problems associated with their empirics. We are able to show how non-monotonicity may lead to paradoxical results and policies. Further we examine the empirics of Abasolo and Tsuchiya [Abasolo, I., Tsuchiya, A., 2004. Exploring social welfare functions and violation of monotonicity: an example from inequalities in health. Journal of Health Economics 23, 313-329] and provide an alternative explanation to the observed patterns in the data that do not violate monotonicity. Finally we briefly mention why the Atkinson-Sen framework may be more appropriate as a way forward.

  14. 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.

  15. Non-Monotonicity of Excited State Populations Observed in a Cu-He Hollow Cathode Discharge

    Institute of Scientific and Technical Information of China (English)

    ZHENG Xu-Tao

    2005-01-01

    @@ An interesting non-monotonic structure in the distribution of excited state populations is observed in a Cu-He hollow cathode discharge, and this is explained by l-changing collisions of the excited states with background gas atoms. At helium pressure of 0.1 Torr and cathode current of 200-300mA, relative populations of He I 1snp 1p (n = 2-16) states are measured with the corresponding VUV radiation intensities, and are plotted against excitation energies. As energy levels increase, populations of high-n (n > 10) states are found to decrease much more quickly than low-n (n < 7) populations. For intermediate states (n = 7-10), the declining tendency is interfered by population transfers from 1sns 1 S states due to l-changing collisions, and an obvious non-monotonic structure is formed at relatively low electric current. Measurements have also been carried out for He Ⅱ np 2 p (n = 2-14) series, in which the l-changing collisions are overwhelmed by Stark quenching of the n2S states and thus population interference does not occur.

  16. Effective interaction in asymmetric charged binary mixtures: the non-monotonic behaviour with the colloidal charge.

    Science.gov (United States)

    Peláez-Fernández, M; Callejas-Fernández, J; Moncho-Jordá, A

    2012-11-01

    In this work we study the effective force between charged spherical colloids induced by the presence of smaller charged spheres using Monte Carlo simulations. The analysis is performed for two size ratios, q = R(s)/R(b), two screened direct repulsions, κ, and two small particle packing fractions, Ø(s). We specially focus on the effect of the charge of the big colloids (Z(b)), and observe that the repulsion between big particles shows a non-monotonic behaviour: for sufficiently small charge, we find an anomalous regime where the total repulsion weakens by increasing the big colloid charge. For larger charges, the system recovers the usual behaviour and the big-big interaction becomes more repulsive increasing Z(b). This effect is linked to the existence of strong attractive depletion interactions caused by the small-big electrostatic repulsion. We have also calculated the effective force using the Ornstein-Zernike equation with the HNC closure. In general, this theory agrees with the simulation results, and is able to capture this non-monotonic behaviour.

  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

    2012-08-17

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Minelli, Matteo; Doghieri, Ferruccio [Dipartimento di Ingegneria Civile, Chimica, Ambientale e dei Materiali (DICAM), Centro Interdipartimentale per la Ricerca Industriale - Meccanica Avanzata e Materiali (CIRI-MAM), Alma Mater Studiorum - Università di Bologna, via Terracini 28 - (Italy)

    2014-05-15

    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.

  19. Crystallographic Refinement by Incorporation of Molecular Dynamics : Thermostable Serine Protease Thermitase Complexed with Eglin c

    NARCIS (Netherlands)

    Gros, Piet; Fujinaga, Masao; Dijkstra, Bauke W.; Kalk, Kor H.; Hol, W G J

    1989-01-01

    In order to investigate the principles of protein thermostability, the crystal structure of thermitase from Thermoactinomyces vulgaris, a thermostable member of the subtilisin family of serine proteases, has been determined in a complex with eglin c. Eglin c is a serine protease inhibitor from the l

  20. Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene

    Science.gov (United States)

    The thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease (ND) for village flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of the thermostable NDV as a vaccine vector, an infectious clone of the...

  1. On the Non-Monotonic Variation of the Opposition Surge Morphology with Albedo Exhibited by Satellites' Surface

    Science.gov (United States)

    Deau, E. A.; Spilker, L. J.; Flandes, A.

    2011-01-01

    We used well know phase functions of satellites and rings around the giant planets of our Solar System to study the morphology of the opposition effect (at phase angles alpha morphological model to retrieve the morphological parameters of the surge (A and HWHM). These parameters are found to have a non-monotonic variation with the single scattering albedo, similar to that observed in asteroids, which is unexplained so far. The non-monotonic variation is discussed in the framework of the coherent backscattering and shadow hiding mechanisms.

  2. A Nonmonotonous Damage Model to Characterize Mullins and Residual Strain Effects of Rubber Strings Subjected to Transverse Vibrations

    Directory of Open Access Journals (Sweden)

    Alex Elías-Zúñiga

    2013-01-01

    Full Text Available This work focuses on the formulation of a constitutive equation to predict Mullins and residual strain effects of buna-N, silicone, and neoprene rubber strings subjected to small transverse vibrations. The nonmonotone behavior exhibited by experimental data is captured by the proposed material model through the inclusion of a phenomenological non-monotonous softening function that depends on the strain intensity between loading and unloading cycles. It is shown that theoretical predictions compare well with uniaxial experimental data collected from transverse vibration tests.

  3. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Iron ions increase the thermostability of phycocyanin of Spirulina maxima

    Science.gov (United States)

    Li, Jian-Hong; Tai, Zi-Hou; Tseng, Chao-Tsi

    1998-03-01

    A spectral method to investigate the effect of Fe3+, Fe2+ on the thermostability of phycocyanin (PC) of Spirulina maxima showed that iron ions provent decrease of visible light absorbance and fluorescence intensity of PC. Increase in denaturation temperature caused by Fe3+ was observed by the micro-differential scanning calorimetric method. All results showed iron ions maintain the aggregation stability of the PC. The absorption spectrum of phycocyanobilin (PCB, a prosthetic group of PC) with Fe3+ in chloroform was quite different from that of free PCB.

  5. Fluctuation formula for nonreversible dynamics in the thermostated Lorentz gas

    Science.gov (United States)

    Dolowschiák, M.; Kovács, Z.

    2002-12-01

    We investigate numerically the validity of the Gallavotti-Cohen fluctuation formula in the two- and three-dimensional periodic Lorentz gas subjected to constant electric and magnetic fields and thermostated by the Gaussian isokinetic thermostat. The magnetic field breaks the time reversal symmetry, and by choosing its orientation with respect to the lattice, one can have either a generalized reversing symmetry or no reversibility at all. Our results indicate that the scaling property described by the fluctuation formula may be approximately valid for large fluctuations even in the absence of reversibility.

  6. Verification of mesoscopic models of viscoelastic fluids with a non-monotonic flow curve

    Science.gov (United States)

    Kuznetsova, Julia L.; Skul'skiy, Oleg I.

    2016-02-01

    The non-monotonic flow curve of a 1 wt.% polyacrylonitrile solution in dimethyl sulfoxide is described by two mesoscopic models: the modified Vinogradov-Pokrovsky model and the model proposed by Remmelgas, Harrison and Leal. To obtain an adequate description of the experimental curve, we have selected suitable internal parameters for these models. Analytical solutions for the Couette-Poiseuille flow problems are determined in parametric form, which allows us to plot the distribution of stress components and anisotropy tensor as well as the velocity profiles containing closed loops and weak tangential discontinuities. It is shown that both models predict a similar qualitative picture of structure evolution, but exhibit a significant discrepancy in the quantitative description of the magnitude of molecular chain stretching.

  7. Magnetohydrodynamic calculations with a nonmonotonic q profile and equilibrium, sheared toroidal flow

    Energy Technology Data Exchange (ETDEWEB)

    Held, E.D. [Univ. of Wisconsin, Madison, WI (United States). Center for Plasma Theory and Computation; Leboeuf, J.N.; Carreras, B.A. [Oak Ridge National Lab., TN (United States). Fusion Energy Div.

    1998-07-01

    The linear and nonlinear stability of a nonmonotonic q profile is examined using a reduced set of magnetohydrodynamic (MHD) equations with an equilibrium, sheared toroidal flow. The reversed shear profile is shown to be unstable to a rich variety of resistive MHD modes including pressure-driven instabilities and tearing instabilities possessing a tearing/interchange character at low Lundquist number, S, and taking on a double/triple tearing structure at high S. Linear calculations show that the destabilizing effect of toroidal velocity shear on tearing modes is enhanced at finite pressure seen previously for tearing modes at high S. Nonlinear calculations show the generation of a large, m = 1, n = 0, Reynolds-stress-driven poloidal flow in the absence of significant flow damping. Calculations in which the poloidal flow was heavily damped show that sub-Alfvenic, sheared toroidal flows have a minimal effect on weakly-coupled, localized instabilities.

  8. 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.

  9. Nonmonotonic effects of perpendicular magnetic anisotropy on current-driven vortex wall motions in magnetic nanostripes

    Science.gov (United States)

    Su, Yuan-Chang; Lei, Hai-Yang; Hu, Jing-Guo

    2015-09-01

    In a magnetic nanostripe, the effects of perpendicular magnetic anisotropy (PMA) on the current-driven horizontal motion of vortex wall along the stripe and the vertical motion of the vortex core are studied by micromagnetic simulations. The results show that the horizontal and vertical motion can generally be monotonously enhanced by PMA. However, when the current is small, a nonmonotonic phenomenon for the horizontal motion is found. Namely, the velocity of the horizontal motion firstly decreases and then increases with the increase of the PMA. We find that the reason for this is that the PMA can firstly increase and then decrease the confining force induced by the confining potential energy. In addition, the PMA always enhances the driving force induced by the current. Project supported by the National Natural Science Foundation of China (Grant Nos. 11247026 and 11374253).

  10. Semantic Matchmaking as Non-Monotonic Reasoning: A Description Logic Approach

    CERN Document Server

    Di Noia, T; Donini, F M; 10.1613/jair.2153

    2011-01-01

    Matchmaking arises when supply and demand meet in an electronic marketplace, or when agents search for a web service to perform some task, or even when recruiting agencies match curricula and job profiles. In such open environments, the objective of a matchmaking process is to discover best available offers to a given request. We address the problem of matchmaking from a knowledge representation perspective, with a formalization based on Description Logics. We devise Concept Abduction and Concept Contraction as non-monotonic inferences in Description Logics suitable for modeling matchmaking in a logical framework, and prove some related complexity results. We also present reasonable algorithms for semantic matchmaking based on the devised inferences, and prove that they obey to some commonsense properties. Finally, we report on the implementation of the proposed matchmaking framework, which has been used both as a mediator in e-marketplaces and for semantic web services discovery.

  11. 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.

  12. 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.

  13. 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.

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

    Science.gov (United States)

    Praxedes, A. P. P.; Webler, G. D.; Souza, S. T.; Ribeiro, A. S.; Fonseca, E. J. S.; de Oliveira, I. N.

    2016-05-01

    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.

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

    DEFF Research Database (Denmark)

    Schjær-Jacobsen, Hans

    2012-01-01

    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...... 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...... by probability distributions is readily done by means of Monte Carlo simulation. Calculation of non-monotonic functions of possibility distributions is done within the theoretical framework of fuzzy intervals, but straight forward application of fuzzy arithmetic in general results in overestimation of interval...

  16. Multistability and Instability of Neural Networks With Discontinuous Nonmonotonic Piecewise Linear Activation Functions.

    Science.gov (United States)

    Nie, Xiaobing; Zheng, Wei Xing

    2015-11-01

    In this paper, we discuss the coexistence and dynamical behaviors of multiple equilibrium points for recurrent neural networks with a class of discontinuous nonmonotonic piecewise linear activation functions. It is proved that under some conditions, such n -neuron neural networks can have at least 5(n) equilibrium points, 3(n) of which are locally stable and the others are unstable, based on the contraction mapping theorem and the theory of strict diagonal dominance matrix. The investigation shows that the neural networks with the discontinuous activation functions introduced in this paper can have both more total equilibrium points and more locally stable equilibrium points than the ones with continuous Mexican-hat-type activation function or discontinuous two-level activation functions. An illustrative example with computer simulations is presented to verify the theoretical analysis.

  17. Entering and exiting the protein-polyelectrolyte coacervate phase via nonmonotonic salt dependence of critical conditions.

    Science.gov (United States)

    Antonov, Margarita; Mazzawi, Malek; Dubin, Paul L

    2010-01-11

    Critical conditions for coacervation of poly(dimethyldiallylammonium chloride) (PDADMAC) with bovine serum albumin were determined as a function of ionic strength, pH, and protein/polyelectrolyte stoichiometry. The resultant phase boundaries, clearly defined with this narrow molecular weight distribution PDADMAC sample, showed nonmonotonic ionic strength dependence, with the pH-induced onset of coacervation (at pH(phi)) occurring most readily at 20 mM NaCl. The corresponding onset of soluble complex formation, pH(c), determined using high-precision turbidimetry sensitive to changes of less than 0.1% transmittance units, mirrored the ionic strength dependence of pH(phi). This nonmonotonic binding behavior is attributable to simultaneous screening of short-range attraction and long-range repulsion. The similarity of pH(c) and pH(phi) was explained by the effect of salt on protein binding, and consequently on the number of bound proteins relative to that required for charge neutralization of the complex, a requirement for phase separation. Expansion of the coacervation regime with chitosan, a polycation with charge spacing similar to that of PDADMAC, could be due to either the charge mobility or chain stiffness of the former. The pH(phi) versus I phase boundary for PDADMAC correctly predicted entrance into and egress from the coacervation region by addition of either salt or water. The ability to induce or suppress coacervation via protein/polyelectrolyte stoichiometry r was found to be consistent with the proposed model. The results indicate that the conjoint effects of I, r, and pH on coacervation could be represented by a three-dimensional phase boundary.

  18. 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...

  19. Nonmonotonic Dose Responses as They Apply to Estrogen, Androgen, and Thyroid Pathways and EPA Testing and Assessment Procedures

    Science.gov (United States)

    A state of the science document providing a judgment on the degree to which nonmonotonic dose-responses are evidenced in the scientific literature and to evaluate the extent to which they may impact U.S. EPA’s chemical testing and risk assessment.

  20. GLOBAL CONVERGENCE RESULTS OF A THREE TERM MEMORY GRADIENT METHOD WITH A NON-MONOTONE LINE SEARCH TECHNIQUE

    Institute of Scientific and Technical Information of China (English)

    Sun Qingying

    2005-01-01

    In this paper, a new class of three term memory gradient method with nonmonotone line search technique for unconstrained optimization is presented. Global convergence properties of the new methods are discussed. Combining the quasi-Newton method with the new method, the former is modified to have global convergence property. Numerical results show that the new algorithm is efficient.

  1. Positive Steady States of a Prey-predator Model with Diffusion and Non-monotone Conversion Rate

    Institute of Scientific and Technical Information of China (English)

    Rui PENG; Ming Xin WANG; Wen Yan CHEN

    2007-01-01

    In this paper,we study the positive steady states of a prey-predator model with di .usion throughout and a non-monotone conversion rate under the homogeneous Dirichlet boundary condition. We obtain some results of the existence and non-existence of positive steady states.The stability and uniqueness of positive steady states are also discussed.

  2. Virus-specific thermostability and heat inactivation profiles of alphaviruses.

    Science.gov (United States)

    Park, So Lee; Huang, Yan-Jang S; Hsu, Wei-Wen; Hettenbach, Susan M; Higgs, Stephen; Vanlandingham, Dana L

    2016-08-01

    Serological diagnosis is a critical component for disease surveillance and is important to address the increase in incidence and disease burden of alphaviruses, such as the chikungunya (CHIKV) and Ross River (RRV) viruses. The gold standard for serological diagnosis is the plaque reduction neutralization test (PRNT), which demonstrates the neutralizing capacity of serum samples after the removal of complement activity and adventitious viruses. This procedure is normally performed following inactivation of the virus at 56°C for 30min. Although this protocol has been widely accepted for the inactivation of envelope RNA viruses, recent studies have demonstrated that prolonged heat inactivation is required to completely inactivate two alphaviruses, Western equine encephalitis virus and CHIKV. Incomplete inactivation of viruses poses a laboratory biosafety risk and can also lead to spurious test results. Despite its importance in ensuring the safety of laboratory personnel as well as test integrity, systematic investigation on the thermostability of alphaviruses has not been performed. In this study, the temperature tolerance and heat inactivation profiles of RRV, Barmah Forest, and o'nyong-nyong viruses were determined. Variations in thermostability were observed within the Semliki forest serocomplex. Therefore, evidence-based heat inactivation procedures for alphaviruses are recommended.

  3. Highly thermostable xylanase purified from Rhizomucor miehei NRL 3169.

    Science.gov (United States)

    Fawzi, E M

    2011-03-01

    A thermostable xylanase was purified and characterized from the thermophilic fungus Rhizomucor miehei (Cooney & Emerson) Schipper. The enzyme was purified to homogeneity by ammonium sulfate precipitation, sephadex G-100 gel filtration and diethylaminoethyl cellulose anion exchange chromatography with a 29.1-fold. The enzyme was highly active within a range of pH from 5.0 to 6.5. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 70°C and 75°C and the half-life of the xylanase at 90°C was 30 min. Km and Vmax values at 50°C of the purified enzyme were 0.055 mg/ml and 113.5 μmol min⁻¹ mg⁻¹ respectively. The enzyme was activated by Ca²+, Cu²+, K+ and Na+. On the other hand, Ag²+, Hg²+, Ba²+, and Zn²+ inhibited the enzyme. The molecular weight of the xylanase was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study is among the first works to examine and describe a secreted highly thermostable endoxylanase from the Rhizomucor miehei fungus. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for industrial and commercial application in pulp bleaching.

  4. 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.

  5. Optical Kerr effect of tRNA solution induced by femtosecond laser pulses

    Science.gov (United States)

    Kucia, Weronika E.; Sharma, Gargi; Joseph, Cecil S.; Sarbak, Szymon; Oliver, Cameron; Dobek, Andrzej; Giles, Robert H.

    2016-10-01

    The optical Kerr effect (OKE) in a transfer ribonucleic acid (tRNA) solution induced by femtosecond pulses of linearly polarized pump light (λi = 800 nm) and sounded by probe light (λp = 800 nm) was studied. The measurements were performed to find nonlinear optical parameters describing a single molecule (molecular Kerr constant K, mean nonlinear third order optical polarizability cpi) and to compare them with our previous OKE results obtained in ns and ps time range. The OKE experiment has proven to be an efficient method to obtain the nonlinear parameters of single molecules in solution, which reflects dynamic structure changes.

  6. 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.

  7. Structure and functional characterization of Vibrio parahaemolyticus thermostable direct hemolysin.

    Science.gov (United States)

    Yanagihara, Itaru; Nakahira, Kumiko; Yamane, Tsutomu; Kaieda, Shuji; Mayanagi, Kouta; Hamada, Daizo; Fukui, Takashi; Ohnishi, Kiyouhisa; Kajiyama, Shin'ichiro; Shimizu, Toshiyuki; Sato, Mamoru; Ikegami, Takahisa; Ikeguchi, Mitsunori; Honda, Takeshi; Hashimoto, Hiroshi

    2010-05-21

    Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 A resolution. The TDH tetramer forms a central pore with dimensions of 23 A in diameter and approximately 50 A in depth. Pi-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

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

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    -administrable, can be distributed independently of functioning freezers and refrigerators, and can be designed to induce mucosal and/or cell-mediated immunity, which is attractive for a number of diseases requiring stimulation of local mucosal immunity for protection. However, the design and delivery of thermostable...... dry powder-based vaccines represents a technological challenge: It calls for careful formulation and dosage form design, combined with cheap and efficient delivery devices, which must be engineered via a thorough understanding of the physiological barrier and the requirements for induction of mucosal...... 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. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    Science.gov (United States)

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    2015-06-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Identification of thermostable beta-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger

    DEFF Research Database (Denmark)

    Pedersen, Mads; Lauritzen, H.K.; 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...

  11. Identification of thermostable beta-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger

    DEFF Research Database (Denmark)

    Pedersen, Mads; Lauritzen, H.K.; 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...

  12. 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...

  13. Improved thermostability of Bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge

    NARCIS (Netherlands)

    Leemhuis, H; Rozeboom, HJ; Dijkstra, BW; Dijkhuizen, L; Dijkstra, Bauke W.

    2004-01-01

    Cyclodextrin glycosyltransferase (CGTase) catalyzes the formation of cyclodextrins from starch. Among the CGTases with known three-dimensional structure, Thennoanaerobacterium thermosulfurigenes CGTase has the highest thermostability. By replacing amino acid residues in the B-domain of Bacillus circ

  14. Universal, non-monotonic structure in the saturation curves of a linear Paul trap

    Science.gov (United States)

    Wells, James; Kwolek, Jonathan; Goodman, Douglas; Blümel, Reinhold; Smith, Winthrop

    2016-05-01

    A common technique to measure ion-atom collision rates in a hybrid trap if the ions have no optical transitions (e.g. alkalis) is to monitor the fluorescence of the neutrals in the presence of a saturated linear Paul trap (LPT). We present numerical simulations, analytical calculations, and experimental results that show that the steady-state ion capacity of an LPT, Ns, exhibits nonlinear, nonmonotonic behavior as a function of ion loading rate, Λ. The steady state as a function of loading rate, Ns(Λ) , shows four distinct regions. In Region I, at the lowest Λ, Ns(Λ) increases monotonically. Then, Ns(Λ) reaches a plateau in Region II, before decreasing to a local minimum in Region III. Finally, in Region IV, Ns(Λ) once again increases monotonically. This behavior appears universal to any Paul trap, regardless of geometry or species trapped. We examine this behavior experimentally as a function of the q stability parameter of the Paul trap and simulate numerically the effect of the particular trap geometry on the onset of each of the four regions. Funding from NSF Grant PHY-1307874.

  15. 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.

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

    Science.gov (United States)

    Sinha, Samiran; Saha, Krishna K; Wang, Suojin

    2014-06-01

    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.

  17. A Bivariate Pseudo-Likelihood for Incomplete Longitudinal Binary Data with Nonignorable Non-monotone Missingness

    Science.gov (United States)

    Sinha, Sanjoy K.; Troxel, Andrea B.; Lipsitz, Stuart R.; Sinha, Debajyoti; Fitzmaurice, Garrett M.; Molenberghs, Geert; Ibrahim, Joseph G.

    2010-01-01

    Summary For analyzing longitudinal binary data with nonignorable and non-monotone missing responses, a full likelihood method is complicated algebraically, and often requires intensive computation, especially when there are many follow-up times. As an alternative, a pseudo-likelihood approach has been proposed in the literature under minimal parametric assumptions. This formulation only requires specification of the marginal distributions of the responses and missing data mechanism, and uses an independence working assumption. However, this estimator can be inefficient for estimating both time-varying and time-stationary effects under moderate to strong within-subject associations among repeated responses. In this article, we propose an alternative estimator, based on a bivariate pseudo-likelihood, and demonstrate in simulations that the proposed method can be much more efficient than the previous pseudo-likelihood obtained under the assumption of independence. We illustrate the method using longitudinal data on CD4 counts from two clinical trials of HIV-infected patients. PMID:21155748

  18. Non-monotonic dependence of Pickering emulsion gel rheology on particle volume fraction.

    Science.gov (United States)

    Kaganyuk, M; Mohraz, A

    2017-03-29

    The microstructure of Pickering emulsion gels features a tenuous network of faceted droplets, bridged together by shared monolayers of particles. In this investigation, we use standard oscillatory rheometry in conjunction with confocal microscopy to gain a more comprehensive understanding of the role particle bridged interfaces have on the rheology of Pickering emulsion gels. The zero-shear elastic modulus of Pickering emulsion gels shows a non-monotonic dependence on particle loading, with three separate regimes of power-law and linear gel strengthening, and subsequent gel weakening. The transition from power-law to linear scaling is found to coincide with a peak in the volume fraction of particles that participate in bridging, which we indirectly calculate using measureable quantities, and the transition to gel weakening is shown to result from a loss in network connectivity at high particle loadings. These observations are explained via a simple representation of how Pickering emulsion gels arise from an initial population of partially-covered droplets. Based on these considerations, we propose a combined variable related to the initial droplet coverage, to be used in reporting and rationalizing the rheology of Pickering emulsion gels. We demonstrate the applicability of this variable with Pickering emulsions prepared at variable fluid ratios and with different-sized colloidal particles. The results of our investigation have important implications for many technological applications that utilize solid stabilized multi-phase emulsions and require a priori knowledge or engineering of their flow characteristics.

  19. Dynamical Behaviors of Multiple Equilibria in Competitive Neural Networks With Discontinuous Nonmonotonic Piecewise Linear Activation Functions.

    Science.gov (United States)

    Nie, Xiaobing; Zheng, Wei Xing

    2016-03-01

    This paper addresses the problem of coexistence and dynamical behaviors of multiple equilibria for competitive neural networks. First, a general class of discontinuous nonmonotonic piecewise linear activation functions is introduced for competitive neural networks. Then based on the fixed point theorem and theory of strict diagonal dominance matrix, it is shown that under some conditions, such n -neuron competitive neural networks can have 5(n) equilibria, among which 3(n) equilibria are locally stable and the others are unstable. More importantly, it is revealed that the neural networks with the discontinuous activation functions introduced in this paper can have both more total equilibria and locally stable equilibria than the ones with other activation functions, such as the continuous Mexican-hat-type activation function and discontinuous two-level activation function. Furthermore, the 3(n) locally stable equilibria given in this paper are located in not only saturated regions, but also unsaturated regions, which is different from the existing results on multistability of neural networks with multiple level activation functions. A simulation example is provided to illustrate and validate the theoretical findings.

  20. Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination.

    Directory of Open Access Journals (Sweden)

    Chia-Jung Chang

    Full Text Available Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43% and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR, the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.

  1. Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

    Energy Technology Data Exchange (ETDEWEB)

    Hawwa, Renda; Aikens, John; Turner, Robert J.; Santarsiero, Bernard D.; Mescar, Andrew D.; (Lybradyn Inc.); (UIC)

    2009-08-31

    A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

  2. 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.

  3. NONMONOTONIC REDUCED PROJECTED HESSIAN METHOD VIA AN AFFINE SCALING INTERIOR MODIFIED GRADIENT PATH FOR BOUNDED-CONSTRAINED OPTIMIZATION

    Institute of Scientific and Technical Information of China (English)

    Peihua GUO; Detong ZHU

    2008-01-01

    The authors propose an affine scaling modified gradient path method in association with reduced projective Hessian and nonmonotonic interior backtracking line search techniques for solving the linear equality constrained optimization subject to bounds on variables. By employing the QR decomposition of the constraint matrix and the eigensystem decomposition of reduced projective Hes-sian matrix in the subproblem, the authors form affine scaling modified gradient curvilinear path very easily. By using interior backtracking line search technique, each iterate switches to trial step of strict interior feasibility. The global convergence and fast local superlinear/quadratical convergence rates of the proposed algorithm are established under some reasonable conditions. A nonmonotonic criterion should bring about speeding up the convergence progress in some ill-conditioned cases. The results of numerical experiments are reported to show the effectiveness of the proposed algorithm.

  4. 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.

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

    Science.gov (United States)

    Sandvik, Elizabeth L.; Fazen, Christopher H.; Henry, Theresa C.; Mok, Wendy W.K.; Brynildsen, Mark P.

    2015-01-01

    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. PMID:26593926

  6. 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.

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

    Science.gov (United States)

    Balan, Anuradha; Ibrahim, Darah; Abdul Rahim, Rashidah; Ahmad Rashid, Fatimah Azzahra

    2012-01-01

    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 BaCl(2), CaCl(2), and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16) and olive oil with optimal activity (100%) compared to other substrates.

  8. Expression and Characterization of Recombinant Thermostable Alkaline Phosphatase from a Novel Thermophilic Bacterium Thermus thermophilus XM

    Institute of Scientific and Technical Information of China (English)

    Jianbo LI; Limei XU; Feng YANG

    2007-01-01

    A gene (tap) encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co2+, Fe2+, Mg2+, or Mn2+ but was strongly inhibited by 2.0 mM Fe2+. Under optimal conditions, the Michaelis constant (Km) for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.

  9. QSAR study on the non-monotonic dose-response curve of PCBs in chicken embryo hepatocyte bioassay

    Institute of Scientific and Technical Information of China (English)

    MU YunSong; ZHANG AiQian; GAO ChangAn; PENG SuFen; WANG LianSheng

    2009-01-01

    Endocrine disrupting chemicals (EDCs) in the natural environment exhibit a unique non-monotonic dose-response curve and it is impossible to select one simple index to characterize the bilogogical activity of these compounds. Quantitative structure-activity relationship (QSAR) study on non-monotonic dose-response curve has become a real challenge presently. In order to explore the possible mechanism for the non-monotonic dose-response curve of polychlorinated biphenyls con-geners (PCBs) in chicken embryo hepatocyte bioassay, AM1 method of ChemOffice was adopted to calculate necessary structure descriptors for PCBs, while the interactions between PCBs and simulated AhR ligand binding domain (LBD) were analyzed by using FlexX in SYBYL7.0. Different binding modes for PCBs have been distinguished not only from aligned conformation but also from free binding energy. Some QSAR models were established separately for both low and high doses ranges, revealing that receptor binding may predominate in the interference of the physiological function of cytochrome P4501A-P4501A in the low doses range. But with the higher doses range, the EROD suppression might he related to acute toxicity owing to molecular polarity or distribution of charges and consequently damage structure and function of chicken embryo hepatocyte.

  10. QSAR study on the non-monotonic dose-response curve of PCBs in chicken embryo hepatocyte bioassay

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Endocrine disrupting chemicals (EDCs) in the natural environment exhibit a unique non-monotonic dose-response curve and it is impossible to select one simple index to characterize the bilogogical activity of these compounds. Quantitative structure-activity relationship (QSAR) study on non-monotonic dose-response curve has become a real challenge presently. In order to explore the possible mechanism for the non-monotonic dose-response curve of polychlorinated biphenyls congeners (PCBs) in chicken embryo hepatocyte bioassay, AM1 method of ChemOffice was adopted to calculate necessary structure descriptors for PCBs, while the interactions between PCBs and simulated AhR ligand binding domain (LBD) were analyzed by using FlexX in SYBYL7.0. Different binding modes for PCBs have been distinguished not only from aligned conformation but also from free binding energy. Some QSAR models were established separately for both low and high doses ranges, revealing that receptor binding may predominate in the interference of the physiological function of cytochrome P4501A-P4501A in the low doses range. But with the higher doses range, the EROD suppression might be related to acute toxicity owing to molecular polarity or distribution of charges and consequently damage structure and function of chicken embryo hepatocyte.

  11. Inhibitory properties underlying non-monotonic input-output relationship in low-frequency spherical bushy neurons of the gerbil.

    Science.gov (United States)

    Kuenzel, Thomas; Nerlich, Jana; Wagner, Hermann; Rübsamen, Rudolf; Milenkovic, Ivan

    2015-01-01

    Spherical bushy cells (SBCs) of the anteroventral cochlear nucleus (AVCN) receive input from large excitatory auditory nerve (AN) terminals, the endbulbs of Held, and mixed glycinergic/GABAergic inhibitory inputs. The latter have sufficient potency to block action potential firing in vivo and in slice recordings. However, it is not clear how well the data from slice recordings match the inhibition in the intact brain and how it contributes to complex phenomena such as non-monotonic rate-level functions (RLF). Therefore, we determined the input-output relationship of a model SBC with simulated endbulb inputs and a dynamic inhibitory conductance constrained by recordings in brain slice preparations of hearing gerbils. Event arrival times from in vivo single-unit recordings in gerbils, where 70% of SBC showed non-monotonic RLF, were used as input for the model. Model output RLFs systematically changed from monotonic to non-monotonic shape with increasing strength of tonic inhibition. A limited range of inhibitory synaptic properties consistent with the slice data generated a good match between the model and recorded RLF. Moreover, tonic inhibition elevated the action potentials (AP) threshold and improved the temporal precision of output functions in a SBC model with phase-dependent input conductance. We conclude that activity-dependent, summating inhibition contributes to high temporal precision of SBC spiking by filtering out weak and poorly timed EPSP. Moreover, inhibitory parameters determined in slice recordings provide a good estimate of inhibitory mechanisms apparently active in vivo.

  12. A pathway EM-algorithm for estimating vaccine efficacy with a non-monotone validation set.

    Science.gov (United States)

    Yang, Yang; Halloran, M Elizabeth; Chen, Yanjun; Kenah, Eben

    2014-09-01

    Here, we consider time-to-event data where individuals can experience two or more types of events that are not distinguishable from one another without further confirmation, perhaps by laboratory test. The event type of primary interest can occur only once. The other types of events can recur. If the type of a portion of the events is identified, this forms a validation set. However, even if a random sample of events are tested, confirmations can be missing nonmonotonically, creating uncertainty about whether an individual is still at risk for the event of interest. For example, in a study to estimate efficacy of an influenza vaccine, an individual may experience a sequence of symptomatic respiratory illnesses caused by various pathogens over the season. Often only a limited number of these episodes are confirmed in the laboratory to be influenza-related or not. We propose two novel methods to estimate covariate effects in this survival setting, and subsequently vaccine efficacy. The first is a pathway expectation-maximization (EM) algorithm that takes into account all pathways of event types in an individual compatible with that individual's test outcomes. The pathway EM iteratively estimates baseline hazards that are used to weight possible event types. The second method is a non-iterative pathway piecewise validation method that does not estimate the baseline hazards. These methods are compared with a previous simpler method. Simulation studies suggest mean squared error is lower in the efficacy estimates when the baseline hazards are estimated, especially at higher hazard rates. We use the pathway EM-algorithm to reevaluate the efficacy of a trivalent live-attenuated influenza vaccine during the 2003-2004 influenza season in Temple-Belton, Texas, and compare our results with a previously published analysis. © 2014, The International Biometric Society.

  13. Dealing with non-unique and non-monotonic response in particle sizing instruments

    Science.gov (United States)

    Rosenberg, Phil

    2017-04-01

    A number of instruments used as de-facto standards for measuring particle size distributions are actually incapable of uniquely determining the size of an individual particle. This is due to non-unique or non-monotonic response functions. Optical particle counters have non monotonic response due to oscillations in the Mie response curves, especially for large aerosol and small cloud droplets. Scanning mobility particle sizers respond identically to two particles where the ratio of particle size to particle charge is approximately the same. Images of two differently sized cloud or precipitation particles taken by an optical array probe can have similar dimensions or shadowed area depending upon where they are in the imaging plane. A number of methods exist to deal with these issues, including assuming that positive and negative errors cancel, smoothing response curves, integrating regions in measurement space before conversion to size space and matrix inversion. Matrix inversion (also called kernel inversion) has the advantage that it determines the size distribution which best matches the observations, given specific information about the instrument (a matrix which specifies the probability that a particle of a given size will be measured in a given instrument size bin). In this way it maximises use of the information in the measurements. However this technique can be confused by poor counting statistics which can cause erroneous results and negative concentrations. Also an effective method for propagating uncertainties is yet to be published or routinely implemented. Her we present a new alternative which overcomes these issues. We use Bayesian methods to determine the probability that a given size distribution is correct given a set of instrument data and then we use Markov Chain Monte Carlo methods to sample this many dimensional probability distribution function to determine the expectation and (co)variances - hence providing a best guess and an uncertainty for

  14. 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

  15. 耐高温植酸酶的热稳定性评估方法分析%Assessments of the thermostability of thermostable phytase

    Institute of Scientific and Technical Information of China (English)

    王海燕; 汤海鸥; 王晓睿; 杨禄良; 刘影

    2013-01-01

    耐高温植酸酶因热稳定性好、经饲料制粒后保存率高而应用日益广泛,但是目前尚无评估耐高温植酸酶热稳定性的统一的、公认的标准方法。文章结合目前评估植酸酶热稳定性的方法及本实验室的实际检测结果,分别介绍了在实验室和制粒条件下的评估情况,以便为植酸酶的热稳定性评估提供参考依据。%Thermostable phytase has become increasingly prevalent with its high heat-resistance and higher survival rate after feed pelleting, but currently there was no unified and accepted standard for assessing the thermostability of thermostable phytase. In this article we introduced the assessment under the lab and granulation condition, combined with the assessment for the thermostability of phytase and our actual detection results, we wish to arouse further communica-tion and exploration of this kind of assessments.

  16. Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases.

    Science.gov (United States)

    Panigrahi, Priyabrata; Chand, Deepak; Mukherji, Ruchira; Ramasamy, Sureshkumar; Suresh, C G

    2015-11-01

    Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in β-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.

  17. 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.

  18. Enhancing RGI lyase thermostability by targeted single point mutations

    DEFF Research Database (Denmark)

    Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten

    2013-01-01

    experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C......, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify......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...

  19. Two strategies to engineer flexible loops for improved enzyme thermostability

    Science.gov (United States)

    Yu, Haoran; Yan, Yihan; Zhang, Cheng; Dalby, Paul A.

    2017-02-01

    Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes.

  20. Enhancing thermostability of β-mannanase by protective additives

    Institute of Scientific and Technical Information of China (English)

    Zhaohui LIU; Wei QI; Weina WU; Yue LIU; Zhimin HE

    2008-01-01

    The effects of some sugars (glucose, mannose, fructose, sucrose and chitosan) and polyols (glycol, gly-cerol and sorbitol) as protective additive on the thermo-stability of β-mannanase were studied. The optimal reaction temperatures of β-mannanase and the ther-modynamics and the deactivation kinetics with or without additives were also investigated. The experimental results show that sucrose, chitosan and sorbitol could apparently improve the thermal stability of β-mannanase when their concentration was kept at 2 g/L. The optimal combina-tion additive proportion was sucrose: chitosan : sorbi-tol = 1 : 2 : 2 (molar ratio) using the orthogonal experimental design. The sucrose, chitosan, glycerol, sor-bitol and the combination additive might increase the optimal reaction temperature from 50℃ to about 60℃ due to their good protection effect. The thermal deactiva-tion curves of βmannanase accorded with the kinetic rules of first order reaction, and the corresponding kinetic and thermodynamic parameters were calculated. Meanwhile, the protective mechanism of the additives against deactivation of enzyme was also discussed.

  1. Characterization of a thermostable hepatitis B vaccine formulation.

    Science.gov (United States)

    Braun, LaToya Jones; Jezek, Jan; Peterson, Sabrina; Tyagi, Anil; Perkins, Shalimar; Sylvester, David; Guy, Mark; Lal, Manjari; Priddy, Scott; Plzak, Heidi; Kristensen, Debra; Chen, Dexiang

    2009-07-23

    Cold chain requirements for vaccine storage and distribution are both economic and logistical burdens for immunization programs, especially those in lower-resource settings. Inadvertent exposure of vaccines to both heat and freezing temperatures within such cold chains are frequently occurring problems in both developing and industrialized countries. Here we report on a new hepatitis B vaccine formulation that is stable against repeated freezing at -20 degrees C and is also stable for 12 months at 37 degrees C. The thermostable vaccine contains all the components of the original vaccine plus 7.5% (v/v) propylene glycol, 40mM phosphate, and 40mM histidine with a final pH of 5.2. The propylene glycol is responsible for the freeze stability while the other components are essential for the heat stability. This formulation was found to be well tolerated in rabbits without any significant local or systemic side effects. The improved stability of this hepatitis B vaccine could be a key factor in ensuring vaccine effectiveness, extending immunization coverage, simplifying immunization logistics, and reducing the costs associated with the cold chain.

  2. Understanding thermostability and pH dependent properties of proteins

    DEFF Research Database (Denmark)

    Galberg, Pernille

    , 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...... activity study and compared to wild type (WT). BCX N35D/A115E was created by site-directed mutagenesis followed by expression and purification. The kinetic characterization for BCX WT and N35D/A115E protein were determined as a function of pH using o-nitrophenyl-β-D-xylobioside as a substrate. The N35D....../A115E substitutions caused a 1.4 pH unit decrease in the pH activity optimum but the overall activity of the mutant was reduced by approximately 75 % compared to WT protein at their respective pH optima. Additionally BCX was used as a model for evaluating PROPKA. Six designed mutants were generated...

  3. A thermostable lectin from the rhizomes of Kaempferia parviflora.

    Science.gov (United States)

    Konkumnerd, Wichchulada; Karnchanatat, Aphichart; Sangvanich, Polkit

    2010-08-30

    Kaempferia parviflora, or black galingale (Kra-Chai-Dam), belongs to the Zingiberaceae family and is used as both a food ingredient and a medicinal plant. There are diverse reports on the biological activities of compounds extracted from the plant, such as antimalarial, antifungal and an effective sexual-enhancing role, but not on the lectins. A lectin was isolated from the rhizomes of Kaempferia parviflora using affinity chromatography on Concanavalin A followed by gel filtration chromatography on Sephacryl S-100. The molecular weight of the purified lectin was about 41.7 kDa. This lectin showed haemagglutinating activity against erythrocytes from several sources, with the highest level being against those from rabbits. Moreover, the lectin was thermostable, with significant haemagglutinating activity detectable up to 75 degrees C. The results of trypsin digestion and liquid chromatography/tandem mass spectrometry analysis suggested that this protein could be a member of the lectin/endochitnase1 family. A lectin that showed thermotolerant haemagglutinating activity against erythrocytes from several sources was successfully purified from K. paviflora rhizomes. Peptide sequence analysis indicated that this lectin is similar to lectin/endochitinase 1 (Urtica dioica) or Hevein-like protein (Hevea brasiliensis). Copyright (c) 2010 Society of Chemical Industry.

  4. Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp. 1519.

    Science.gov (United States)

    Modarres, Hassan Pezeshgi; Dorokhov, Boris D; Popov, Vladimir O; Ravin, Nikolai V; Skryabin, Konstantin G; Dal Peraro, Matteo

    2015-05-19

    The physical chemical principles underlying enzymatic thermostability are keys to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also an important factor for engineering more thermoresistant enzymes to be used in the industrial setting, such as, for instance, DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, we first address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance thermostability of the enzyme through bioinformatics analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.

  5. Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Prakash Chandra Rathi

    Full Text Available Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004 and fair (R2 = 0.46 correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

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

    Science.gov (United States)

    Jia, Lei; Yarlagadda, Ramya; Reed, Charles C

    2015-01-01

    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.

  7. 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.

  8. 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...

  9. 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.

  10. 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.

  11. Multiple periodic solutions of a delayed predator-prey model with non-monotonic functional response and stage structure.

    Science.gov (United States)

    Liu, Yingyuan; Zhang, Xiaolan; Zhou, Tiejun

    2014-01-01

    The paper studies a periodic and delayed predator-prey system with non-monotonic functional responses and stage structure. In the system, both the predator and prey are divided into immature individuals and mature individuals by two fixed ages. It is assumed that the immature predators cannot attack preys, and the case of the mature predators attacking the immature preys is also ignored. Based on Mawhin's coincidence degree, sufficient conditions are obtained for the existence of two positive periodic solutions of the system. An example is presented to illustrate the feasibility of the main results.

  12. 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.

  13. Periodic Solutions and Homoclinic Bifurcations of Two Predator-Prey Systems with Nonmonotonic Functional Response and Impulsive Harvesting

    Directory of Open Access Journals (Sweden)

    Mingzhan Huang

    2014-01-01

    Full Text Available Two predator-prey models with nonmonotonic functional response and state-dependent impulsive harvesting are formulated and analyzed. By using the geometry theory of semicontinuous dynamic system, we obtain the existence, uniqueness, and stability of the periodic solution and analyse the dynamic phenomenon of homoclinic bifurcation of the first system by choosing the harvesting rate β as control parameter. Besides, we also study the homoclinic bifurcation of the second system about parameter δ on the basis of the theory of rotated vector field. Finally, numerical simulations are presented to illustrate the results.

  14. Asymptotic behavior and dynamic stability of phase mixtures for the equations of Navier Stokes with nonmonotonic pressure

    Science.gov (United States)

    Tsyganov, Eugene

    2007-09-01

    We investigate the asymptotic behavior of the solutions of the compressible Navier Stokes equations with nonmonotonic pressure when the initial data is large and discontinuous. We provide sufficient conditions on the pressure function for different boundary-value problems that guarantee strong convergence of the volume variable as time approaches infinity and show that, typically, fairly arbitrary discontinuous static phase mixtures can be realized as time-asymptotic limits from smooth initial data. It is required in the analysis that we improve known existence theories, which typically have small data or time-dependent bounds.

  15. A transition model for quality-of-life data with non-ignorable non-monotone missing data.

    Science.gov (United States)

    Liao, Kaijun; Freres, Derek R; Troxel, Andrea B

    2012-12-10

    In this paper, we consider a full likelihood method to analyze continuous longitudinal responses with non-ignorable non-monotone missing data. We consider a transition probability model for the missingness mechanism. A first-order Markov dependence structure is assumed for both the missingness mechanism and observed data. This process fits the natural data structure in the longitudinal framework. Our main interest is in estimating the parameters of the marginal model and evaluating the missing-at-random assumption in the Effects of Public Information Study, a cancer-related study recently conducted at the University of Pennsylvania. We also present a simulation study to assess the performance of the model.

  16. Thermostability of in vitro evolved Bacillus subtilis lipase A: a network and dynamics perspective.

    Directory of Open Access Journals (Sweden)

    Ashutosh Srivastava

    Full Text Available Proteins in thermophilic organisms remain stable and function optimally at high temperatures. Owing to their important applicability in many industrial processes, such thermostable proteins have been studied extensively, and several structural factors attributed to their enhanced stability. How these factors render the emergent property of thermostability to proteins, even in situations where no significant changes occur in their three-dimensional structures in comparison to their mesophilic counter-parts, has remained an intriguing question. In this study we treat Lipase A from Bacillus subtilis and its six thermostable mutants in a unified manner and address the problem with a combined complex network-based analysis and molecular dynamic studies to find commonality in their properties. The Protein Contact Networks (PCN of the wild-type and six mutant Lipase A structures developed at a mesoscopic scale were analyzed at global network and local node (residue level using network parameters and community structure analysis. The comparative PCN analysis of all proteins pointed towards important role of specific residues in the enhanced thermostability. Network analysis results were corroborated with finer-scale molecular dynamics simulations at both room and high temperatures. Our results show that this combined approach at two scales can uncover small but important changes in the local conformations that add up to stabilize the protein structure in thermostable mutants, even when overall conformation differences among them are negligible. Our analysis not only supports the experimentally determined stabilizing factors, but also unveils the important role of contacts, distributed throughout the protein, that lead to thermostability. We propose that this combined mesoscopic-network and fine-grained molecular dynamics approach is a convenient and useful scheme not only to study allosteric changes leading to protein stability in the face of negligible

  17. POTENTIAL OF THERMOSTABLE CELLULASES IN BIOPROCESSING OF SWITCHGRASS TO ETHANOL

    Directory of Open Access Journals (Sweden)

    Kasivishavanathan Muthukumarappan

    2011-04-01

    Full Text Available Switchgrass (Panicum virgatum, a perennial grass native to North America, is a promising energy crop for bioethanol production. The aim of this study was to optimize the enzymatic saccharification of thermo-mechanically pretreated switchgrass using a thermostable cellulase from Geobacillus sp. in a three-level, four-variable central composite design of response surface methodology. Different combinations of solids loadings (5 to 20%, enzyme loadings (5 to 20 FPU g-1 DM, temperature (50 to 70 oC, and time (36 to 96 h were investigated in a total of 30 experiments to model glucose release from switchgrass. All four factors had a significant impact on the cellulose conversion yields with a high coefficient of determination of 0.96. The use of higher solids loadings (20% and temperatures (70 oC during enzymatic hydrolysis proved beneficial for the significant reduction of hydrolysis times (2.67-times and enzyme loadings (4-times, with important implications for reduced capital and operating costs of ethanol production. At 20% solids, the increase of temperature of enzymatic hydrolysis from 50 oC to 70 oC increased glucose concentrations by 34%. The attained maximum glucose concentration of 23.52 g L-1 translates into a glucose recovery efficiency of 46% from the theoretical yield. Following red yeast fermentation, a maximum ethanol concentration of 11 g L-1 was obtained, accounting for a high glucose to ethanol fermentation efficiency of 92%. The overall conversion efficiency of switchgrass to ethanol was 42%.

  18. 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.

  19. On the formation of localized peaks and non-monotonic tailing of breakthrough curves

    Science.gov (United States)

    Siirila, Erica R.; Sanchez-Vila, Xavier; Fernàndez-Garcia, Daniel

    2014-05-01

    While breakthrough curve (BTC) analysis is a traditional tool in hydrogeology to obtain hydraulic parameters, in recent years emphasis has been placed on analyzing the shape of the receding portion of the curve. A number of field and laboratory observations have found a constant BTC slope in log-log space, and thus it has been hypothesized that a power law behavior is representative of real aquifers. Usually, monotonicity of the late-time BTC slope is just assumed, meaning that local peaks in the BTC are not considered, and that a local (in time) increase or decrease of BTC slope is also not considered. We contend that local peaks may exist but are sometimes not reported for a number of reasons. For example, when BTCs are obtained from actual measurements, sub-sampling may mask non-monotonicity, or small peaks may be reported as measurement errors and thus smoothed out or removed. When numerical analyses of synthetic aquifers are performed, the simulation method may yield artificially monotonous curves as a consequence of the methods used. For example, Eulerian methods may suffer from numerical dispersion, where curves tend to become over-smoothed while Lagrangian methods may suffer from artificial BTC oscillations stemming from the reconstruction of concentrations from a limited number of particles. A paradigm shift in terms of the BTC shape must also accompany two major advancements within the hydrogeology field: 1) the increase of high frequency data and progression of data collection techniques that diminish the problems of under-sampling BTCs and 2) advancements in supercomputing and numerical simulation allowing for higher resolution of flow and transport problems. As more information is incorporated into BTCs and/or they are obtained in more spatial locations, it is likely that classical definitions of BTC shapes will no longer be adequate descriptors for future treatment of contaminant transport problems. For example, the presence of localized peaks in BTCs

  20. Global stability analysis of two-strain epidemic model with bilinear and non-monotone incidence rates

    Science.gov (United States)

    Baba, Isa Abdullahi; Hincal, Evren

    2017-05-01

    In this article we studied an epidemic model consisting of two strains with different types of incidence rates; bilinear and non-monotone. The model consists of four equilibrium points: disease-free equilibrium, endemic with respect to strain 1, endemic with respect to strain 2, and endemic with respect to both strains. The global stability analysis of the equilibrium points was carried out through the use of Lyapunov functions. Two basic reproduction ratios R 1 0 and R 2 0 are found, and we have shown that if both are less than one, the disease dies out, and if both are greater than one epidemic occurs. Furthermore, epidemics occur with respect to any strain with a basic reproduction ratio greater than one and disease dies out with respect to any strain with a basic reproduction ratio less than one. It was also shown that any strain with highest basic reproduction ratio will automatically outperform the other strain, thereby eliminating it. Numerical simulations were carried out to support the analytic result and to show the effect of the parameter k in the non-monotone incidence rate, which describes the psychological effect of general public towards infection.

  1. On the motion of a sessile drop on an incline: Effect of non-monotonic thermocapillary stresses

    Science.gov (United States)

    Mamalis, Dimitrios; Koutsos, Vasileios; Sefiane, Khellil

    2016-12-01

    We studied the short-time contact-line dynamics of a self-rewetting sessile droplet sliding "freely" on a silicone oil layer, on an inclined, uniformly heated substrate under non-isothermal conditions (liquid-solid). The effect of thermocapillarity and the contribution of surface tension gradients (Marangoni effect) to the droplet motion was investigated. The temperature of the substrate in conjunction with the non-monotonic surface tension/temperature dependence of the deformed self-rewetting droplet was found to significantly affect the early-stage inertial-capillary spreading regime. Infrared (IR) thermography images were also acquired to investigate the generation of thermal patterns at the liquid surface due to the strong surface-tension gradients. Our results demonstrate that the presence of strong surface tension driven flows at the liquid interface combined with droplet deformation (contact-angle hysteresis) gives rise to complex droplet dynamics. The interplay between thermocapillary stresses and body forces results in enhanced spreading rates, temporal non-monotonic dependence of the contact-line speed, as well as the droplet motion overcoming gravity in some instances.

  2. 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.

  3. Equilibrium oxygen storage capacity of ultrathin CeO2-δ depends non-monotonically on large biaxial strain

    Science.gov (United States)

    Balaji Gopal, Chirranjeevi; García-Melchor, Max; Lee, Sang Chul; Shi, Yezhou; Shavorskiy, Andrey; Monti, Matteo; Guan, Zixuan; Sinclair, Robert; Bluhm, Hendrik; Vojvodic, Aleksandra; Chueh, William C.

    2017-05-01

    Elastic strain is being increasingly employed to enhance the catalytic properties of mixed ion-electron conducting oxides. However, its effect on oxygen storage capacity is not well established. Here, we fabricate ultrathin, coherently strained films of CeO2-δ between 5.6% biaxial compression and 2.1% tension. In situ ambient pressure X-ray photoelectron spectroscopy reveals up to a fourfold enhancement in equilibrium oxygen storage capacity under both compression and tension. This non-monotonic variation with strain departs from the conventional wisdom based on a chemical expansion dominated behaviour. Through depth profiling, film thickness variations and a coupled photoemission-thermodynamic analysis of space-charge effects, we show that the enhanced reducibility is not dominated by interfacial effects. On the basis of ab initio calculations of oxygen vacancy formation incorporating defect interactions and vibrational contributions, we suggest that the non-monotonicity arises from the tetragonal distortion under large biaxial strain. These results may guide the rational engineering of multilayer and core-shell oxide nanomaterials.

  4. Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java

    Directory of Open Access Journals (Sweden)

    Lucia Dhiantika Witasari

    2015-11-01

    Full Text Available Thermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR. Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermostable enzymes, the thermostable DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermostable DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using spesific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermostable DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermostable DNA polymerase genes from other Bacillus.Key words : Thermostable DNA Pol I, Brevibacillus sp., PCR, cloning

  5. Final Technical/Scientific Report: Commodity Scale Thermostable Enzymatic Transformations

    Energy Technology Data Exchange (ETDEWEB)

    James J. Lalonde; Brian Davison

    2003-08-30

    The conversion of corn starch to high fructose corn-syrup sweetener is a commodity process, producing over 3 billion kg/y. In the last step of the process, an enzyme catalyst is used to convert glucose to the much sweeter sugar fructose. Due to incomplete conversion in the last step, the syrup must be purified using a chromatographic separation technique, which results in equal quantities of water being added to the syrup, and finally the water must be evaporated (up to 1 lb of water/lb of syrup). We have estimated the energy requirement in the evaporation step to be on the order of 13 billion BTU's/y. This process inefficiency could be eliminated if a thermostable form of glucose isomerase (GI), the enzyme catalyst used in the final step, was developed. Our chosen strategy was to develop an immobilized form of the enzyme in which the protein is first crystallized and then chemically cross-linked to form an insoluble particle. This so-called cross-linked enzyme crystal (CLE C(reg. sign)) technology had been shown to be a powerful method for enzyme stabilization for several other protein catalysts. In this work we have developed more than 30 CLEC preparations of glucose isomerase and tested them for activity and stability. We found these preparations to be highly active, with a 10-50 fold rate per gram of catalyst increase over existing commercial catalysts. The initial rates were also higher at higher temperatures as expected, however the efficiency of the CLEC GI preparations unexpectedly rapidly decreased to a low constant value with use at the higher temperatures. At this point, the source of this activity loss is unclear, however during this loss, the catalyst is found to form a solid mass indicating either breakage of the chemical cross-links or simple aggregation of the particles. It is likely that the increased mass transfer resistance due to this agglomeration is a major component of the activity loss. This research suggests that one potentially

  6. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).

    Science.gov (United States)

    Zhao, Hong-Yan; Wu, Li-Ying; Liu, Gang; Feng, Hong

    2016-12-01

    To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

  7. Highly thermostable GH39 ß-xylosidase from a Geobacillus sp. strain WSUCF1

    Science.gov (United States)

    Background Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and ß-xylosidase. ß-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable ß-xylosidases have been a focus of attention as industrially important enzymes due to th...

  8. 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

  9. 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

    1994-01-01

    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 alpha-amylas

  10. Characterization of a new Bacillus stearothermophilus isolate : a highly thermostable α-amylase-producing strain

    NARCIS (Netherlands)

    Wind, R.D.; Buitelaar, R.M.; Eggink, G.; Huizing, H.J.; Dijkhuizen, L.

    1994-01-01

    A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known α-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable α-amylase. The half-time of inactivation of this α-amylase was 5.1 h

  11. 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

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

    NARCIS (Netherlands)

    Lončar, Nikola; Fraaije, Marco W

    2014-01-01

    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 overexpre

  13. Directed evolution of a histone acetyltransferase--enhancing thermostability, whilst maintaining catalytic activity and substrate specificity.

    Science.gov (United States)

    Leemhuis, Hans; Nightingale, Karl P; Hollfelder, Florian

    2008-11-01

    Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

  14. 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-states...

  15. 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...

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

    NARCIS (Netherlands)

    Lončar, Nikola; Fraaije, Marco W

    2015-01-01

    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 overexpre

  17. Fluctuation theorem applied to the Nosé-Hoover thermostated Lorentz gas

    Science.gov (United States)

    Gilbert, Thomas

    2006-03-01

    We present numerical evidence supporting the validity of the Gallavotti-Cohen fluctuation theorem applied to the driven Lorentz gas with Nosé-Hoover thermostating. It is moreover argued that the asymptotic form of the fluctuation formula is independent of the amplitude of the driving force in the limit where it is small.

  18. DESIGN OF CANDIDA ANTARCTICA LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

    Directory of Open Access Journals (Sweden)

    Usman Sumo Friend Tambunan

    2014-01-01

    Full Text Available Candida Antarctica Lipase B (CALB is extensively studied in enzymatic production of biodiesel, pharmaceutical products, detergents and other chemicals. One drawback of using CALB is its relatively low optimum temperature at 313 K (40°C. The objective of this research is to design CALB mutant with improved thermostability by introducing extra disulfide bond. Molecular dynamic simulation was conducted to get better insight into the process of thermal denaturation or unfolding in CALB. Thermal denaturation of CALB was accelerated by conducting simulation at high temperature. Molecular dynamic simulation of CALB was performed with GROMACS software package at 300-700 K. Prediction of possible mutation was done using “Disulfide by DesignTM” software. Selection of mutated residues was based on flexibility analysis of CALB. From those analyses, three mutants were designed, which are Mutant-1 (73LeuCys/151AlaCys, Mutant-2 (155TrpCys/294GluCys and Mutant-3 (43ThrCys/67SerCys. Parameters that were used to compare the thermostability of mutant with wild type enzyme were Root Mean Square Deviations (RMSD, Solvent Accessible Surface Area (SASA, Radius of gyration (Rg and secondary structure. Molecular dynamic simulation conducted on those three mutants showed that Mutant-1 has better thermostability compared to wild type CALB. We proposed the order of mutant thermostability improvement as follows: Mutant-1, Mutant-2 and Mutant-3, with Mutant-1 having better potential thermostability improvement and Mutant-3, the least stable.

  19. 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.

  20. [Expression of thermostable recombiant Luciola lateralis luciferase and development of heat-stable pyrosequencing system].

    Science.gov (United States)

    Xu, Shu; Zou, Bingjie; Wang, Jianping; Wu, Haiping; Zhou, Guohua

    2012-06-01

    Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.

  1. 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.

  2. 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.

  3. Nonmonotonic size dependence in the hole mobility of methoxide-stabilized PbSe quantum dot solids.

    Science.gov (United States)

    Scheele, Marcus; Engel, Jesse H; Ferry, Vivian E; Hanifi, David; Liu, Yi; Alivisatos, A Paul

    2013-08-27

    We present a facile procedure to fabricate p-type PbSe-based quantum dot solids with mobilities as large as 0.3 cm(2) V(-1)s(-1). Upon partial ligand exchange of oleate-capped PbSe quantum dots with the methoxide ion, we observe a pronounced red shift in the excitonic transition in conjunction with a large increase in conductivity. We show that there is little correlation between these two phenomena and that the electronic coupling energy in PbSe quantum dot solids is much smaller than often assumed. However, we observe for the first time a nonmonotonic size dependence of the hole mobility, illustrating that coupling can nonetheless be dominant in determining the transport characteristics. We attribute these effects to a decrease in charging energy and interparticle spacing, leading to enhanced electronic coupling on one hand and enhanced dipole interactions on the other hand, which is held responsible for the majority of the red shift.

  4. Mechanistic modeling of fingering, nonmonotonicity, fragmentation, and pulsation within gravity/buoyant destabilized two-phase/unsaturated flow

    Science.gov (United States)

    Glass, Robert J.; Yarrington, Lane

    2003-03-01

    Fingering, nonmonotonicity, fragmentation, and pulsation within gravity/buoyant destabilized two-phase/unsaturated flow systems has been widely observed with examples in homogeneous to heterogeneous porous media, in single fractures to fracture networks, and for both wetting and nonwetting invasion. To model this phenomena, we consider a mechanistic approach based on forms of modified invasion percolation (MIP) that include gravity, the influence of the local interfacial curvature along the phase-phase interface, and the simultaneous invasion and reinvasion of both wetting and nonwetting fluids. We present example simulations and compare them to experimental data for three very different situations: (1) downward gravity-driven fingering of water into a dry, homogeneous, water-wettable, porous medium; (2) upward buoyancy-driven migration of gas within a water saturated, heterogeneous, water-wettable, porous medium; and (3) downward gravity-driven fingering of water into a dry, water-wettable, rough-walled fracture.

  5. 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

    2017-01-30

    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.

  6. Argumentation-Based Non-Monotonic Reasoning of Agents%基于论辩的Agent非单调推理

    Institute of Scientific and Technical Information of China (English)

    廖备水; 代建华

    2012-01-01

    现有的Agent信念修正、慎思、手段-目的推理等理论和方法大多基于经典一阶逻辑,对不完全的、不一致的知识,缺乏有效的处理机制.基于论辩的Agent非单调推理(包括认识推理和实践推理)理论和方法有望弥补这个不足.不过,作为一个新的研究方向,其基本概念、理论、方法及存在的关键性问题尚有待于澄清和梳理.文中首先介绍论辩的基本概念.在此基础上,分析基于论辩的Agent非单调推理的最新研究进展.最后,讨论存在的关键性问题并指出可能的研究方向.%Most of existing theories and methods for belief revision, deliberation, means-ends reasoning etc. are based on classical first order logic, and therefore effective mechanisms are absent in handling incomplete and inconsistent knowledge. Argumentation-based non-monotonic reasoning, including epistemic reasoning and practical reasoning, has become a promising theory to solve the above-mentioned problem. However, as an emerging research area, the basic notions, theories, methods, as well as the existing research problems, are still unclear. In this paper, after presenting the basic notions of argumentation, the recent development of argumentation-based non-monotonic reasoning of agents is analyzed. Finally, some challenging problems are discussed, and the possible future work is pointed out.

  7. Analyzing the impact of direct subsidies on the performance of the Greek olive farms with a non-monotonic efficiency effects model

    NARCIS (Netherlands)

    Zhu, X.; Karagiannis, G.; Oude Lansink, A.G.J.M.

    2008-01-01

    We analyse the impacts of the CAP reforms on technical efficiency of Greek olive farms. We use a production frontier function and a non-monotonic inefficiency effects model which incorporates the influences of exogenous variables on the mean and the variances of farm efficiency. We formulate policy

  8. 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.

  9. 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...... light scattering (DLS), showed a slight increase after heating at high temperature (80°C). Optical waveguide lightmode spectroscopy (OWLS) studies showed facile adsorption of BSM onto poly(dimethylsiloxane) (PDMS) surface (>180ng/cm2) at room temperature due to its amphiphilic characteristics. Adsorbed...

  10. Crystal structure of thermostable catechol 2,3-dioxygenase determined by multiwavelength anomalous dispersion method

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The selenomethionyl derivative of the thermostable catechol 2,3-dioxygenase (SeMet-TC23O) is expressed,purified and crystallized. By using multiwave length anomalous dispersion (MAD) phasing techniques, the crystal structure of TC23O at 0.3 nm resolutions is determined.TC23O is a homotetramer. Each monomer is composed of N-terminal and C-terminal domains (residues 1~153 and 153~319, respectively). The two domains are proximately symmetric by a non-crystallographic axis. Each domain contains two characteristic motifs which are found in almost all of extradial dioxygenases.Kevwords: multiwavelength anomalous dispersion (MAD), X-ray diffraction, thermostable catechol 2,3-dioxygenase, crystal structure,synchrotron light source.

  11. Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus

    DEFF Research Database (Denmark)

    Zhang, Ye-Wang; Tiwari, Manish Kumar; Gao, Hui;

    2012-01-01

    .6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of Lr......Nox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications...

  12. A Highly Thermostable Ceramic-Grafted Microporous Polyethylene Separator for Safer Lithium-Ion Batteries.

    Science.gov (United States)

    Zhu, Xiaoming; Jiang, Xiaoyu; Ai, Xinping; Yang, Hanxi; Cao, Yuliang

    2015-11-04

    The safety concern is a critical obstacle to large-scale energy storage applications of lithium-ion batteries. A thermostable separator is one of the most effective means to construct the safe lithium-ion batteries. Herein, we demonstrate a novel ceramic (SiO2)-grafted PE separator prepared by electron beam irradiation. The separator shows similar thickness and pore structure to the bare separator, while displaying strong dimensional thermostability, as the shrinkage ratio is only 20% even at an elevated temperature of 180 °C. Besides, the separator is highly electrochemically inert, showing no adverse effect on the energy and power output of the batteries. Considering the excellent electrochemical and thermal stability, the SiO2-grafted PE separator developed in this work is greatly beneficial for constructing safer lithium-ion batteries.

  13. Investigation of Thermostability of a Composite Resistive Material with Nanodimensional Carbon Fillers

    Science.gov (United States)

    Malinovskaya, T. D.; Vlasov, V. A.; Volokitin, G. G.; Melentyev, S. V.

    2014-06-01

    Thermostability of resistive materials based on polyurethane used as heat-liberating elements in the design of heating elements of thermoactive formworks is investigated. The application of polyurethane as a binder provides solid contact of polymer molecules with nanodimensional carbon fillers and their uniform distribution in a composite material. The influence of thermal treatment and dispersed fillers on the stability of electrophysical and thermophysical properties of carbon-filled polyurethane coatings is established.

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

    OpenAIRE

    Pendru eRaghunath

    2015-01-01

    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 ...

  15. Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin.

    OpenAIRE

    Nishibuchi, M.; Fasano, A; Russell, R G; Kaper, J B

    1992-01-01

    Vibrio parahaemolyticus produces a thermostable direct hemolysin (TDH) that has been implicated in the pathogenesis of diarrheal disease caused by this organism. However, previous studies attempting to demonstrate the contribution of the hemolysin to virulence have been inconclusive. We investigated this putative virulence factor by using an isogenic TDH-negative (TDH-) strain constructed by specifically inactivating the two copies of the tdh gene encoding TDH. The enterotoxigenicities of the...

  16. 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.

  17. Fluctuation formula in the Nosé-Hoover thermostated Lorentz gas

    Science.gov (United States)

    Dolowschiák, M.; Kovács, Z.

    2005-02-01

    In this paper we examine numerically the Gallavotti-Cohen fluctuation formula for phase-space contraction rate and entropy production rate fluctuations in the Nosé-Hoover thermostated periodic Lorentz gas. Our results indicate that while the phase-space contraction rate fluctuations violate the fluctuation formula near equilibrium states, the entropy production rate fluctuations obey this formula near and far from equilibrium states as well.

  18. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

    Science.gov (United States)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

    2015-09-01

    Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

  19. Isozymic variations in specific and nonspecific esterase and its thermostability in silkworm, Bombyx mori L.

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Biswas, Tapati Datta; Nayak, Sandeepta Kumar; Saha, A K; Majumdar, M K

    2012-09-01

    Esterase isozymic variations were documented in the haemolymph of developed multivoltine and bivoltine silkworm breeds during unfavorable seed crop seasons of May - September using á- and â- napthylacetate separately to identify specific and nonspecific esterase having thermotolerant potentiality. Variations existed in the isozyme pattern with three bands (Est-2, 3 and 4) in pure Nistari race and other developed multivoltine and bivoltine breeds. Est-2 and Est-3 were non-specific esterases as they were observed when both á- and â-napthylacetate was used as substrates separately. Est-4 band was observed only with á-napthylacetate as substrate and was therefore confirmed to be specific á-esterase band in the haemolymph of silkworm, Bombyx mori L. Zymograms showed that the non-specific esterase band (Est-3) with R1 of 0.43 and specific á-esterase band (Est-4) with R(f) of 0.32 predominately withstood a temperature of 70 +/- 2 degrees C for a duration of 10 min and were confirmed as thermostable esterases in haemolymph of silkworm, Bombyx mori L. This also categorized the presence of thermostable esterases in developed multivoltine and bivoltine breeds of silkworm, even though the qualitative activity was more in the former than the latter. The qualitative presence of thermostable esterases and their activity could be adopted as an indicative biochemical marker in relation to thermotolerance in silkworm.

  20. 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.

  1. Thermostable α-amylase immobilization: Enhanced stability and performance for starch biocatalysis.

    Science.gov (United States)

    Kumar, Gudi Satheesh; Rather, Gulam Mohmad; Gurramkonda, Chandrasekhar; Reddy, Bontha Rajasekhar

    2016-01-01

    The uses of thermostable starch hydrolytic biocatalysts are steadily increasing for the industrial application because of their obvious need for biocatalytic performance at elevated temperatures. The starch liquefaction and saccharification can be carried out simultaneously by the use of thermostable starch hydrolytic biocatalysts, thus minimizing the unit operations, time, and efforts. The cost factor hampers the industrialization of expensive soluble (free) enzymes for biocatalytic applications and the immobilization of enzymes offers promising alternative to the hurdle. The present investigation was aimed for immobilization of thermostable α-amylase using calcium alginate, and statistical optimization studies were carried out for enhanced biocatalytic performance. Initially, one-parameter at a time optimization studies were carried out for identification of significant factors influencing the immobilization. Furthermore, a statistical approach, response surface methodology, was applied for immobilization of α-amylase. The immobilized α-amylase in alginate microbeads showed enhanced stability to temperature and reusable property for up to seven cycles (with the retention of 50% initial activity). Finally, the kinetic behavior of free and immobilized enzyme showed the Km value of 1.2% and 2.6% (w/v) and Vmax of 1,020 and 1,030 U, respectively. Fifty percent reduction in affinity of the immobilized enzyme toward substrate was compensated by its longer stability.

  2. 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.

  3. Remarkable improvement of methylglyoxal synthase thermostability by His-His interaction.

    Science.gov (United States)

    Mohammadi, Malihe; Kashi, Mona Atabakhshi; Zareian, Shekufeh; Mirshahi, Manoochehr; Khajeh, Khosro

    2014-01-01

    Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine-histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HH(O)), and another two histidines (TMGS-HHH(O)) were inserted between Arg22 and His23 (H(O)). In comparison with the wild type, TMGS-HH(O) thermostability increased remarkably, whereas TMGS-HHH(O) was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHH(O) was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHH(O). These results demonstrated that histidine-histidine interactions at position 23 in TMGS-HH(O) probably have the main role in TMGS thermostability.

  4. Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability

    Directory of Open Access Journals (Sweden)

    Kian Mau Goh

    2012-04-01

    Full Text Available Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

  5. Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability.

    Science.gov (United States)

    Goh, Poh Hong; Illias, Rosli Md; Goh, Kian Mau

    2012-01-01

    Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

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

    Science.gov (United States)

    Nunes, Daniel; Cruz, Tomás L.; Jespersen, Sune N.; Shemesh, Noam

    2017-04-01

    White Matter (WM) microstructures, such as axonal density and average diameter, are crucial to the normal function of the Central Nervous System (CNS) as they are closely related with axonal conduction velocities. Conversely, disruptions of these microstructural features may result in severe neurological deficits, suggesting that their noninvasive mapping could be an important step towards diagnosing and following pathophysiology. Whereas diffusion based MRI methods have been proposed to map these features, they typically entail the application of powerful gradients, which are rarely 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-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 were here used as a simple model for the microstructure - and that, for axons parallel to the main magnetic field, the axonal density can be extracted. We then experimentally demonstrate in ex-vivo rat spinal cords that its different tracts - characterized by different microstructures - can be clearly contrasted using the MGE-derived maps. When the quantitative results are compared against ground-truth histology, they 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 a potential and usefulness of the approach to map underlying microstructures using a simple and time-efficient MRI sequence. We further show that a simple general-linear-model can predict the average axonal diameters from the four model parameters, and

  7. Thermocapillary-driven motion of a sessile drop: effect of non-monotonic dependence of surface tension on temperature.

    Science.gov (United States)

    Karapetsas, George; Sahu, Kirti Chandra; Sefiane, Khellil; Matar, Omar K

    2014-04-22

    We study the thermocapillary-driven spreading of a droplet on a nonuniformly heated substrate for fluids associated with a non-monotonic dependence of the surface tension on temperature. We use lubrication theory to derive an evolution equation for the interface that accounts for capillarity and thermocapillarity. The contact line singularity is relieved by using a slip model and a Cox-Voinov relation; the latter features equilibrium contact angles that vary depending on the substrate wettability, which, in turn, is linked to the local temperature. We simulate the spreading of droplets of fluids whose surface tension-temperature curves exhibit a turning point. For cases wherein these turning points correspond to minima, and when these minima are located within the droplet, then thermocapillary stresses drive rapid spreading away from the minima. This gives rise to a significant acceleration of the spreading whose characteristics resemble those associated with the "superspreading" of droplets on hydrophobic substrates. No such behavior is observed for cases in which the turning point corresponds to a surface tension maximum.

  8. Stability and Bifurcation Analysis of a Delayed Leslie-Gower Predator-Prey System with Nonmonotonic Functional Response

    Directory of Open Access Journals (Sweden)

    Jiao Jiang

    2013-01-01

    Full Text Available A delayed Leslie-Gower predator-prey model with nonmonotonic functional response is studied. The existence and local stability of the positive equilibrium of the system with or without delay are completely determined in the parameter plane. Using the method of upper and lower solutions and monotone iterative scheme, a sufficient condition independent of delay for the global stability of the positive equilibrium is obtained. Hopf bifurcations induced by the ratio of the intrinsic growth rates of the predator and prey and by delay, respectively, are found. Employing the normal form theory, the direction and stability of Hopf bifurcations can be explicitly determined by the parameters of the system. Some numerical simulations are given to support and extend our theoretical results. Two limit cycles enclosing an equilibrium, one limit cycle enclosing three equilibria and different types of heteroclinic orbits such as connecting two equilibria and connecting a limit cycle and an equilibrium are also found by using analytic and numerical methods.

  9. Nonmonotonic Diameter Dependence of Thermal Conductivity of Extremely Thin Si Nanowires: Competition between Hydrodynamic Phonon Flow and Boundary Scattering.

    Science.gov (United States)

    Zhou, Yanguang; Zhang, Xiaoliang; Hu, Ming

    2017-02-08

    By carefully and systematically performing Green-Kubo equilibrium molecular dynamics simulations, we report that the thermal conductivity (κ) of Si nanowires (NWs) does not diverge but converges and increases steeply when NW diameter (D) becomes extremely small (dκ/dD < 0), a long debate of one-dimensional heat conduction in history. The κ of the thinnest possible Si NWs reaches a superhigh level that is as large as more than 1 order of magnitude higher than its bulk counterpart. The abnormality is explained in terms of the dominant normal (N) process (energy and momentum conservation) of low frequency acoustic phonons that induces hydrodynamic phonon flow in the Si NWs without being scattered. With D increasing, the downward shift of optical phonons triggers strong Umklapp (U) scattering with acoustic phonons and attenuates the N process, leading to the regime of phonon boundary scattering (dκ/dD < 0). The two competing mechanisms result in nonmonotonic diameter dependence of κ with minima at critical diameter of 2-3 nm. Our results unambiguously demonstrate the converged κ and the clear trend of κ ∼ D for extremely thin Si NWs by fully elucidating the competition between the hydrodynamic phonon flow and phonon boundary scattering.

  10. A weighted combination of pseudo-likelihood estimators for longitudinal binary data subject to nonignorable non-monotone missingness

    Science.gov (United States)

    Troxel, Andrea B.; Lipsitz, Stuart R.; Fitzmaurice, Garrett M.; Ibrahim, Joseph G.; Sinha, Debajyoti; Molenberghs, Geert

    2010-01-01

    SUMMARY For longitudinal binary data with non-monotone non-ignorably missing outcomes over time, a full likelihood approach is complicated algebraically, and with many follow-up times, maximum likelihood estimation can be computationally prohibitive. As alternatives, two pseudo-likelihood approaches have been proposed that use minimal parametric assumptions. One formulation requires specification of the marginal distributions of the outcome and missing data mechanism at each time point, but uses an “independence working assumption,” i.e., an assumption that observations are independent over time. Another method avoids having to estimate the missing data mechanism by formulating a “protective estimator.” In simulations, these two estimators can be very inefficient, both for estimating time trends in the first case and for estimating both time-varying and time-stationary effects in the second. In this paper, we propose use of the optimal weighted combination of these two estimators, and in simulations we show that the optimal weighted combination can be much more efficient than either estimator alone. Finally, the proposed method is used to analyze data from two longitudinal clinical trials of HIV-infected patients. PMID:20205269

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

    Science.gov (United States)

    Dagan, Guy; Koren, Ilan; Altaratz, Orit; Heiblum, Reuven H.

    2017-06-01

    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.

  12. Cross-Diffusion Induced Turing Instability and Amplitude Equation for a Toxic-Phytoplankton-Zooplankton Model with Nonmonotonic Functional Response

    Science.gov (United States)

    Han, Renji; Dai, Binxiang

    2017-06-01

    The spatiotemporal pattern induced by cross-diffusion of a toxic-phytoplankton-zooplankton model with nonmonotonic functional response is investigated in this paper. The linear stability analysis shows that cross-diffusion is the key mechanism for the formation of spatial patterns. By taking cross-diffusion rate as bifurcation parameter, we derive amplitude equations near the Turing bifurcation point for the excited modes in the framework of a weakly nonlinear theory, and the stability analysis of the amplitude equations interprets the structural transitions and stability of various forms of Turing patterns. Furthermore, we illustrate the theoretical results via numerical simulations. It is shown that the spatiotemporal distribution of the plankton is homogeneous in the absence of cross-diffusion. However, when the cross-diffusivity is greater than the critical value, the spatiotemporal distribution of all the plankton species becomes inhomogeneous in spaces and results in different kinds of patterns: spot, stripe, and the mixture of spot and stripe patterns depending on the cross-diffusivity. Simultaneously, the impact of toxin-producing rate of toxic-phytoplankton (TPP) species and natural death rate of zooplankton species on pattern selection is also explored.

  13. 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.

  14. 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.

  15. Non-monotonic dynamics of water in its binary mixture with 1,2-dimethoxy ethane: A combined THz spectroscopic and MD simulation study

    Science.gov (United States)

    Das Mahanta, Debasish; Patra, Animesh; Samanta, Nirnay; Luong, Trung Quan; Mukherjee, Biswaroop; Mitra, Rajib Kumar

    2016-10-01

    A combined experimental (mid- and far-infrared FTIR spectroscopy and THz time domain spectroscopy (TTDS) (0.3-1.6 THz)) and molecular dynamics (MD) simulation technique are used to understand the evolution of the structure and dynamics of water in its binary mixture with 1,2-dimethoxy ethane (DME) over the entire concentration range. The cooperative hydrogen bond dynamics of water obtained from Debye relaxation of TTDS data reveals a non-monotonous behaviour in which the collective dynamics is much faster in the low Xw region (where Xw is the mole fraction of water in the mixture), whereas in Xw ˜ 0.8 region, the dynamics gets slower than that of pure water. The concentration dependence of the reorientation times of water, calculated from the MD simulations, also captures this non-monotonous character. The MD simulation trajectories reveal presence of large amplitude angular jumps, which dominate the orientational relaxation. We rationalize the non-monotonous, concentration dependent orientational dynamics by identifying two different physical mechanisms which operate at high and low water concentration regimes.

  16. The nonmonotonic concentration dependence of the mean activity coefficient of electrolytes is a result of a balance between solvation and ion-ion correlations.

    Science.gov (United States)

    Vincze, Julianna; Valiskó, Mónika; Boda, Dezso

    2010-10-21

    We propose a simple model to explain the nonmonotonic concentration dependence of the mean activity coefficient of simple electrolytes without using any adjustable parameters. The primitive model of electrolytes is used to describe the interaction between ions computed by the adaptive grand canonical Monte Carlo method. For the dielectric constant of the electrolyte, we use experimental concentration dependent values. This is included through a solvation term in our treatment to describe the interaction between ions and water that changes as the dielectric constant changes with concentration. This term is computed by a Born-treatment fitted to experimental hydration energies. Our results for LiCl, NaCl, KCl, CsCl, NaBr, NaI, MgCl(2), CaCl(2), SrCl(2), and BaCl(2) demonstrate that the principal reason of the nonmonotonic behavior of the activity coefficient is a balance between the solvation and ion-ion correlation terms. This conclusion differs from previous studies that assumed that it is the balance of hard sphere repulsion and electrostatic attraction that produces the nonmonotonic behavior. Our results indicate that the earlier assumption that solvation can be taken into account by a larger, "solvated" ionic radius should be reconsidered. To explain second order effects (such as dependence on ionic size), we conclude that explicit water models are needed.

  17. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    Science.gov (United States)

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  18. 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 po

  19. The performance of monotonic and new non-monotonic gradient ascent reconstruction algorithms for high-resolution neuroreceptor PET imaging

    Energy Technology Data Exchange (ETDEWEB)

    Angelis, G I; Kotasidis, F A; Matthews, J C [Imaging, Proteomics and Genomics, MAHSC, University of Manchester, Wolfson Molecular Imaging Centre, Manchester (United Kingdom); Reader, A J [Montreal Neurological Institute, McGill University, Montreal (Canada); Lionheart, W R, E-mail: georgios.angelis@mmic.man.ac.uk [School of Mathematics, University of Manchester, Alan Turing Building, Manchester (United Kingdom)

    2011-07-07

    Iterative expectation maximization (EM) techniques have been extensively used to solve maximum likelihood (ML) problems in positron emission tomography (PET) image reconstruction. Although EM methods offer a robust approach to solving ML problems, they usually suffer from slow convergence rates. The ordered subsets EM (OSEM) algorithm provides significant improvements in the convergence rate, but it can cycle between estimates converging towards the ML solution of each subset. In contrast, gradient-based methods, such as the recently proposed non-monotonic maximum likelihood (NMML) and the more established preconditioned conjugate gradient (PCG), offer a globally convergent, yet equally fast, alternative to OSEM. Reported results showed that NMML provides faster convergence compared to OSEM; however, it has never been compared to other fast gradient-based methods, like PCG. Therefore, in this work we evaluate the performance of two gradient-based methods (NMML and PCG) and investigate their potential as an alternative to the fast and widely used OSEM. All algorithms were evaluated using 2D simulations, as well as a single [{sup 11}C]DASB clinical brain dataset. Results on simulated 2D data show that both PCG and NMML achieve orders of magnitude faster convergence to the ML solution compared to MLEM and exhibit comparable performance to OSEM. Equally fast performance is observed between OSEM and PCG for clinical 3D data, but NMML seems to perform poorly. However, with the addition of a preconditioner term to the gradient direction, the convergence behaviour of NMML can be substantially improved. Although PCG is a fast convergent algorithm, the use of a (bent) line search increases the complexity of the implementation, as well as the computational time involved per iteration. Contrary to previous reports, NMML offers no clear advantage over OSEM or PCG, for noisy PET data. Therefore, we conclude that there is little evidence to replace OSEM as the algorithm of choice

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

    Science.gov (United States)

    Germán, Gabriel; Herrera-Aguilar, Alfredo; Hidalgo, Juan Carlos; Sussman, Roberto A.

    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 epsilon(phi) and its derivatives epsilon'(phi) and epsilon''(phi), thereby extracting general conditions on the tensor-to-scalar ratio r and the running nsk at phiH where the perturbations are produced, some 50-60 e-folds before the end of inflation. We find quite generally that for models where epsilon(phi) 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 ξ2(phi). To accommodate a large tensor-to-scalar ratio that meets the limiting values allowed by the Planck data, we study a non-monotonic epsilon(phi) decreasing during most part of inflation. Since at phiH the slow-roll parameter epsilon(phi) is increasing, we thus require that epsilon(phi) develops a maximum for phi > phiH after which epsilon(phi) 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 Δphie ≡ |phiH-phie| is obtained with a sufficiently thin profile for epsilon(phi) which, however, should not conflict with the second slow-roll parameter η(phi). As a consequence of this analysis we find bounds for Δphie, rH and for the scalar spectral index nsH. Finally we provide examples where these considerations are explicitly realised.

  1. Thermostability analysis of line-tension-associated nucleation at a gas-liquid interface

    Science.gov (United States)

    Singha, Sanat Kumar; Das, Prasanta Kumar; Maiti, Biswajit

    2017-01-01

    The influence of line tension on the thermostability of a droplet nucleated from an oversaturated vapor at the interface of the vapor and another immiscible liquid is investigated. Along with the condition of mechanical equilibrium, the notion of extremization of the reversible work of formation is considered to obtain the critical parameters related to heterogeneous nucleation. From the energetic formulation, the critical reversible work of formation is found to be greater than that of homogeneous nucleation for high value of the positive line tension. On the other hand, for high value of the negative line tension, the critical reversible work of formation becomes negative. Therefore, these thermodynamic instabilities under certain substrate wettability situations necessitate a free-energetics-based stability of the nucleated droplet, because the system energy is not minimized under these conditions. This thermostability is analogous to the transition-based stability proposed by Widom [B. Widom, J. Phys. Chem. 99, 2803 (1995)], 10.1021/j100009a041 in the case of partial wetting phenomena along with the positive line tension. The thermostability analysis limits the domain of the solution space of the present critical-value problem as the thermodynamic transformation in connection with homogeneous and workless nucleation is considered. Within the stability range of the geometry-based wetting parameters, three limiting modes of nucleation, i.e., total-dewetting-related homogeneous nucleation, and total-wetting-associated and total-submergence-associated workless nucleation scenarios, are identified. Either of the two related limiting wetting scenarios of workless nucleation, namely, total wetting and total submergence, is found to be favorable depending on the geometry-based wetting conditions. The line-tension-associated nucleation on a liquid surface can be differentiated from that on a rigid substrate, as in the former, the stability based on mechanical

  2. Cloning, overexpression, and characterization of a novel alkali-thermostable xylanase from Geobacillus sp. WBI.

    Science.gov (United States)

    Mitra, Suranjita; Mukhopadhyay, Bidhan Chandra; Mandal, Anisur Rahaman; Arukha, Ananta Prasad; Chakrabarty, Kuheli; Das, Gourab Kanti; Chakrabartty, Pran Krishna; Biswas, Swadesh Ranjan

    2015-04-01

    An endo-β-1,4-xylanase gene xynA of a thermophilic Geobacillus sp. WBI from "hot" compost was isolated by PCR amplification. The gene encoding 407 residues were overexpressed in E. coli and purified by Ni-NTA chromatography. The purified enzyme (47 kDa) had a broad pH optimum of 6.0 to 9.0, and was active between 50 and 90 °C. The enzyme retained 100% of its activity when incubated at 65 °C for 1 h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0. The K(m) and V(max) of the enzyme were 0.9 mg ml(-1) and 0.8 µmol ml(-1) min(-1), respectively. In molecular dynamics simulation at 338 K (65 °C), the enzyme was found to be stable. At an elevated temperature (450 K) specific α-helix and β-turns of the proteins were most denatured. The denaturation was less in WBI compared with its highest homolog G. stearothermophilus T-6 xylanase with difference of six residues. The results predict that these regions are responsible for the improved thermostability observed over related enzymes. The present work encourages further experimental demonstration to understand how these regions contribute thermostability to WBI xylanase. The study noted that WBI produces a xylanase with unique characteristics, specifically alkali-thermostability.

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

    Indian Academy of Sciences (India)

    GHOLAMREZA FARNOOSH; KHOSRO KHAJEH; ALI MOHAMMAD LATIFI; HOSSEIN AGHAMOLLAEI

    2016-12-01

    The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the mostfrequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has beenshown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineeringof de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rationalmethod of protein engineering. In this study, Disulphide by Design software, homology modelling and moleculardynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge toimprove protein thermostability. The thermostability of the wild-type and three selected mutant enzymes wereevaluated by half-life, ΔG inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Dataanalysis 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/E124Cmutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results werecorrelated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsicfluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi alsoincreased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results weresubmitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing adisulphide bridge after bioinformatics design, even though this design would not be always successful.

  4. Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design

    Science.gov (United States)

    Lv, Jinzhi; Li, Qingbin; Tian, Jian; Wu, Ningfeng

    2016-01-01

    Pullulanase (EC 3.2.1.41) plays an important role in the specific hydrolysis of branch points in amylopectin. Enhancing its thermostability is required for its industrial application. In this study, rational protein design was used to improve the thermostability of PulB from Bacillus naganoensis (AB231790.1), which has strong enzymatic properties. Three positive single-site mutants (PulB-D328H, PulB-N387D, and PulB-A414P) were selected from six mutants. After incubation at 65°C for 5 min, the residual activities of PulB-D328H, PulB-N387D, and PulB-A414P were 4.5-, 1.7-, and 1.47-fold higher than PulB-WT, and their Tm values (the temperature at which half protein molecule denature) were 1.8°C, 0.4°C, and 0.9°C higher than PulB-WT, respectively. Then the final combined mutant PulB-328/387/414 was constructed. The t1/2 of it was 12.9-fold longer than that of PulB-WT at 65°C and the total increase in Tm of it (5.0°C) was almost 60% greater than the sum of individual increases (3.1°C). In addition, kinetic studies revealed that the kcat and the kcat/Km of PulB-328/387/414 increased by 38.8% and 12.9%. The remarkable improvement in thermostability and the high catalytic efficiency of PulB-328/387/414 make it suitable for industrial applications. PMID:27764201

  5. A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase.

    Science.gov (United States)

    Fei, Baojin; Xu, Hui; Cao, Yu; Ma, Shuhan; Guo, Hongxiu; Song, Tao; Qiao, Dairong; Cao, Yi

    2013-05-01

    Despite recent advances in our understanding of the importance of protein surface properties for protein thermostability,there are seldom studies on multi-factors rational design strategy, so a more scientific, simple and effective rational strategy is urgent for protein engineering. Here, we first attempted to use a three-factors rational design strategy combining three common structural features, protein flexibility, protein surface, and salt bridges. Escherichia coli AppA phytase was used as a model enzyme to improve its thermostability. Moreover, the structure and enzyme features of the thermostable mutants designed by our strategy were analyzed roundly. For the single mutants, two (Q206E and Y311K), in five exhibited thermostable property with a higher success rate of prediction (40 %). For the multiple mutants, the themostable sites were combined with another site, I427L, we obtained by directed evolution, Q206E/I427L, Y311K/I427L, and Q206E/Y311K/I427L, all exhibited thermostable property. The Y311K/I427L doubled thermostability (61.7 %, and was compared to 30.97 % after being heated at 80 °C for 10 min) and catalytic efficiency (4.46 was compared to 2.37) improved more than the wild-type AppA phytase almost without hampering catalytic activity. These multi-factors of rational design strategy can be applied practically as a thermostabilization strategy instead of the conventional single-factor approach.

  6. Factors influencing the activity and thermostability of immobilized porcine pancreatic lipase.

    Science.gov (United States)

    Kéry, V; Haplová, J; Tihlárik, K; Schmidt, S

    1990-01-01

    Lipase from porcine pancreas was immobilized on cellulose beads having various degrees of hydrophobicity, by covalent linking and by hydrophobic adsorption. Lipolytic activity was measured in heterogeneous organic-aqueous systems of various hydrophobicities using olive oil as a substrate. The main factors influencing lipase activity were hydrophobicity of the reaction mixture and of the carrier. Carriers with increased hydrophobicity enhanced lipase activity more than less hydrophobic ones. Lipase immobilized covalently on cellulose beads was less active than that adsorbed onto tritylcellulose but was considerably more thermostable.

  7. Partial Purification and Characterization of a Thermostable Actinomycete beta-Amylase.

    Science.gov (United States)

    Obi, S K; Odibo, F J

    1984-03-01

    A thermostable amylase, possibly a beta-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60 degrees C and pH 7 and by retention of 70% activity at 70 degrees C (30 min). It was stimulated by Mn and Fe but strongly inhibited by Hg. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.

  8. Synthesis of Thermostable Azo-type Photoswitches towards Photoreaulatina Nucleic Acid Structures

    Institute of Scientific and Technical Information of China (English)

    WANG Qi; GAO Shuang; ZHOU Kai; CHEN Wenbin; NIU Congwei; XI Zhen

    2009-01-01

    In order to design efficient and thermostable azo-type regulators,a series of azo-type compounds were synthesized and characterized.While introducing an inductive electron-withdrawing group to an azobenzene para or meta-position,the obtained compound can be an excellent photoswitch.3,3'-Azo-di-benzyl alcohol was designed and synthesized as one of therrnostable and efficient photoswitches,which can efficiently reversibly photoregulate the nucleic acid structure with its cis-isomer being sufficiently stable at physiological temperature.

  9. 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.

  10. Synthesis, characterization and thermostability of barium β-diketonate with tetraethylenepentamine ligand

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    The metal-organic chemical vapor deposition (MOCVD) technique is a promising process for high-temperature superconductor YBa2Cu3O7-δ(YBCO) preparation. In this technique, it is a challenge to obtain barium precursors with high volatility. In addition, the purity, evaporation characteristics and thermostability of adopted precursors in the whole process would decide the quality and reproducible results of YBCO film. In the present report, the barium precursor containing 2,2,6,6-tetramethylheptane-3,5-dionate...

  11. Nonmonotonic logics and algebras

    Institute of Scientific and Technical Information of China (English)

    CHAKRABORTY Mihir Kr; GHOSH Sujata

    2008-01-01

    Several nonmonotonie logic systems together with their algebraic semantics are discussed. NM-algebra is defined.An elegant construction of an NM-algebra starting from a Boolean algebra is described which gives rise to a few interesting algebraic issues.

  12. 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; Fukui, Toshiaki; Xing, Xin-Hui

    2013-04-01

    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 K(m) 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.

  13. Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

    Science.gov (United States)

    Gharib, Ghazaleh; Rashid, Naeem; Bashir, Qamar; Gardner, Qura-Tul Ann Afza; Akhtar, Muhammad; Imanaka, Tadayuki

    2016-01-01

    Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

  14. Structure of a diguanylate cyclase from Thermotoga maritima: insights into activation, feedback inhibition and thermostability.

    Directory of Open Access Journals (Sweden)

    Angeline Deepthi

    Full Text Available Large-scale production of bis-3'-5'-cyclic-di-GMP (c-di-GMP would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.

  15. Thermostable alkaline halophilic-protease production by Natronolimnobius innermongolicus WN18.

    Science.gov (United States)

    Selim, Samy; Hagagy, Nashwa; Abdel Aziz, Mohamed; El-Meleigy, El Syaed; Pessione, Enrica

    2014-01-01

    This study reports the production and biochemical characterisation of a thermostable alkaline halophilic protease from Natronolimnobius innermongolicus WN18 (HQ658997), isolated from soda Lake of Wadi An-Natrun, Egypt. The enzyme was concentrated by spinning through a centriplus, centrifugal ultrafiltration Millipore membrane with a total yield of 25%. The relative molecular mass of this protease determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ranged from 67 to 43 kDa. The extracellular protease of N. innermongolicus WN18 was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 2.5 M NaCl. This enzyme was stable in a broad pH range (6-12) with an optimum pH of 9-10 for azocasein hydrolysis. This extracellular protease, therefore, could be defined as thermostable and haloalkaliphilic with distinct properties that make the enzyme applicable for different industrial purposes.

  16. A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis.

    Science.gov (United States)

    Ren, Xiaodong; Yu, Dawei; Yu, Lei; Gao, Gui; Han, Siping; Feng, Yan

    2007-05-10

    Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.

  17. [The applications of thermostable ligase chain reaction in facilitating DNA recombination].

    Science.gov (United States)

    Xiangda, Zhou; Xiao, Song; Cong, Huai; Haiyan, Sun; Hongyan, Chen; Daru, Lu

    2016-02-01

    The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

  18. [A new balloon-expandable plastic endoprosthesis. Initial report of experience with the malleable thermostent].

    Science.gov (United States)

    Beck, A

    1990-07-01

    A new system of balloon-expandable stents for different purposes is presented. A special plastic material that can be shaped by a hot balloon technique or other internal or external heating modalities has been developed. The plastic material - a distant derivative of polyurethane - is caprolactone, which is soft from 52 degrees C to 70 degrees C. Using balloon techniques, the "thermo-stent" can be modeled to suit the form of the vessels, the bile ducts or the bronchial tree, as required. The balloon can be heated simply by means of a warm NaCl solution, electric matter in the balloon itself or microwaves. The plastic material can even be heated directly, which is especially beneficial if the stent needs to be thicker, e.g. in the bronchial tree, by an electric network within the plastic material, which heats the material to the necessary temperature by electric current. When the balloon is cooled after the dilatation the new form, the plastic has been modeled to is maintained exactly. The advantages of this thermo-stent will be the perfect adaptation to every individual situation in the intraluminal vessels, the bile ducts, and even the bronchi. The problems encountered hitherto with conventional metallic stents, e.g. high thrombogenicity, risk of metal intoxication or metallic rupture of filaments, have not be seen so far in animal experiments.

  19. Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.

    Science.gov (United States)

    Ozawa, Kazumichi; Iwasa, Hisanori; Sasaki, Noriko; Kinoshita, Nao; Hiratsuka, Atsunori; Yokoyama, Kenji

    2017-01-01

    FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.

  20. Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiu-yan; RUAN Hui; MU Lin; HE Guo-qing; TANG Xing-jun; CHEN Qi-he

    2006-01-01

    In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-l,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher (28%) for the former or much lower (21.6%)for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.

  1. [Thermostability testing of iron(II) sulphate for formulation of drug product].

    Science.gov (United States)

    Laczkovich, Orsolya; Révész, Piroska; Pallagi, Edina; Eros, István

    2003-01-01

    The aim of the experimental study was to determine which iron(II) sulphate form, the monohydrate or the heptahydrate, is more suitable for formulation of a retard dosage form with melt technology, and to control the suitable compound from a thermostability aspect according to the ICH (International Conference on Harmonization). Iron(II) sulphate is susceptible to oxidation and a rise in temperature increases the rate of this redox change. The presence of Fe3+ is definitely harmful in the case of enteral administration. First, the activation energy of the oxidation process was quantified for both iron(II) sulphate forms. It was found that the monohydrate is oxidized four times more slowly than the monohydrate form. It follows from this that the monohydrate well resists short-lived high temperature. The thermostability tests on the monohydrate showed that samples, stored at high temperature and high relative humidity for a long time, oxidized only slowly. On the basis of all these findings, the monohydrate form may be suggested for melt technology.

  2. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    Science.gov (United States)

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  3. Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

    Directory of Open Access Journals (Sweden)

    Michael J Moser

    Full Text Available Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR. Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth and two-enzyme (MMLV RT/Taq RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

  4. 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.

  5. Overexpression and biochemical characterization of a thermostable phytase from Bacillus subtilis US417 in Pichia pastoris.

    Science.gov (United States)

    Hmida-Sayari, Aïda; Elgharbi, Fatma; Farhat, Ameny; Rekik, Hatem; Blondeau, Karine; Bejar, Samir

    2014-09-01

    The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50-65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100% of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.

  6. 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.

  7. Characterization of a thermostable endoglucanase produced by Isoptericola variabilis sp. IDAH9

    Directory of Open Access Journals (Sweden)

    Maryam Azizi

    2015-12-01

    Full Text Available Abstract This study aimed to isolate and evaluate the cellulase activity of cellulolytic bacteria in hot springs of Dehloran, Ilam province, Iran. Water and sludge samples were collected from the hot springs and the bacterial enrichment was performed in a medium containing rice barn and carboxymethyl cellulose (CMC. The cultures were incubated at 50 °C in aerobic conditions. The bacteria were isolated on CMC agar (1% medium. Cellulase assay of the isolates was measured by the evaluation of endoglucanase enzyme activity, which is also called as carboxymethyl cellulase (CMCase. The isolated thermotolerant bacteria were then identified and optimized for the production of CMCase. Moreover, stabilizing elements of the enzyme were identified with in silico approach. The chosen isolate was identified as Isoptericola variabilis sp. IDAH9. The identified strain produced the most thermostable CMCase at a concentration of 5.6 g/L of ammonium sulfate, 9 g/L CMCase or 12 g/L rice bran, 0/6% Tween-80, and 0.2% sucrose. The produced enzyme showed 80% of the residual activity after 1 h of incubation at 65 °C. In silico data indicated that the remaining residual activity was due to the redundant stabilizing elements in the protein structure. Consequently, I. variabilis can be isolated from the extreme environment and has a thermostable endoglucanase which may be used for various applications after studying them.

  8. A thermostable GH45 endoglucanase from yeast: impact of its atypical multimodularity on activity

    Directory of Open Access Journals (Sweden)

    Couturier Marie

    2011-12-01

    Full Text Available Abstract Background The gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1, designated PpCel45A, was identified in the Pichia pastoris GS115 genome. Results PpCel45A (full-length open reading frame, and three derived constructs comprising (i the catalytic module with its proximal CBM1, (ii the catalytic module only, and (iii the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C, which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five. Conclusions The present study has revealed the specific features of the first characterized endo β-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.

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

    Energy Technology Data Exchange (ETDEWEB)

    Longo, Gabriel S. [Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), CONICET, La Plata (Argentina); Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208 (United States); Olvera de la Cruz, Monica [Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208 (United States); Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Department of Chemistry, Northwestern University, Evanston, Illinois 60208 (United States); Szleifer, I., E-mail: igal@northwestern.edu [Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208 (United States); Department of Chemistry, Northwestern University, Evanston, Illinois 60208 (United States)

    2014-09-28

    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

  10. A New Topological Degree Theory for Perturbations of Demicontinuous Operators and Applications to Nonlinear Equations with Nonmonotone Nonlinearities

    Directory of Open Access Journals (Sweden)

    Teffera M. Asfaw

    2016-01-01

    Full Text Available Let X be a real reflexive locally uniformly convex Banach space with locally uniformly convex dual space X⁎. Let T:X⊇DT→2X⁎ be maximal monotone of type Γdϕ (i.e., there exist d≥0 and a nondecreasing function ϕ:0,∞→0,∞ with ϕ(0=0 such that 〈v⁎,x-y〉≥-dx-ϕy for all x∈DT, v⁎∈Tx, and y∈X,L:X⊃D(L→X⁎ be linear, surjective, and closed such that L-1:X⁎→X is compact, and C:X→X⁎ be a bounded demicontinuous operator. A new degree theory is developed for operators of the type L+T+C. The surjectivity of L can be omitted provided that RL is closed, L is densely defined and self-adjoint, and X=H, a real Hilbert space. The theory improves the degree theory of Berkovits and Mustonen for L+C, where C is bounded demicontinuous pseudomonotone. New existence theorems are provided. In the case when L is monotone, a maximality result is included for L and L+T. The theory is applied to prove existence of weak solutions in X=L20,T;H01Ω of the nonlinear equation given by ∂u/∂t-∑i=1N(∂/∂xiAix,u,∇u+Hλx,u,∇u=fx,t,  x,t∈QT;  ux,t=0,  x,t∈∂QT; and ux,0=ux,T,  x∈Ω, where λ>0, QT=Ω×0,T, ∂QT=∂Ω×0,T, Aix,u,∇u=∂/∂xiρx,u,∇u+aix,u,∇u  (i=1,2,…,N, Hλx,u,∇u=-λΔu+gx,u,∇u, Ω is a nonempty, bounded, and open subset of RN with smooth boundary, and ρ,ai,g:Ω¯×R×RN→R satisfy suitable growth conditions. In addition, a new existence result is given concerning existence of weak solutions for nonlinear wave equation with nonmonotone nonlinearity.

  11. Thermostable Eu(III)-nanorod luminophores with effective photosensitized energy transfer

    Energy Technology Data Exchange (ETDEWEB)

    Onodera, Hiromitsu [Laser Systems Inc, 1-4-1-10 Nijyuyonken, Nishi-ku, Sapporo, Hokkaido 063-0801 (Japan); Faculty School of Engineering, Hokkaido University, North-13 West-8, Kita-ku, Sapporo, Hokkaido 060-8628 (Japan); Nakajima, Ayako; Nakanishi, Takayuki; Fushimi, Koji [Faculty School of Engineering, Hokkaido University, North-13 West-8, Kita-ku, Sapporo, Hokkaido 060-8628 (Japan); Hasegawa, Yasuchika, E-mail: hasegaway@eng.hokudai.ac.jp [Faculty School of Engineering, Hokkaido University, North-13 West-8, Kita-ku, Sapporo, Hokkaido 060-8628 (Japan)

    2015-11-05

    The thermostable Eu(III)-doped nanorod aggregates composed of luminescent Eu(III) complexes and diphenylphosphinic acids (DPP) are reported. Prepared Eu(III)-nanorod aggregates [Eu(hfa){sub x}(TPPO){sub y}(DPP){sub z}]{sub n} (hfa: hexafluoroacetylacetionate, TPPO: triphenylphosphine oxide), Eu(III)-nanoparticle aggregates [Eu(hfa){sub x}(DPP){sub y}]{sub n}, and standard Eu(III)-nanoparticles [Eu(DPP){sub x}]{sub n} were characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM) measurements. The decomposition temperatures of [Eu(hfa){sub x}(DPP){sub y}]{sub n} and [Eu(hfa){sub x}(TPPO){sub y}(DPP){sub z}]{sub n} for thermogravimetric analysis (TGA) were found to be over > 500 °C. Photosensitized luminescence was successfully observed for Eu(III)-nanorod and Eu(III)-nanoparticle aggregates excited at the specific π–π* transition band for organic hfa ligands (λ = 340 nm), which is similar to the inter-ligand charge transfer (ILCT) band in the solid-state, were successfully observed. The photosensitized energy transfer efficiency of Eu(III)-nanorod aggregates of [Eu(hfa){sub x}(TPPO){sub y}(DPP){sub z}]{sub n} was estimated to be 91%. In this study, thermostable Eu(III)-nanorod aggregate luminophore composed of Eu(III) complexes and DPP joint compounds are demonstrated. - Graphical abstract: The thermostable Eu(III)-doped nanorod aggregates composed of luminescent Eu(III) complexes and diphenylphosphinic acids (DPP) are reported. Prepared Eu(III)-nanorod aggregates [Eu(hfa){sub x}(TPPO){sub y}(DPP){sub z}]{sub n} (hfa: hexafluoroacetylacetionate, TPPO: triphenylphosphine oxide), Eu(III)-nanoparticle aggregates [Eu(hfa){sub x}(DPP){sub y}]{sub n}, and standard Eu(III)-nanoparticles [Eu(DPP){sub x}]{sub n} were characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM) measurements. The decomposition temperatures of [Eu(hfa){sub x}(DPP){sub y}]{sub n} and [Eu(hfa){sub x}(TPPO){sub y}(DPP){sub z}]{sub n} for

  12. Temperature Dependence of the Stability of Ion Pair Interactions, and its Implications on the Thermostability of Proteins from Thermophiles

    Indian Academy of Sciences (India)

    SWETHA BIKKINA; AGASTYA P BHATI; SILADITYA PADHI; U DEVA PRIYAKUMAR

    2017-03-01

    An understanding of the determinants of the thermal stability of thermostable proteins is expected to enable design of enzymes that can be employed in industrial biocatalytic processes carried out at high temperatures. A major factor that has been proposed to stabilize thermostable proteins is the high occurrenceof salt bridges. The current study employs free energy calculations to elucidate the thermodynamics of the formation of salt bridge interactions and the temperature dependence, using acetate and methylguanidium ionsas model systems. Three different orientations of the methylguanidinium approaching the carboxylate grouphave been considered for obtaining the free energy profiles. The association of the two ions becomes more favorable with an increase in temperature. The desolvation penalty corresponding to the association of the ionpair is the lowest at high temperatures. The occurrence of bridging water molecules between the ions ensures that the ions are not fully desolvated, and this could provide an explanation for the existence of internal watermolecules in thermostable proteins reported recently. The findings provide a detailed picture of the interactions that make ion pair association at high temperatures a favorable process, and reaffirm the importance of saltbridges in the design of thermostable proteins.

  13. ADOPTING SELECTED HYDROGEN BONDING AND IONIC INTERACTIONS FROM ASPERGILLUS FUMIGATUS PHYTASE STRUCTURE IMPROVES THE THERMOSTABILITY OF ASPERGILLUS NIGER PHYA PHYTASE

    Science.gov (United States)

    Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatu...

  14. Maltose effects on barley malt diastatic power enzyme activity and thermostability at high isothermal mashing temperature: I. ß-amylase

    Science.gov (United States)

    The hypothesis that maltose would increase the thermostability of barley malt beta-amylase activity during isothermal mashing was tested at 68, 73 and 78°C and compared to isothermal mashing at 63°C. Finely ground malts of the two-row cultivar Harrington and the six-row cultivar Morex were incubated...

  15. Maltose effects on barley malt diastatic power enzyme activity and thermostability at high isothermal mashing temperature: II. Alpha-amylase

    Science.gov (United States)

    Maltose, the primary product of starch degradation during mashing, has the potential as a compatible solute to affect the activity of and increase the thermostability of barley malt alpha-amylase activity at high temperatures used in mashing and temperatures above those normally used in mashing. To ...

  16. Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface.

    Science.gov (United States)

    Han, Zhen-lin; Han, Shuang-yan; Zheng, Sui-ping; Lin, Ying

    2009-11-01

    To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60 degrees C for 5 h and increased synthetic activity in organic solvent at 60 degrees C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.

  17. 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.

  18. Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1.7 A resolution.

    Science.gov (United States)

    Hwang, K Y; Song, H K; Chang, C; Lee, J; Lee, S Y; Kim, K K; Choe, S; Sweet, R M; Suh, S W

    1997-04-30

    alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of alpha-1, 4-glucosidic linkages of starch components, glycogen, and various oligosaccharides. Thermostable alpha-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose. Thermostable alpha-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability. This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins. The three-dimensional structure of thermostable alpha-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography. The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with F0 > 2 sigma F0 between 8.0 and 1.7 A resolution, with root mean square deviations of 0.013 A from ideal bond lengths and 1.72 degrees from ideal bond angles. The final model consists of 469 amino acid residues and 294 water molecules. Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192. Like other alpha-amylases, the polypeptide chain folds into three distinct domains. The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (beta/alpha)8-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between the third beta-strand and the third alpha-helix of domain A. The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel beta-barrel. Neither calcium ion nor chloride ion is located near the active site. This study reveals the architecture of the thermostable alpha-amylase from Bacillus licheniformis. By homology with other alpha-amylases, important active site residues can be identified as Asp231, Glu261, and Asp

  19. Improving the thermostability and enhancing the Ca(2+) binding of the maltohexaose-forming α-amylase from Bacillus stearothermophilus.

    Science.gov (United States)

    Li, Zhu; Duan, Xuguo; Wu, Jing

    2016-03-20

    The thermostability of the maltohexaose-forming α-amylase from Bacillus stearothermophilus (AmyMH) without added Ca(2+) was improved through structure-based rational design in this study. Through comparison of a homologous model structure of AmyMH with the crystal structure of the thermostable α-amylase from Bacillus licheniformis, Ser242, which located at the beginning of fourth α-helix of the central (β/α)8 barrel was selected for mutation to improve thermostability. In addition, an amide-containing side chain (Asn193) and a loop in domain B (ΔIG mutation), which have been proven to be important for thermostability in corresponding position of other α-amylases, were also investigated. Five mutants carrying the mutations ΔIG, N193F, S242A, ΔIG/N193F, and ΔIG/N193F/S242A were generated and their proteins characterized. The most thermostable mutant protein, ΔIG/N193F/S242A, exhibited a 26-fold improvement in half-life at 95°C compared to the wild-type enzyme without added Ca(2+). Mutant ΔIG/N193F/S242A also exhibited substantially better activity and stability in the presence of the chelator EDTA, demonstrating enhanced Ca(2+) binding. These results suggest that mutant ΔIG/N193F/S242A has potential for use in the industrial liquefaction of starch. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. 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

  1. Characterization of a Novel Thermostable Oligopeptidase from Geobacillus thermoleovorans DSM 15325.

    Science.gov (United States)

    Jasilionis, Andrius; Kuisiene, Nomeda

    2015-07-01

    A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His(400)-Glu(401)-X-XHis (404)). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepFBa from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0-8.0, at pH 7.3 and 40°C, showing the Km, Vmax, and kcat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10(-6) M, 2.65 ± 0.03 × 10(-3) micrometer/min, and 5.99 ± 0.07 s(-1), respectively. Peptidase remained stable at a broad pH range of 5.0-8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50°C and 60°C, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60°C for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.

  2. Non-monotonic course of protein solubility in aqueous polymer-salt solutions can be modeled using the sol-mxDLVO model.

    Science.gov (United States)

    Herhut, Marcel; Brandenbusch, Christoph; Sadowski, Gabriele

    2016-02-01

    Protein purification is often performed using cost-intensive chromatographic steps. To discover economic alternatives (e.g., crystallization), knowledge on protein solubility as a function of temperature, pH, and additives in solution as well as their concentration is required. State-of-the-art models for predicting protein solubility almost exclusively consider aqueous salt systems, whereas "salting-in" and "salting-out" effects induced by the presence of an additional polymer are not considered. Thus, we developed the sol-mxDLVO model. Using this newly developed model, protein solubility in the presence of one salt and one polymer, especially the non-monotonic course of protein solubility, could be predicted. Systems considered included salts (NaCl, Na-p-Ts, (NH(4))(2) SO(4)) and the polymer polyethylene glycol (MW: 2000 g/mol, 12000 g/mol) and proteins lysozyme from chicken egg white (pH 4 to 5.5) and D-xylose ketol-isomerase (pH 7) at 298.15 K. The results show that by using the sol-mxDLVO model, protein solubility in polymer-salt solutions can be modeled in good agreement with the experimental data for both proteins considered. The sol-mxDLVO model can describe the non-monotonic course of protein solubility as a function of polymer concentration and salt concentration, previously not covered by state-of-the-art models.

  3. Non-monotonic magnetoresistance in an AlGaN/GaN high-electron-mobility transistor structure in the ballistic region

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Y. T.; Woo, T. P.; Lo, S. T. [National Taiwan University, Taipei, Taiwan (China); Kim, G. H. [Sungkyunkwan University, Suwon (Korea, Republic of); Liang, C. T. [Sungkyunkwan University, Suwon (Korea, Republic of); National Taiwan University, Taipei, Taiwan (China)

    2014-05-15

    In this report, we will discuss the nonmonotonic magnetoresistance (MR) in an AlGaN/GaN high-electron-mobility transistor (HEMT) in a perpendicular magnetic field B in the ballistic region (k{sub B}T τ/h > 1) and in the weakly-disordered limit (k{sub F}l = 159 >> 1), where k{sub B}, T, τ , h, k{sub F} , and l represent the Boltzmann constant, temperature, elastic scattering time, reduced Planck constant, Fermi wave vector and mean free path, respectively. The MR shows a local maximum between the weak localization (WL) and the Shubnikov-de Haas regions. In the low magnetic field regime, the quantum correction to the conductivity is proportional to T{sup -3/2}, which is consistent with a recent theory [T. A. Sedrakyan, and M. E. Raikh, Phys. Rev. Lett. 100, 106806 (2008)]. According to our results, as the temperature is increased, the position of the MR maximum in B increases. These results cannot be explained by present theories. Moreover, in the high-magnetic-field regime, neither the magnetic and nor the temperature dependences of the observed MR is consistent with present theories. We, therefore, suggest that while some features of the observed nonmonotonic MR can be successfully explained, further experimental and theoretical studies are necessary to obtain a thorough understanding of the MR effects.

  4. Structural Adaptation of a Thermostable Biotin-binding Protein in a Psychrophilic Environment

    Science.gov (United States)

    Meir, Amit; Bayer, Edward A.; Livnah, Oded

    2012-01-01

    Shwanavidin is an avidin-like protein from the marine proteobactrium Shewanella denitrificans, which exhibits an innate dimeric structure while maintaining high affinity toward biotin. A unique residue (Phe-43) from the L3,4 loop and a distinctive disulfide bridge were shown to account for the high affinity toward biotin. Phe-43 emulates the function and position of the critical intermonomeric Trp that characterizes the tetrameric avidins but is lacking in shwanavidin. The 18 copies of the apo-monomer revealed distinctive snapshots of L3,4 and Phe-43, providing rare insight into loop flexibility, binding site accessibility, and psychrophilic adaptation. Nevertheless, as in all avidins, shwanavidin also displays high thermostability properties. The unique features of shwanavidin may provide a platform for the design of a long sought after monovalent form of avidin, which would be ideal for novel types of biotechnological application. PMID:22493427

  5. Structure and Function of Thermostable Direct Hemolysin (TDH) from Vibrio Parahaemolyticus

    Science.gov (United States)

    Hashimoto, Hiroshi; Yamane, Tsutomu; Ikeguchi, Mitsunori; Nakahira, Kumiko; Yanagihara, Itaru

    Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic food-borne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside the pore. Molecular dynamics (MD) simulations suggested that water molecules permeate freely through the central and side channel pores. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Pendru eRaghunath

    2015-01-01

    Full Text Available 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 haemolysin (TDH or TDH-related haemolysin (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.

  8. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction.

    Science.gov (United States)

    Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F

    2017-01-01

    Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.

  9. Characterization of the peroxidase activity of CYP119, a thermostable P450 from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Rabe, Kersten S; Kiko, Kathrin; Niemeyer, Christof M

    2008-02-15

    We report the cloning, expression, and purification of CYP119, a thermostable enzyme previously thought to derive from Sulfolobus solfataricus. Sequence analysis suggested that, in contrast to the conclusions of earlier studies, the enzyme stems from the closely related Sulfolobus acidocaldarius, and we were indeed able to clone the gene from the genomic DNA of this organism. For the first time, we report here on the peroxidase activity of this enzyme and the optimization of the associated reaction parameters. The optimized reaction conditions were then applied to the biocatalytic epoxidation of styrene. The values obtained for k(cat) (78.2+/-20.6 min(-1)) and K(M) (9.2+/-4.3 mM) indicated an approximately 100-fold increased catalytic activity over previously reported results.

  10. THERMOSTABILITY OF ANTIOXIDANT AND DETERIORATIVE ENZYMES FROM SOURSOP AND CASHEW APPLE JUICES

    Directory of Open Access Journals (Sweden)

    MARCELA CRISTINA RABELO

    2016-01-01

    Full Text Available ABSTRACT This work aimed to evaluate the thermostability of antioxidant (superoxide dismutase - SOD, catalase - CAT and ascorbate peroxidase - APX and deteriorative (guaiacol peroxidase - G-POD, polyphenoloxidase - PPO, pectin-methylesterase - PME and polygalacturonase - PG enzymes from soursop and cashew apple juices. Juices were prepared homogenizing ripe fruit pulps and submitting to different thermal treatments (55, 65, 75, 85 and 95°C for different time period (1, 3, 5, 10, 15, 20 and 30 min then enzymatic activities were evaluated. The treatments at 55°C for soursop juice and at 75°C for cashew apple juice presented the best results, considering the low residual activities for deteriorative enzymes and the retained activity of SOD. Our results suggest appropriate technological condition to thermal processing from soursop and cashew apple juice whereby ensuring quality beyond their functionality.

  11. Thermo-stability of ultra-fine non-equilibrium microstructures

    Institute of Scientific and Technical Information of China (English)

    Huibin Wu; Shanwu Yang; Aimin Guo; Shaoqiang Yuan; Chengjia Shang; Xinlai He

    2004-01-01

    The evolution of the microstructures and hardness of a bainitic plate steel during tempering at 650℃ has been investigated.The steel was manufactured by RPC (relaxation-precipitation controlling phase transformation) technique. A part of the plate was reheated to 930℃ and held for 1 h before quenched into water (RQ). No obvious change was detected by means of optical microscopy in the RPC steel tempering for 0.5 h, while dislocation cells were formed inside the bainite laths, accompanied by an obvious drop of hardness. The bainite laths started to coalesce in some regions, but the sample hardness kept nearly constant during tempering from 1 to 7 h. With further tempering, polygonal ferrite was formed in local regions while the hardness decreased dramatically. The RQ samples softened faster during tempering and finally transformed into the polygonal ferrite completely. These results indicate that the thermo-stability of fine non-equilibrium microstructures is tightly related to their history.

  12. Thermostable β-galactosidases for the synthesis of human milk oligosaccharides

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Nyffenegger, Christian; Mikkelsen, Jørn Dalgaard;

    2016-01-01

    T; Gal-β(1,4)-GlcNAc-β(1,3)-Gal-β(1,4)-Glc). In order to reduce reaction times and be able to work at temperatures, which are less welcoming to microbial growth, the current study investigates the possibility of using thermostable β-galactosidases for synthesis of LNnT and N-acetyllactosamine (Lac......NAc, resulting in 5-6 times higher reaction yields and significantly shorter reaction times.......Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk. Using lactose as a cheap donor, some β-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto- N-neotetraose (LNn...

  13. A highly efficient and thermostable α-amylase from soya bean seeds.

    Science.gov (United States)

    Prakash, Om; Jaiswal, Nivedita

    2010-12-01

    The α-amylase from soya bean seeds was purified by affinity precipitation, resulting in approx. 20-fold purification with approx. 84% recovery. The purified α-amylase had an optimum pH of 5.5, optimum temperature of 75 °C, Arrhenius energy of activation of 6.03 kcal/mol (1 kcal≈4.184 kJ) and a Km of 2.427 mg/ml (starch substrate). The enzyme had maximum substrate specificity for starch. Among the various metal ions tested, Co2+ and Mn2+ were found to be strong activators. The effect of thiol group modifying agents showed that the thiols of soya bean α-amylase are not directly involved in catalysis. The thermostability of the enzyme makes it suitable for starch liquefaction and the detergent industry respectively.

  14. Two-dimensional self-organization of the light-harvesting polypeptides/BChl a complex into a thermostable liposomal membrane

    NARCIS (Netherlands)

    Iida, K; Kiriyama, H; Fukai, A; Konings, WN; Nango, M

    2001-01-01

    The detergent-isolated light-harvesting polypeptide (LR)/bacteriochlorophyll alpha (BChl alpha) complex from the photosynthetic bacterium Rhodospirillum rubrum was organized in thermostable liposomal membranes comprising membrane-spanning tetraether lipids from Sulfolobus acidocaldarius to develop a

  15. Engineering a more thermostable blue light photo receptor Bacillus subtilis YtvA LOV domain by a computer aided rational design method.

    Directory of Open Access Journals (Sweden)

    Xiangfei Song

    Full Text Available The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the T m of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.

  16. CYCLODEXTRIN FORMATION BY THE THERMOSTABLE ALPHA-AMYLASE OF THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1 AND RECLASSIFICATION OF THE ENZYME AS A CYCLODEXTRIN GLYCOSYLTRANSFERASE

    NARCIS (Netherlands)

    WIND, RD; LIEBL, W; BUITELAAR, RM; PENNINGA, D; SPREINAT, A; DIJKHUIZEN, L; BAHL, H

    Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase). Product analysis after incubation of the enzyme

  17. Purification and properties of thermostable tryptophanase from an obligately symbiotic thermophile, Symbiobacterium thermophilum.

    Science.gov (United States)

    Suzuki, S; Hirahara, T; Horinouchi, S; Beppu, T

    1991-12-01

    A thermostable tryptophanase was extracted from a thermophilic bacterium, Symbiobacterium thermophilum strain T, which is obligately symbiotic with the thermophilic Bacillus strain S. The enzyme was purified 21-fold to homogeneity with 19% recovery by a series of chromatographies using anion-exchange, hydroxylapatite, hydrophobic interaction, and MonoQ anion-exchange columns. The molecular weight of the purified enzyme was estimated to be approximately 210,000 by gel filtration, while the molecular weight of its subunit was 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits. The isoelectric point of the enzyme was 4.9. The tryptophanase was stable to heating at 65 degrees C for 20 min and the optimum temperature for the enzyme activity for 20 min reaction was 70 degrees C. The optimum pH was 7.0. The NH2-terminal amino acid sequence of this tryptophanase shows similarity to that of Escherichia coli K-12, despite a great difference in the thermostability of these two enzymes. The purified enzyme catalyzed the degradation (alpha, beta-elimination) of L-tryptophan into indole, pyruvate, and ammonia in the presence of pyridoxal-5'-phosphate. The Km value for L-tryptophan was 1.47 mM. 5-Hydroxy-L-tryptophan, 5-methyl-DL-tryptophan, L-cysteine, S-methyl-L-cysteine, and L-serine were also used as substrates and converted to pyruvate. The reverse reaction of alpha, beta-elimination of this tryptophanase produced L-tryptophan from indole and pyruvate in the presence of a high concentration of ammonium acetate.

  18. Thermostability of photosynthesis in two new chlorophyll b-less rice mutants

    Institute of Scientific and Technical Information of China (English)

    LIN; Zhifang; PENG; Changlian; Xu; Xinlan; LIN; Guizhu; ZH

    2005-01-01

    Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of Fo rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H2O2 accumulation in leaves were found in the two Chl b-less mutants, whereas in the wild type plant, less H2O2 was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and Fv/Fm was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature.

  19. The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design.

    Science.gov (United States)

    Kim, Su Jin; Lee, Jeong Ah; Joo, Jeong Chan; Yoo, Young Je; Kim, Yong Hwan; Song, Bong Keun

    2010-01-01

    Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild-type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins.

  20. Expression and Characterization of a Thermostable Acyl-peptide Releasing Enzyme ST0779 from Sulfolobus tokodaii

    Institute of Scientific and Technical Information of China (English)

    LI Rong; ZHANG Fei; CAO Shu-gui; XIE Gui-qiu; GAO Ren-jun

    2012-01-01

    Acyl-peptide releasing enzyme(AARE) belongs to a serine peptidase family and catalyzes the NH2-terminal hydrolysis of Nα-acylpeptides to release Nα-acylated amino acids.ORF0779(ORF=open reading frame)from thermophilic archaea Sulfolobus tokodaii(ST0779) was cloned and expressed in E.coti BL21 and the expressed protein was identified as a thermostable AARE.The target protein could be optimally overexpressed in E.coli at 30 ℃ for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside(IPTG).The crude enzyme was heated at 70 ℃ for 30 min,and then the target protein could account for above 40% of the total protein.The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity.The optimal temperature and pH for ST0779 were 70 ℃and 8.0 when Ac-Ala3 was used as substrate.The half-life of the enzyme(0.2 mg/mL) at 90 ℃ was about 16 h,indicating that the enzyme exhibits a favorable thermostability.The activity of ST0779 could still remain over 85% after being treated at 25 ℃ in different buffers with pH range from 6.0 to10.0 for 24 h,which indicates ST0779 is stable in neutral or slight alkali environment.Under neutral or slightly alkali conditions,the enzyme exhibits really high catalytic efficiency against acyl-peptide,and the optimal substrate is Ac-Ala3.Most metal ions have no inhibition effect on the activity of ST0779,while 4% activity of ST0779 is inhibited in the presence of K+.This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.

  1. Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption.

    Science.gov (United States)

    Mehta, Deepika; Satyanarayana, T

    2014-05-01

    The gene (1,542 bp) encoding thermostable Ca(2+)-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t 1/2 of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir-Hinshelwood kinetic analysis in terms of equilibrium parameter (K R) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t 1/2 of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs.

  2. 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.

  3. A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP

    Directory of Open Access Journals (Sweden)

    Yogesh eChander

    2014-08-01

    Full Text Available Meeting the goal of providing point of care (POC tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations . Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular

  4. Puriifcation and characterization of a thermostable glutamate dehydrogenase from a thermophilic microorganism from Deception Island, Antarctica

    Institute of Scientific and Technical Information of China (English)

    Patricio A Flores M; Freddy Boehmwald; Jenny M Blamey A

    2016-01-01

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate toα-ketoglutarate and ammonium ions. Currently the determination of ammonium and glutamate is carried out using a bovine GDH enzyme, which lacks optimal thermostability for long term storage at room temperature. From samples of Deception Island, Antarctica, we obtained the thermophilic bacteria PID 15 belonging to theBacillus genera with high GDH speciifc activity. This new enzyme exhibited NAD+dependent activity and no activity was observed when NADP+was used. This enzyme shows a speciifc activity of 4.7 U∙mg-1 for the oxidative deamination reaction and 15.4 U∙mg-1 for the reduction ofα-ketoglutarate. This enzyme has an optimum temperature of 65°C and pH of 8.5 for the oxidative deamination. For the reduction ofα-ketoglutarate, the optimum temperature is 60°C, with a pH of 8.0. One of the most important characteristics of this enzyme is its ability to retain more than 60% of its activity when it is incubated for 8 h at 65°C. The enzyme is also able to retain full activity when it is incubated for 48 d at 4°C and over 80% of its activity when it is incubated at 25°C. Characterization of its kinetics suggests that it primarily catalyzes the formation of α-ketoglutarate. This enzyme has an important biological role in the catabolism of glutamate and may have some interesting biotechnological applications based on its thermostable properties.

  5. 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.

  6. Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate.

    Directory of Open Access Journals (Sweden)

    Dimitra Zarafeta

    Full Text Available With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.

  7. Purification and Characterization of a Novel Thermostable Chitinase from Thermomyces lanuginosus SY2 and Cloning of Its Encoding Gene

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 min. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable ehitinases so far isolated in fungi. Ca2+, Ba2+, Na2+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, Hg2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplieation. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.

  8. Cloning, expression and characteristics of a novel alkalistable and thermostable xylanase encoding gene (Mxyl retrieved from compost-soil metagenome.

    Directory of Open Access Journals (Sweden)

    Digvijay Verma

    Full Text Available BACKGROUND: The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. METHODOLOGY/PRINCIPAL FINDINGS: Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1 was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids displayed homology with glycosyl hydrolase (GH family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3. The recombinant xylanase (rMxyl exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. CONCLUSION/SIGNIFICANCE: This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues.

  9. Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

    Science.gov (United States)

    2012-04-26

    12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS prolidase, organophosphate , OP nerve agent, thermostable enzyme, Pyrococcus furiosus...Singh, 2009). Annually, there are an estimated 3 million poisonings and 300,000 human deaths owing to OP compounds (Singh, 2009). There is a need... organophosphate anhydrolase/prolidase. Biochemistry, 49, 547-59. Wang, Q., Sun, M., Zhang, H. & Huang, C. 1998. Purification and properties of soman

  10. 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.

  11. Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: thermodynamics and temperature dependence.

    Science.gov (United States)

    Huang, Rong; Yang, Qingjun; Feng, Hong

    2015-02-01

    Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (kcat/Km) in a wide range of temperatures, mainly due to an increase of k1 (substrate diffusion) and k2 (acylation) for V149I and of k2 and k3 (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (ΔGa°) of activation for thermal denaturation may govern the thermostability. The value of ΔGa° is increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.

  12. 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 (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.

  13. Coexistence and local μ-stability of multiple equilibrium points for memristive neural networks with nonmonotonic piecewise linear activation functions and unbounded time-varying delays.

    Science.gov (United States)

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

    2016-12-01

    In this paper, the coexistence and dynamical behaviors of multiple equilibrium points are discussed for a class of memristive neural networks (MNNs) with unbounded time-varying delays and nonmonotonic piecewise linear activation functions. By means of the fixed point theorem, nonsmooth analysis theory and rigorous mathematical analysis, it is proven that under some conditions, such n-neuron MNNs can have 5(n) equilibrium points located in ℜ(n), and 3(n) of them are locally μ-stable. As a direct application, some criteria are also obtained on the multiple exponential stability, multiple power stability, multiple log-stability and multiple log-log-stability. All these results reveal that the addressed neural networks with activation functions introduced in this paper can generate greater storage capacity than the ones with Mexican-hat-type activation function. Numerical simulations are presented to substantiate the theoretical results. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. 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.

  15. Thermostability of Well-Ordered HIV Spikes Correlates with the Elicitation of Autologous Tier 2 Neutralizing Antibodies.

    Directory of Open Access Journals (Sweden)

    Yu Feng

    2016-08-01

    Full Text Available In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were "stress-tested" at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (Tm. Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design.

  16. 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.

  17. A Thermostable Crude Endoglucanase Produced by Aspergillus fumigatus in a Novel Solid State Fermentation Process Using Isolated Free Water

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    Abdul A. N. Saqib

    2012-01-01

    Full Text Available Aspergillus fumigatus was grown on chopped wheat straw in a solid state fermentation (SSF process carried out in constant presence of isolated free water inside the fermentation chamber. The system allowed maintaining a constant vapor pressure inside the fermentor throughout the fermentation process. Crude endoglucanase produced by A. fumigatus under such conditions was more thermostable than previously reported enzymes of the same fungal strain which were produced under different conditions and was also more thermostable than a number of other previously reported endoglucanases as well. Various thermostability parameters were calculated for the crude endoglucanase. Half lives (T1/2 of the enzyme were 6930, 866, and 36 min at 60°C, 70°C, and 80°C, respectively. Enthalpies of activation of denaturation (ΔHD* were 254.04, 253.96, and 253.88 K J mole−1, at 60°C, 70°C and 80°C, respectively, whereas entropies of activation of denaturation (ΔSD* and free energy changes of activation of denaturation (ΔGD* were 406.45, 401.01, and 406.07 J mole−1 K−1 and 118.69, 116.41, and 110.53 K J mole−1 at 60°C, 70°C and 80°C, respectively.

  18. Investigation of thermostable metalloproteins in Perna perna mussels from differentially contaminated areas in Southeastern Brazil by bioanalytical techniques.

    Science.gov (United States)

    Lavradas, Raquel Teixeira; Rocha, Rafael Christian Chávez; Saint' Pierre, Tatiana Dillenburg; Godoy, José Marcus; Hauser-Davis, Rachel Ann

    2016-03-01

    Metallomic studies regarding environmental contamination by metals are of value in elucidating metal uptake, trafficking, accumulation and metabolism in biological systems. Many proven bioindicator species, such as bivalves, have not yet, however, been well-characterized regarding their metalloprotein expression in response to environmental contaminants. In this context, the aim of the present study was to investigate metalloprotein expressions in the thermostable protein fraction of muscle tissue and digestive glands from mussels (Perna perna) from three differentially metal-contaminated sites in Southeastern Brazil in comparison with a reference site. The thermostable protein fractions were analyzed by SDS-PAGE and SEC-HPLC-ICP-MS. Metal content was also determined in both the crude and the purified extracts. Several inter-organ differences were observed, which is to be expected, while inter-site differences regarding thermostable protein content were also verified, indicating accumulation of these elements in muscle tissue and digestive glands and disruption of homeostasis of essential elements, with detoxification attempts by metal-bound proteins, since all metalloproteins present in both matrices eluted bound to at least one non-essential metal. These results are also noteworthy with regard to the adopted reference site, that also seems to be contaminated by toxic metals.

  19. Characterization of a thermostable raw-starch hydrolyzing α-amylase from deep-sea thermophile Geobacillus sp.

    Science.gov (United States)

    Jiang, Tao; Cai, Menghao; Huang, Mengmeng; He, Hao; Lu, Jian; Zhou, Xiangshan; Zhang, Yuanxing

    2015-10-01

    A deep-sea thermophile, Geobacillus sp. 4j, was identified to grow on starch and produce thermostable amylase. N-terminally truncated form of Geobacillus sp. 4j α-amylase (Gs4j-amyA) was fused at its N-terminal end with the signal peptide of outer membrane protein A (OmpA) of Escherichia coli. The enzyme was over-expressed in E. coli BL21 with a maximum extracellular production of 130U/ml in shake flask. The yield of the transformant increased 22-fold as compared with that of the wild strain. The recombinant enzyme purified to apparent homogeneity by metal-affinity chromatography, exhibited a molecular mass of 62kDa. It displayed the maximal activity at 60-65°C and pH 5.5. Its half-life (t1/2) at 80°C was 4.25h with a temperature deactivation energy of 166.3kJ/mol. Compared to three commonly used commercial α-amylases, the Gs4j-amyA exhibited similar thermostable performance to BLA but better than BAA and BSA. It also showed a universally efficient raw starch hydrolysis performance superior to commercial α-amylases at an acidic pH approaching nature of starch slurry. As a new acidic-resistant thermostable α-amylase, it has the potential to bypass the industrial gelatinization step in raw starch hydrolysis.

  20. Solid bioneedle-delivered influenza vaccines are highly thermostable and induce both humoral and cellular immune responses.

    Directory of Open Access Journals (Sweden)

    Peter C Soema

    Full Text Available The potential of bioneedles to deliver influenza vaccines was investigated. Four influenza vaccine formulations were screened to determine the optimal formulation for use with bioneedles. The stability of the formulations after freeze-drying was checked to predict the stability of the influenza vaccines in the bioneedles. Subunit, split, virosomal and whole inactivated influenza (WIV vaccine were formulated and lyophilized in bioneedles, and subsequently administered to C57BL/6 mice. Humoral and cellular immune responses were assessed after vaccination. The thermostability of lyophilized vaccines was determined after one-month storage at elevated temperatures. Bioneedle influenza vaccines induced HI titers that are comparable to those induced by intramuscular WIV vaccination. Delivery by bioneedles did not alter the type of immune response induced by the influenza vaccines. Stability studies showed that lyophilized influenza vaccines have superior thermostability compared to conventional liquid vaccines, and remained stable after one-month storage at 60°C. Influenza vaccines delivered by bioneedles are a viable alternative to conventional liquid influenza vaccines. WIV was determined to be the most potent vaccine formulation for administration by bioneedles. Lyophilized influenza vaccines in bioneedles are independent of a cold-chain, due to their increased thermostability, which makes distribution and stockpiling easier.

  1. Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization.

    Science.gov (United States)

    Sharma, Nirmal Kant; Thakur, Shikha; Thakur, Neerja; Savitri; Bhalla, Tek Chand

    2016-03-01

    Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg(2+), Ag(+) and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

  2. Thermostability of photosystem I trimers and monomers from the cyanobacterium Thermosynechococcus elongatus

    Science.gov (United States)

    Shubin, Vladimir V.; Terekhova, Irina V.; Bolychevtseva, Yulia V.; El-Mohsnawy, Eithar; Rögner, Matthias; Mäntele, Werner; Kopczak, Marta J.; Džafić, Enela

    2017-05-01

    The performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermostability of photosystem I (PSI) complexes Terasaki et al. (2007), Iwuchukwu et al. (2010), Kothe et al. (2013) . To assess the thermostability of PSI complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus heating induced perturbations on the level of secondary structure of the proteins were studied. Changes were monitored by Fourier transform infrared (FT-IR) spectra in the mid-IR region upon slow heating (1 °C per minute) of samples in D2O phosphate buffer (pD 7.4) from 20 °C to 100 °C. These spectra showed distinct changes in the Amide I region of PSI complexes as a function of the rising temperature. Absorbance at the Amide I maximum of PSI monomers (centered around 1653 cm- 1), gradually dropped in two temperature intervals, i.e. 60-75 and 80-90 °C. In contrast, absorbance at the Amide I maximum of PSI trimers (around 1656 cm- 1) dropped only in one temperature interval 80-95 °C. The thermal profile of the spectral shift of α-helices bands in the region 1656-1642 cm- 1 confirms the same two temperature intervals for PSI monomers and only one interval for trimers. Apparently, the observed absorbance changes at the Amide I maximum during heating of PSI monomers and trimers are caused by deformation and unfolding of α-helices. The absence of absorbance changes in the interval of 20-65 °C in PSI trimers is probably caused by a greater stability of protein secondary structure as compared to that in monomers. Upon heating above 80 °C a large part of α-helices both in trimers and monomers converts to unordered and aggregated structures. Spectral changes of PSI trimers and monomers heated up to 100 °C are irreversible due to protein denaturation and non-specific aggregation of complexes leading to new absorption bands at 1618-1620 cm- 1. We propose that monomers shield the denaturation sensitive sides at the

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

    Energy Technology Data Exchange (ETDEWEB)

    Barros, Bráulio Silva, E-mail: brauliobarros@ect.ufrn.br [School of Science and Technology, Federal University of Rio Grande do Norte (UFRN), Campus Universitário Lagoa Nova, 59078-970 Natal, RN (Brazil); Chojnacki, Jaroslaw [Department of Inorganic Chemistry, Chemical Faculty, Gdansk University of Technology, G. Narutowicza 11/12, 80-233 Gdansk (Poland); Macêdo Soares, Antonia Alice; Kulesza, Joanna; Lourenço da Luz, Leonis; Júnior, Severino Alves [Department of Fundamental Chemistry, Federal University of Pernambuco (UFPE), Av. Prof. Moraes Rego, 1235 – Cidade Universitária, 50670-901 Recife, PE (Brazil)

    2015-07-15

    The reaction between Zn(NO{sub 3}){sub 2}·6H{sub 2}O or Zn(CH{sub 3}COO){sub 2}·2H{sub 2}O and isophthalic acid (1,3-H{sub 2}bdc) in the presence of benzimidazole (Hbzim) in dimethylformamide (DMF)/ethanol (EtOH)/H{sub 2}O solvent mixture at room temperature yielded two structurally different coordination polymers: [Zn{sub 2}(1,3-bdc){sub 2}(Hbzim){sub 2}] (1) and [Zn{sub 2}(1,3-bdc)(bzim){sub 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{sub 2}.2H{sub 2}O and 1,3-H{sub 2}bdc at 90 °C resulted in the formation of (3), a 3D coordination polymer Zn(HCOO){sub 3}(Me{sub 2}NH{sub 2}{sup +}). 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.

  4. Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

    Science.gov (United States)

    Wang, Kun; Tian, Jian; Turunen, Ossi; Huang, Huoqing; Shi, Pengjun; Hua, Huifang; Wang, Caihong; Wang, Shuanghe

    2014-01-01

    Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability. PMID:24463976

  5. In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria.

    Science.gov (United States)

    Tajima, Kenji; Han, Xuerong; Hashimoto, Yoshiki; Satoh, Yasuharu; Satoh, Toshifumi; Taguchi, Seiichi

    2016-12-01

    Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. 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.

  7. Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680.

    Science.gov (United States)

    Wang, Zong-Da; Wang, Bao-Juan; Ge, Ya-Dong; Pan, Wei; Wang, Jie; Xu, Lei; Liu, Ai-Min; Zhu, Guo-Ping

    2011-03-01

    A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

  8. 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

    2014-07-31

    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.

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

    Science.gov (United States)

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

    2012-01-01

    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: C2221, with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P41212, 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. PMID:22442220

  10. Hydrolysis of isoflavone glycosides by a thermostable β-glucosidase from Pyrococcus furiosus.

    Science.gov (United States)

    Yeom, Soo-Jin; Kim, Bi-Na; Kim, Yeong-Su; Oh, Deok-Kun

    2012-02-15

    The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.

  11. 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.

  12. 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.

  13. A thermostable bacterial cocaine esterase rapidly eliminates cocaine from brain in nonhuman primates.

    Science.gov (United States)

    Howell, L L; Nye, J A; Stehouwer, J S; Voll, R J; Mun, J; Narasimhan, D; Nichols, J; Sunahara, R; Goodman, M M; Carroll, F I; Woods, J H

    2014-07-01

    A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a K(M) of 1.33+0.085 μM. In vivo evaluation of CocE has shown protection against convulsant and lethal effects of cocaine in rodents, confirming the therapeutic potential of CocE against cocaine overdose. However, the current study is the first to evaluate the effects of CocE on cocaine brain levels. Positron emission tomogrpahy neuroimaging of [(11)C]cocaine was used to evaluate the time course of cocaine elimination from brain in the presence and absence of CocE in nonhuman primates. Systemic administration of CocE eliminated cocaine from the rhesus-monkey brain approximately three times faster than control conditions via peripheral actions through attenuating the input function from blood plasma. The efficiency of this process is sufficient to alleviate or prevent adverse central nervous system effects induced by cocaine. Although the present study used tracer doses of cocaine to access brain clearance, these findings further support the development of CocE for the treatment of acute cocaine toxicity.

  14. Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability.

    Science.gov (United States)

    Le Bail, P; Rondeau, C; Buléon, A

    2005-03-01

    Crystalline amylose complexes were prepared with decanal, 1-butanol, menthone and alpha-naphtol. Their crystalline structure and the related helical conformation, determined by wide angle X-ray diffraction (WAXD) and 13C CPMAS solid state NMR, were assigned to V6I, V6II, V6III and V8 types, respectively. It was possible to propose some hypotheses on the possible nature of interactions and especially intra-/inter-helical inclusion. Some shifts in the NMR C1 carbon signals were attributed to the presence of ligand in specific sites inside the structure for a same type of V6 helical conformation. Moreover, the crystallinity and polymorphic changes induced by desorption/rehydration were studied. A general increase of the carbon resonances sharpness upon rehydration has been observed, but also a V6II-V6I transition when decreasing the water content. Differential scanning calorimetry (DSC) experiments were also performed to approach the thermostability of the four types of complex and also the way they form again after melting/cooling sequences.

  15. Novel thermostable endo-xylanase cloned and expressed from bacterium Geobacillus sp. WSUCF1.

    Science.gov (United States)

    Bhalla, Aditya; Bischoff, Kenneth M; Uppugundla, Nirmal; Balan, Venkatesh; Sani, Rajesh K

    2014-08-01

    A gene encoding a GH10 endo-xylanase from Geobacillus sp. WSUCF1 was cloned and expressed in Escherichia coli. Recombinant endo-xylanase (37kDa) exhibited high specific activity of 461.0U/mg of protein. Endo-xylanase was optimally active on birchwood xylan at 70°C and pH 6.5. The endo-xylanase was found to be highly thermostable at 50 and 60°C, retaining 82% and 50% of its original activity, respectively, after 60h. High xylan conversions (92%) were obtained with oat-spelt xylan hydrolysis. Higher glucan and xylan conversions were obtained on AFEX-treated corn stover with an enzyme cocktail containing WSUCF1 endo-xylanase (71% and 47%) as compared to enzyme cocktail containing commercial fungal endo-xylanase (64% and 41%). High specific activity, active at high pH's, wide substrate specificity, and higher hydrolytic activity on recalcitrant lignocellulose, make this endo-xylanase a suitable candidate for biofuel and bioprocess industries.

  16. Characteristics of thermostable amylopullulanase of Geobacillus thermoleovorans and its truncated variants.

    Science.gov (United States)

    Nisha, M; Satyanarayana, T

    2015-05-01

    The far-UV CD spectroscopic analysis of the secondary structure in the temperature range between 30 and 90°C revealed a compact and thermally stable structure of C-terminal truncated amylopullulanase of Geobacillus thermoleovorans NP33 (gt-apuΔC) with a higher melting temperature [58°C] than G. thermoleovorans NP33 amylopullulanase (gt-apu) [50°C] and the N-terminal truncated amylopullulanase from G. thermoleovorans NP33 (gt-apuΔN) [55°C]. A significant decline in random coils in gt-apuΔC and gt-apuΔN suggested an improvement in conformational stability, and thus, an enhancement in their thermal stability. The improvement in the thermostability of gt-apuΔC was corroborated by the thermodynamic parameters for enzyme inactivation. The Trp fluorescence emission (335 nm) and the acrylamide quenching constant (22.69 M(-1)) of gt-apuΔC indicated that the C-terminal truncation increases the conformational stability of the protein with the deeply buried tryptophan residues. The 8-Anilino Naphthalene Sulfonic acid (ANS) fluorescence experiments indicated the unfolding of gt-apu to expose its hydrophobic surface to a greater extent than the gt-apuΔC and gt-apuΔN.

  17. Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

    Science.gov (United States)

    Zhu, Yanbing; Zheng, Wenguang; Ni, Hui; Liu, Han; Xiao, Anfeng; Cai, Huinong

    2015-10-01

    A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

  18. Potential antitumor therapeutic application of Grimontia hollisae thermostable direct hemolysin mutants.

    Science.gov (United States)

    Huang, Sheng-Cih; Wang, Yu-Kuo; Huang, Wan-Ting; Kuo, Tsam-Ming; Yip, Bak-Sau; Li, Tien-Hsiung Thomas; Wu, Tung-Kung

    2015-04-01

    We report on the preparation of a new type of immunotoxin by conjugation of an epidermal growth factor receptor (EGFR)-binding peptide and an R46E mutation of thermostable direct hemolysin from Grimontia hollisae, (Gh-TDH(R) (46E) /EB). The hybrid immunotoxin was purified to homogeneity and showed a single band with slight slower mobility than that of Gh-TDH(R) (46E) . Cytotoxicity assay of Gh-TDH(R) (46E) /EB on EGFR highly, moderately, low, and non-expressed cells, A431, MDA-MB-231, HeLa, and HEK293 cells, respectively, showed apparent cytotoxicity on A431 and MDA-MB-231 cells but not on HeLa or HEK293 cells. In contrast, no cytotoxicity was observed for these cells treated with either Gh-TDH(R) (46E) or EB alone, indicating enhanced cytotoxic efficacy of Gh-TDH(R) (46E) by the EGFR binding moiety. Further antitumor activity assay of Gh-TDH(R) (46E) /EB in a xenograft model of athymic nude mice showed obvious shrinkage of tumor size and degeneration, necrosis, and lesions of tumor tissues compared to the normal tissues. Therefore, the combination of Gh-TDH(R) (46E) with target affinity agents opens new possibilities for pharmacological treatment of cancers and potentiates the anticancer drug's effect.

  19. 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. PMID:26694028

  20. Gene expression and molecular characterization of a thermostable trehalose phosphorylase from Thermoanaerobacter tengcongensis

    Institute of Scientific and Technical Information of China (English)

    REN Yuanyuan; DAI Xiuyu; ZHOU Jian; LIU Jingfang; PEI Huadong; XIANG Hua

    2005-01-01

    A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from a-glucose-1-phosphate (α-GIc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli.The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70℃ (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual activity after incubation at 50℃ for 7 h. Under the optimum reaction conditions, 50 μg crude enzyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-GIc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L trehalose is degraded within the same time period. Dot blotting revealed that the treP gene in T.tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for industrial production of trehalose.

  1. Functional screening of hydrolytic activities reveals an extremely thermostable cellulase from a deep-sea archaeon

    Directory of Open Access Journals (Sweden)

    Benedikt eLeis

    2015-07-01

    Full Text Available Extreme habitats serve as a source of enzymes which are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8 to 70 °C. Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70 °C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12 endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45 % sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92 °C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.

  2. Isolation and characterization of a novel thermostable alpha-amylase from Korean pine seeds.

    Science.gov (United States)

    Azad, Md Abul Kalam; Bae, Jae-Han; Kim, Jong-Sang; Lim, Jin-Kyu; Song, Kyung-Sik; Shin, Beom-Soo; Kim, Hak-Ryul

    2009-10-31

    Amylases have significant importance in broad industrial application including bio-ethanol production. Although amylases are widely distributed in microbes, plants and animals, it has been sought for new amylases from various sources with special industrial potential. In this study we firstly isolated and characterized a novel thermostable alpha-amylase from Korean pine seed. Enzyme was purified to homogeneity level with purification fold of 1286.1 using several techniques such as self-precipitation, (NH(4))(2)SO(4) fractionation, DEAE anion exchange and starch affinity chromatography. The purified alpha-amylase showed two bands in SDS-PAGE with molecular weight of 44 and 45 kDa. The apparent molecular weight of native enzyme was calculated to be 46.7 kDa. Internal peptide sequencing confirmed that the purified alpha-amylase was a novel enzyme. The optimum pH and temperature for enzyme activity were pH 4.5 and 65 degrees C, respectively. This enzyme was fully stable for 48h at 50 degrees C and retained 80% activity up to 96h. The K(m) and V(max) were 0.84 mg/ml and 3.71 micromol/min, respectively. On the basis of high thermal stability and a broad range of pH stability, the pine seed alpha-amylase showed a good prospect of industrial application.

  3. Rhizopus microsporus var. rhizopodiformis: a thermotolerant fungus with potential for production of thermostable amylases.

    Science.gov (United States)

    Peixoto, Simone C; Jorge, João A; Terenzi, Héctor F; Polizeli, Maria de Lourdes T M

    2003-12-01

    The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45 degrees C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of alpha-amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.

  4. Investigation of thermostability of Mo6S3I6 nanowires using Raman spectroscopy

    Directory of Open Access Journals (Sweden)

    Todorović Jelena M.

    2009-01-01

    Full Text Available The thermostability (phase stability of Mo6S3I6 nanowires was investigated by Raman spectroscopy, varying the incident laser power (1-9 mW or by gradual heating of the sample from room temperature to 600°C. We have noticed 18 Raman modes in the room temperature Raman spectra, which is in good agreement with the factor group analysis prediction for P1 space group. We confirmed that the vibrations of Mo6S8 clusters dominate in vibrational properties of the Mo6S3I6 nanostructure, since nanowires Raman spectra are similar to Chevrel phase Raman spectra. During the temperature treatment, it was established that in the temperature range between 300 and 400°C a new Raman mode appeares. This mode can be ascribed to molybdenum oxide (MoO3. With further temperature increase, the intensity of this mode increases, drawing a conclusion that at temperature above 300°C the phase separation takes place in this system followed by a formation of oxide layer.

  5. 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.

  6. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    Science.gov (United States)

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-07-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

  7. 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.

  8. The structure of a thermostable mutant of pro-papain reveals its activation mechanism.

    Science.gov (United States)

    Roy, Sumana; Choudhury, Debi; Aich, Pulakesh; Dattagupta, Jiban K; Biswas, Sampa

    2012-12-01

    Papain is the archetype of a broad class of cysteine proteases (clan C1A) that contain a pro-peptide in the zymogen form which is required for correct folding and spatio-temporal regulation of proteolytic activity in the initial stages after expression. This study reports the X-ray structure of the zymogen of a thermostable mutant of papain at 2.6 Å resolution. The overall structure, in particular that of the mature part of the protease, is similar to those of other members of the family. The structure provides an explanation for the molecular basis of the maintenance of latency of the proteolytic activity of the zymogen by its pro-segment at neutral pH. The structural analysis, together with biochemical and biophysical studies, demonstrated that the pro-segment of the zymogen undergoes a rearrangement in the form of a structural loosening at acidic pH which triggers the proteolytic activation cascade. This study further explains the bimolecular stepwise autocatalytic activation mechanism by limited proteolysis of the zymogen of papain at the molecular level. The possible factors responsible for the higher thermal stability of the papain mutant have also been analyzed.

  9. Hfq regulates the expression of the thermostable direct hemolysin gene in Vibrio parahaemolyticus

    Directory of Open Access Journals (Sweden)

    Mawatari Kazuaki

    2008-09-01

    Full Text Available Abstract Background The hfq gene is conserved in a wide variety of bacteria and Hfq is involved in many cellular functions such as stress responses and the regulation of gene expression. It has also been reported that Hfq is involved in bacterial pathogenicity. However, it is not clear whether Hfq regulates virulence in Vibrio parahaemolyticus. To evaluate this, we investigated the effect of Hfq on the expression of virulence-associated genes including thermostable direct hemolysin (TDH, which is considered to be an important virulence factor in V. parahaemolyticus, using an hfq deletion mutant. Results The production of TDH in the hfq deletion mutant was much higher than in the parental strain. Quantification of tdh promoter activity and mRNA demonstrated that transcription of the tdh gene was up-regulated in the mutant strain. The hfq-complemented strain had a normal (parental amount of tdh expression. The transcriptional activity of tdhA was particularly increased in the mutant strain. These results indicate that Hfq is closely associated with the expression level of the tdh gene. Interestingly, other genes involved in the pathogenicity of V. parahaemolyticus, such as VP1680, vopC, and vopT, were also up-regulated in the mutant strain. Conclusion Hfq regulates the expression of virulence-associated factors such as TDH and may be involved in the pathogenicity of V. parahaemolyticus.

  10. Synthesis of thermostable geopolymer from circulating fluidized bed combustion (CFBC) bottom ashes.

    Science.gov (United States)

    Xu, Hui; Li, Qin; Shen, Lifeng; Wang, Wei; Zhai, Jianping

    2010-03-15

    Circulating fluidized bed combustion (CFBC) bottom ashes (CBAs) are a class of calcined aluminosilicate wastes with a unique thermal history. While landfill disposal of hazardous element-containing CBAs poses serious challenge, these wastes have long been neglected as source materials for geopolymer production. In this paper, geopolymerization of ground CBAs was investigated. Reactivity of the CBAs was analyzed by respective dissolution of the ashes in 2, 5, and 10N NaOH and KOH solutions. Geopolymer pastes were prepared by activating the CBAs by a series of alkalis hydroxides and/or sodium silicate solutions. Samples were cured at 40 degrees C for 168 h, giving a highest compressive strength of 52.9 MPa. Of the optimal specimen, characterization was conducted by TG-DTA, SEM, XRD, as well as FTIR analyses, and thermal stability was determined in terms of compressive strength evolution via exposure to 800 or 1050 degrees C followed by three cooling regimes, i.e. cooling in air, cooling in the furnace, and immerging in water. The results show that CBAs could serve as favorable source materials for thermostable geopolymers, which hold a promise to replace ordinary Portland cement (OPC) and organic polymers in a variety of applications, especially where fire hazards are of great concern.

  11. Thermostability of Cromobacterium viscosum lipase in AOT/isooctane reverse micelle.

    Science.gov (United States)

    Talukder, M M R; Zaman, M M; Hayashi, Y; Wu, J C; Kawanishi, T

    2007-04-01

    The thermostability of Cromobacterium viscosum lipase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was increased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60 degrees C was 28 h, ninefold higher than that in reverse micelles without PEG 400. The lipase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60 degrees C in PEG containing reverse micelles was 0.055 h!1, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lipase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.

  12. Characterisation of three starch degrading enzymes: thermostable β-amylase, maltotetraogenic and maltogenic α-amylases.

    Science.gov (United States)

    Derde, L J; Gomand, S V; Courtin, C M; Delcour, J A

    2012-11-15

    Maltogenic α-amylase from Bacillus stearothermophilus (BStA) is widely used as bread crumb anti-firming enzyme. A maltotetraose-forming α-amylase from Pseudomonas saccharophila (PSA) was recently proposed as alternative, hence the need to compare both exo-acting enzymes with some endo-action component. A purely exo-acting thermostable β-amylase from Clostridium thermosulfurogenes (CTB) was included for reference purposes. Under the experimental conditions used, temperature optima of the enzymes are rather similar (60-65 °C), but temperature stability decreased in the order BStA, PSA and CTB. The action of the enzymes on different substrates and their impact on the rheological behaviour of maize starch suspensions demonstrated that, while CTB acts exclusively through an exo-action mechanism, BStA displayed limited endo-action which became more pronounced at higher temperatures. PSA has more substantial endo-action than BStA, which is rather temperature independent. This is important for their impact in processes such as breadmaking, where temperature is gradually increased.

  13. Optimization and characterization of thermostable β-glucosidase by Scytalidium thermophilum SKESMBKU02

    Directory of Open Access Journals (Sweden)

    S. Santosh Kumar

    2015-06-01

    Full Text Available Scytalidium thermophilum SKESMBKU02, a thermophilic fungus isolated from cattle dung, produced a significant amount of thermostable β-glucosidase in Mandel and Weber medium (45°C, 3 days. Scytalidium thermophilum SKESMBKU02 opted pH – 5, temperature of 45°C for production of β-glucosidases. The glucose and yeast extract are the best carbon and nitrogen sources for β-glucosidase production. It has found that shake flak culture method exerted more β-glucosidase production than the still culture method. The fungus showed maximum dry weight in cellulose as carbon sources, yeast extract and malt extract as nitrogen source, pH of 9.0-10.0 and temperature of 45oC.The crude enzyme was characterized and found that substrate concentration of 6 mM and enzyme concentration of 300 μl are found suitable for the maximum β-glucosidase activity. Stability studies reveals that the enzyme was active at wide range of pH and temperature.

  14. Purification and characterization of a novel thermostable beta-amylase from Clostridium thermosulphurogenes.

    Science.gov (United States)

    Shen, G J; Saha, B C; Lee, Y E; Bhatnagar, L; Zeikus, J G

    1988-01-01

    An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined. The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase. The beta-amylase was enriched in both acidic and hydrophobic amino acids. The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5. The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition. The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins. The enzyme displayed kcat. and Km values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively. The enzyme was antigenically distinct from plant beta-amylases. PMID:2461701

  15. Limitations of yeast surface display in engineering proteins of high thermostability.

    Science.gov (United States)

    Park, Sheldon; Xu, Yao; Stowell, Xiaoran Fu; Gai, Feng; Saven, Jeffery G; Boder, Eric T

    2006-05-01

    Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.

  16. Characterization of a Recombinant Thermostable Arylsulfatase from Deep-Sea Bacterium Flammeovirga pacifica.

    Science.gov (United States)

    Gao, Chao; Jin, Min; Yi, Zhiwei; Zeng, Runying

    2015-11-01

    A novel sulfatase gene, ary423 (1,536 bp ORF), encoding a protein of 511 amino acids with a calculated molecular mass of 56 kDa, was identified from Flammeovirga pacifica, which was isolated from deep-sea sediments of west Pacific Ocean. Amino acid sequence analysis revealed that Ary423 possessed a conserved C-X-A-X-R motif, which was recognized as the sulfatase signature. Phylogenetic analysis suggested that Ary423 belonged to arylsulfatases. After heterologous expression in Escherichia coli cells, the recombinant Ary423 was purified with a Ni(+) affinity column, and was shown to be highly active at a broad range of temperatures from 30° to 70°C, with maximum activity at 40°C. Furthermore, recombinant Ary423 retained more than 70% and 40% of its maximum activity after 12 h of incubation at 50°C and 60°C, respectively, exhibiting good thermostability at high temperatures. The optimal pH for Ary423 was determined to be 8.0 and the activity of Ary423 could be slightly enhanced by Mg(2+). The recombinant enzyme could hydrolyze sulfate ester bonds in pnitrophenyl sulfate (NPS) and Asparagus crude polysaccharides with a specific activity of 64.8 U/mg and 25.4 U/mg, respectively. These favorable properties could make Ary423 attractive for application in the desulfating process of agar production.

  17. Choice and optimization of ratio of components to develop fast-mounted thermostable heat-insulating constructions

    Science.gov (United States)

    Loginova, N. A.; Grigor'ev, S. V.; Lapin, E. E.; Pogorelov, S. I.; Ryzhenkov, A. V.

    2016-05-01

    Fast-mounted heat-insulating constructions based on foamed synthetic rubbers, polyethylene, and polyurethane are characterized by a thermostability up to 150°C and emit toxic substances when burnt. However, there is a need for heat insulation of surfaces with higher coolant temperatures, such as pipelines, equipment of nuclear and thermal power plants, and heating systems with remote heat sources. One of the most promising types of heat insulation materials for creation of fast-mounted heat insulation constructions is the syntactic foams or thin-film multilayer heat-insulating coatings (TFMHIC), which are created using hollow microspheres and various types of binders. The formation of TFMHIC on the heat-insulating surface is carried out mostly by means of spraying methods that have well proven themselves at coating on flat and cylindrical surfaces of large area, but they turned out ineffective for cylindrical surfaces with a diameter of 300 mm and less, since they are characterized by a large degree of carryover of composite material. This article analyzed the binders and microspheres promising to create the fast-mounted heat-insulating constructions based on TFMHIC with high thermostability. Based on the analysis, a conclusion is drawn that organicsilicon binding and glass microspheres are promising for use in the heat-insulating constructions with thermostability up to 300°C. The results of experimental research are given that point to the possibility of predicting the optimal composition of heat-insulating material characterized by a high degree of filling with microspheres with maintaining the mechanical strength, by means of performing the analysis of rheological characteristics of nonpolymerized liquid compositions of heat-insulation material. The index of tensile strength in bending was the criterion for evaluating the mechanical strength of heat-insulating material. The critical volume concentrations of filling the heat-insulating material with glass

  18. Immobilization of a thermostable alpha-amylase by covalent binding to an alginate matrix increases high temperature usability.

    Science.gov (United States)

    Tee, Boon L; Kaletunç, Gönül

    2009-01-01

    Thermostable alpha-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95 degrees C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized alpha-amylase were investigated. Covalently bound thermostable alpha-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95 degrees C in a stirred batch reactor. The free and covalently bound alpha-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (K(m)) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V(max)) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable alpha-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process.

  19. Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design.

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    Sruti Dutta

    Full Text Available Ervatamins (A, B and C are papain-like cysteine proteases from the plant Ervatamia coronaria. Among Ervatamins, Ervatamin-C is a thermostable protease, but it shows lower catalytic efficiency. In contrast, Ervatamin-A which has a high amino acid sequence identity (∼90% and structural homology (Cα rmsd 0.4 Å with Ervatamin-C, has much higher catalytic efficiency (∼57 times. From the structural comparison of Ervatamin-A and -C, two residues Thr32 and Tyr67 in the catalytic cleft of Ervatamin-A have been identified whose contributions for higher activity of Ervatamin-A are established in our earlier studies. In this study, these two residues have been introduced in Ervatamin-C by site directed mutagenesis to enhance the catalytic efficiency of the thermostable protease. Two single mutants (S32T and A67Y and one double mutant (S32T/A67Y of Ervatamin-C have been generated and characterized. All the three mutants show ∼ 8 times higher catalytic efficiency (k cat/K m than the wild-type. The thermostability of all the three mutant enzymes remained unchanged. The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A. By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed. The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

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

    Science.gov (United States)

    Xu, Xin; Li, Qi

    2016-01-01

    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. PMID:27100881

  1. Improvement of activity, thermo-stability and fruit juice clarification characteristics of fungal exo-polygalacturonase.

    Science.gov (United States)

    Amin, Faiza; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

    2017-02-01

    An extracellular exo-polygalacturonase (exo-PG) from Penicillium notatum was immobilized in sodium-alginate matrix through two different protocols, viz. covalent bonding and adsorption to enhance its catalytic activity, thermal stability and life-time properties for industrial applications. Covalent immobilization was more efficient in terms of high relative activity (45.89%) and immobilization yield (71.6%) as compared to adsorption. Immobilized exo-PG derivatives displayed maximum activities at pH 5.5 and 55°C as compared to free enzyme which showed its optimum activity at pH 6.0 and 50°C. The affinity of enzyme towards its substrate (Km(app)) was reduced after immobilization and Vmax of covalently immobilized exo-PG decreased to 66.7% while the Vmax value of adsorbed enzyme increased up to 150% as compared to free counterpart. Both immobilization techniques greatly enhanced the thermal stability profile of the enzyme. At 60°C, immobilized exo-PGs retained more than 90% of their residual activities after 60min of heating, while free enzyme did not show any activity at the same temperature. Thermodynamic properties (i.e., Ea, ΔH*, ΔS*and ΔG*) of the free and immobilized enzymes were also investigated. Sodium-alginate covalently immobilized and adsorbed enzymes showed excellent recycling efficiencies and retained 50.0% and 41.0% of original activities, respectively after seven consecutive batch reactions. Moreover, the immobilized enzymes treatment achieved promising results in turbidity and viscosity reduction as well as clarity amelioration in various fruit juices. Altogether catalytic, thermo-stability and fruit juices clarification characteristics of the immobilized ex-PGs suggest a high potential for biotechnological exploitability. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Thermostable levansucrase from Bacillus subtilis BB04, an isolate of Banana peel

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    Viniti D Vaidya

    2012-04-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} Extensive screening resulted in the isolation of Bacillus sp. from Banana peel that produces considerable amount of thermostable levansucrase of molecular size 52kDa. 16S rRNA sequence analysis suggests that it belongs to Bacillus subtilis and was designated as strain BB04. Levansucrase was sucrose inducible, showed optimum activity at 50°C and pH 6.0. It was stable at pH range 6.0 - 7.0. Ca2+ at 1.0 mmol-1 concentration enhanced levansucrase activity by 24%. However levan production was highest at 40°C and pH 6.0. Cane molasses and juice proved to be good sources of sucrose for levan production. B. subtilis BB04 produced relatively more levan using cane molasses (11.32 gl-1 as sucrose source than in cane juice (4.81 gl-1.

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

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    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.

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

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    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. Structural elements of thermostability in the maltogenic amylase of Geobacillus thermoleovorans.

    Science.gov (United States)

    Mehta, Deepika; Satyanarayana, T

    2015-08-01

    Maltogenic amylase of Geobacillus thermoleovorans (Gt-MamyIII), which has the highest thermostability among bacterial maltogenic amylases, has been used as a model enzyme to understand the role of networked salt bridges in thermoadaptation. The role of intra-chain cross-domain salt bridge networks in the thermostabilization of maltogenic amylase of G. thermoleovorans was confirmed by site-directed mutagenesis and CD analysis. The amino acid pairs in seven salt bridges have been mutated singly and pair-wise, and their free energy of thermal inactivation has been calculated. Among seven, single and double mutations in the amino acids corresponding to four salt bridges (lys306.glu336, arg403.asp65, arg497.glu523 and lys524.glu523) decrease T1/2 and Tm of Gt-MamyIII significantly. Moreover, glu523 forms networked salt bridges with arg497 and lys524. OE1 of glu523 forms electrostatic interactions with NH1 of arg497, NH2 of arg497 and NZ of lys524 at a distance of 2.33, 2.02 and 0.33Å, respectively. The mutations in three buried amino acids led to a decline in T1/2 and Tm. The buried as well as networked cross-domain salt bridges thus appear to play a significant role in the thermostabilization of Gt-MamyIII. The salt bridges lys306.glu336 and arg403.asp65, which are isolated and partially accessible, play a minor role in its thermostabilization.

  6. Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus.

    Science.gov (United States)

    Bechlars, Silke; Wüstenhagen, Doreen A; Drägert, Katja; Dieckmann, Ralf; Strauch, Eckhard; Kubick, Stefan

    2013-12-15

    Vibrio parahaemolyticus is a recognized enteropathogen causing diarrhea in humans and is one of the major causes of seafoodborne gastroenteritis. An important virulence factor is thermostable direct hemolysin (TDH), a pore-forming toxin, which is able to lyse eukaryotic cells. The active toxin is a tetramer of four identical protein subunits, which is secreted by the pathogen after cleavage of a signal peptide. To establish diagnostic detection systems for TDH we expressed the hemolysin with and without the signal peptide in a prokaryotic cell-free system to obtain pure toxin. In order to purify and to facilitate the isolation from cell lysates we synthesized TDH variants with different tags. Important regulatory sequences for cell-free protein synthesis as well as sequences for N-terminal Strep-tag and C-terminal 6xHis-tag were added by a two-step PCR. For the expression in the cell-free system these linear tdh templates were subjected directly to prokaryotic cell extracts. Protein yields were in the range of 500-600 μg/ml for the preproteins and approx. 300-400 μg/ml for the mature proteins. The identities of expressed proteins were further confirmed by SDS-PAGE, immunological and MALDI-TOF mass spectrometric analyses. The functionality of newly synthesized toxin variants was tested by performing qualitative and semiquantitative hemolysis assays. Cell-free produced mature TDH and its variants were active while the preprotein and its derivatives lacked hemolytic activity. A C-terminal 6xHis-tag showed less influence on functionality compared to the N-terminal Strep-tag.

  7. The thermostable direct hemolysin from Grimontia hollisae causes acute hepatotoxicity in vitro and in vivo.

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    Yan-Ren Lin

    Full Text Available BACKGROUND: G. hollisae thermostable direct hemolysin (Gh-TDH is produced by most strains of G. hollisae. This toxin has been reported to be absorbed in the intestines in humans. Secondary liver injury might be caused by venous return of the toxin through the portal system. We aimed to firstly analyze the in vitro and in vivo hepatotoxicity of Gh-TDH. METHODS: Liver cells (primary human non-cancer cell and FL83B mouse cells were treated and mice (BALB/c were fed with this toxin to investigate its hepatotoxicity. Morphological examination and cytotoxicity assays using liver cells were also performed. Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of this protein in liver cells. Mice were subjected to liver function measurements and liver biopsies following toxin treatment and wild-type bacterial infection. PET (positron emission tomography/CT (computed tomography images were taken to assess liver metabolism during acute injury and recovery. RESULTS: The effect of hepatotoxicity was dose and time dependent. Cellular localization showed that the toxin was initially located around the cellular margins and subsequently entered the nucleus. Liver function measurements and liver biopsies of the mice following treatment with toxin or infection with wild-type Grimontia hollisae showed elevated levels of transaminases and damage to the periportal area, respectively. The PET/CT images revealed that the reconstruction of the liver continued for at least one week after exposure to a single dose of the toxin or bacterial infection. CONCLUSIONS: The hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in the periportal area of the liver, and the liver became functionally insufficient.

  8. Catalytic performance and thermostability of chloroperoxidase in reverse micelle: achievement of a catalytically favorable enzyme conformation.

    Science.gov (United States)

    Wang, Yali; Wu, Jinyue; Ru, Xuejiao; Jiang, Yucheng; Hu, Mancheng; Li, Shuni; Zhai, Quanguo

    2011-06-01

    The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40 °C, CPO essentially lost all its activity after 5 h incubation, while 58-76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50 °C, about 44-75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V(pentanol)/V(isooctane)), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k(cat)) was increased in reverse micelle. Moreover, the lower K(m) and higher k(cat)/K(m) demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV-vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.

  9. 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.

  10. Halophilic alkali- and thermostable amylase from a novel polyextremophilic Amphibacillus sp. NM-Ra2.

    Science.gov (United States)

    Mesbah, Noha M; Wiegel, Juergen

    2014-09-01

    Extracellular gluco-amylo-pullulanase from Amphibacillus sp. NM-Ra2 was purified to homogeneity by ethanol precipitation, anion exchange chromatography and gel filtration chromatography. Molecular mass of the enzyme was 50kDa (SDS-PAGE). The enzyme showed maximal activity at 1.9 M NaCl, pH50°C 8.0 and 54°C and was active from 0 to 4.3 M NaCl and 37 to 65°C. The enzyme was inhibited by EDTA and was stable and active in the presence of PMSF, DTT, H2O2, Triton-X-100, Tween 20 and Tween 80. Ca2+ is inessential for activity. The amylase was stimulated with K+ and inhibited with Cu2+ and Mg2+. Hg2+, Zn2+ and Fe2+ had no effect on activity. Amylase was stable and active in the presence of ethanol, methanol and benzene (25%, v/v). The enzyme hydrolyzed linear and branched polysaccharides including pullulan, glycogen and amylopectin, and hydrolyzed raw wheat starch and raw corn starch (14.6% and 13.5% over 2 h). Amylase activity was inhibited by soluble starch concentrations greater than 0.3%. The major products of soluble starch hydrolysis were maltose and maltotriose. The amylase, being halophilic and alkali-thermostable, in addition to being resistant to surfactants, oxidizing agents and organic solvents, can find applications in the starch processing, pharmaceutical, food and paper/pulp industries. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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    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.

  12. 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

    2015-03-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Opperman, Diederik J. [Department of Microbial, Biochemical and Food Biotechnology, BioPAD Metagenomics Platform, University of the Free State, Bloemfontein 9300 (South Africa); Sewell, Bryan T. [Electron Microscope Unit, University of Cape Town, Rondebosch 7701 (South Africa); Litthauer, Derek [Department of Microbial, Biochemical and Food Biotechnology, BioPAD Metagenomics Platform, University of the Free State, Bloemfontein 9300 (South Africa); Isupov, Mikhail N.; Littlechild, Jennifer A. [School of Biosciences, Henry Wellcome Building for Biocatalysis, University of Exeter, Stocker Road, Exeter EX4 4QD (United Kingdom); Heerden, Esta van, E-mail: vheerde.sci@ufs.ac.za [Department of Microbial, Biochemical and Food Biotechnology, BioPAD Metagenomics Platform, University of the Free State, Bloemfontein 9300 (South Africa)

    2010-03-12

    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 {alpha},{beta}-unsaturated carbonyl compounds with a preference towards cyclic substrates however no activity was observed towards {beta}-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.

  14. Thermal optima of photosynthetic functions and thermostability of photochemistry in cork oak seedlings.

    Science.gov (United States)

    Ghouil, Hana; Montpied, Pierre; Epron, Daniel; Ksontini, Mustapha; Hanchi, Belkacem; Dreyer, Erwin

    2003-10-01

    Temperature effects on photosynthesis were studied in seedlings of evergreen Mediterranean cork oak (Quercus suber L.). Responses to changes in temperature and the temperature optima of maximal carboxylation rate (V(cmax)) and maximal light-driven electron flux (J(max)) were estimated from gas exchange measurements and a leaf-level photosynthesis model. The estimated temperature optima were approximately 34 and 33 degrees C for V(cmax) and J(max), respectively, which fall within the lower range of temperature optima previously observed in deciduous tree species. The thermostability of the photosynthetic apparatus was estimated according to the temperature at which basal chlorophyll a fluorescence begins to increase (T(c)). The T(c) was highly variable, increasing from 42 to 51 degrees C when ambient temperature rose from 10 to 40 degrees C, and increasing from 44 to 54 degrees C with decreasing soil water availability while net CO(2) assimilation rate dropped to almost zero. When a heat shock was imposed, an additional small increase in T(c) was observed in drought-stressed and control seedlings. Maximal T(c) values following heat shock were about 56 degrees C, which, to our knowledge, are the highest values that have been observed in tree species. In conclusion, the intrinsic temperature responses of cork oak did not differ from those of other species (similar T(c) under ambient temperature and water availability, and relatively low thermal optima for photosynthetic capacity in seedlings grown at cool temperatures). However, the large ability of cork oak to acclimate to drought and elevated temperature may be an important factor in the tolerance of this evergreen Mediterranean species to summer drought and high temperatures.

  15. 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.

  16. 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...

  17. [Comparison of the thermostability of natural (sulfoprolamine and sodium usnate) and synthetic (climbazole and piroctone olamine) antidandruff agents].

    Science.gov (United States)

    Coiffard, C; Coiffard, L; de Roeck-Holtzhauer, Y

    1999-09-01

    We compared thermostability of various natural (sulfoprolamine and sodium usnate) or synthetic (climbazol and piroctone olamine) antidandruff agents in aqueous diluted solution at pH around 7. Thermodegradation of these solutions was studied by an isothermal method in thermostatically controlled ovens, at three temperatures (50, 70 and 90 degrees C). For each molecule, we determined at 20 degrees C t90% (time necessary to obtain a decrease of 10% of the initial concentration, value which shows the stability of the product). The present study shows that piroctone olamine is the most stable antidandruff agent among those studied.

  18. Uniqueness of limit cycles and multiple attractors in a Gause-type predator-prey model with nonmonotonic functional response and Allee effect on prey.

    Science.gov (United States)

    González-Olivares, Eduardo; González-Yañez, Betsabé; Mena-Lorca, Jaime; Flores, Jose D

    2013-04-01

    The main purpose of this work is to analyze a Gause type predator-prey model in which two ecological phenomena are considered: the Allee effect affecting the prey growth function and the formation of group defence by prey in order to avoid the predation. We prove the existence of a separatrix curves in the phase plane, determined by the stable manifold of the equilibrium point associated to the Allee effect, implying that the solutions are highly sensitive to the initial conditions. Trajectories starting at one side of this separatrix curve have the equilibrium point (0,0) as their ω-limit, while trajectories starting at the other side will approach to one of the following three attractors: a stable limit cycle, a stable coexistence point or the stable equilibrium point (K,0) in which the predators disappear and prey attains their carrying capacity. We obtain conditions on the parameter values for the existence of one or two positive hyperbolic equilibrium points and the existence of a limit cycle surrounding one of them. Both ecological processes under study, namely the nonmonotonic functional response and the Allee effect on prey, exert a strong influence on the system dynamics, resulting in multiple domains of attraction. Using Liapunov quantities we demonstrate the uniqueness of limit cycle, which constitutes one of the main differences with the model where the Allee effect is not considered. Computer simulations are also given in support of the conclusions.

  19. Universal nonmonotonic structure in the saturation curves of magneto-optical-trap-loaded Na+ ions stored in an ion-neutral hybrid trap: Prediction and observation

    Science.gov (United States)

    Blümel, R.; Wells, J. E.; Goodman, D. S.; Kwolek, J. M.; Smith, W. W.

    2015-12-01

    We predict that the maximal, steady-state ion capacity Ns(λ ) of radio-frequency (rf) traps, loaded at a rate of λ particles per rf cycle, shows universal, nonlinear, nonmonotonic behavior as a function of loading rate λ . The shape of Ns(λ ) , characterized by four dynamical regimes, is universal; i.e., it is predicted to manifest itself in all types of rf traps independent of the details of their construction and independent of particle species loaded. For λ ≪ 1 (region I), as expected, Ns(λ ) increases monotonically with λ . However, contrary to intuition, at intermediate λ ˜1 (region II), Ns(λ ) reaches a maximum, followed by a local minimum of Ns(λ ) (region III). For λ ≫1 (region IV), Ns(λ ) again rises monotonically. In region IV, numerical simulations, analytical calculations, and experiments show Ns(λ ) ˜λ2 /3 . We confirm our predictions both experimentally with magneto-optical-trap-loaded Na+ ions stored in a hybrid ion-neutral trap and numerically with the help of detailed ab initio molecular-dynamics simulations.

  20. Non-monotonic, distance-dependent relaxation of water in reverse micelles: propagation of surface induced frustration along hydrogen bond networks.

    Science.gov (United States)

    Biswas, Rajib; Chakraborti, Tamaghna; Bagchi, Biman; Ayappa, K G

    2012-07-07

    Layer-wise, distance-dependent orientational relaxation of water confined in reverse micelles (RM) is studied using theoretical and computational tools. We use both a newly constructed "spins on a ring" (SOR) Ising-type model (with Shore-Zwanzig rotational dynamics) and atomistic simulations with explicit water. Our study explores the effect of reverse micelle size and role of intermolecular correlations, compromised by the presence of a highly polar surface, on the distance (from the interface) dependence of water relaxation. The "spins on a ring" model can capture some aspects of distance dependence of relaxation, such as acceleration of orientational relaxation at intermediate layers. In atomistic simulations, layer-wise decomposition of hydrogen bond formation pattern clearly reveals that hydrogen bond arrangement of water at a certain distance away from the surface can remain frustrated due to the interaction with the polar surface head groups. This layer-wise analysis also reveals the presence of a non-monotonic slow relaxation component which can be attributed to this frustration effect and which is accentuated in small to intermediate size RMs. For large size RMs, the long time component decreases monotonically from the interface to the interior of the RMs with slowest relaxation observed at the interface.

  1. A weighted combination of pseudo-likelihood estimators for longitudinal binary data subject to non-ignorable non-monotone missingness.

    Science.gov (United States)

    Troxel, Andrea B; Lipsitz, Stuart R; Fitzmaurice, Garrett M; Ibrahim, Joseph G; Sinha, Debajyoti; Molenberghs, Geert

    2010-06-30

    For longitudinal binary data with non-monotone non-ignorably missing outcomes over time, a full likelihood approach is complicated algebraically, and with many follow-up times, maximum likelihood estimation can be computationally prohibitive. As alternatives, two pseudo-likelihood approaches have been proposed that use minimal parametric assumptions. One formulation requires specification of the marginal distributions of the outcome and missing data mechanism at each time point, but uses an 'independence working assumption,' i.e. an assumption that observations are independent over time. Another method avoids having to estimate the missing data mechanism by formulating a 'protective estimator.' In simulations, these two estimators can be very inefficient, both for estimating time trends in the first case and for estimating both time-varying and time-stationary effects in the second. In this paper, we propose the use of the optimal weighted combination of these two estimators, and in simulations we show that the optimal weighted combination can be much more efficient than either estimator alone. Finally, the proposed method is used to analyze data from two longitudinal clinical trials of HIV-infected patients. (c) 2010 John Wiley & Sons, Ltd.

  2. Universal non-monotonic structure in the saturation curves of MOT-loaded Na$^+$ ions stored in an ion-neutral hybrid trap: Prediction and observation

    CERN Document Server

    Blümel, R; Goodman, D S; Kwolek, J M; Smith, W W

    2015-01-01

    We predict that the steady-state ion number $N_s$ for radio-frequency (rf) traps, loaded at a rate of $\\lambda$ particles per unit time, shows universal non-monotonic behavior as a function of loading rate $\\lambda$. The shape of $N_s(\\lambda)$, characterized by four dynamical regions, is universal in the sense that it is predicted to manifest itself in all rf traps independently of the details of their construction. For $\\lambda\\ll$ 1 particles / rf cycle (Region I), as expected, $N_s(\\lambda)$ increases monotonically with $\\lambda$. However, contrary to intuition, at intermediate $\\lambda \\sim 1$ particles / rf cycle (Region II), $N_s(\\lambda)$ reaches a maximum, followed by a minimum of $N_s(\\lambda)$ (Region III). For $\\lambda\\gg 1$ particles / rf cycle (Region IV), $N_s(\\lambda)$ again rises monotonically. In Region IV numerical simulations, analytical calculations, and experiments show $N_s(\\lambda)\\sim \\lambda^{2/3}$. We confirm this prediction experimentally with MOT-loaded Na$^+$ ions stored in a hyb...

  3. 法律知识的因果表达和非单调推理模型%Causal Knowledge Representation and Nonmonotonic Reasoning Models in Law Consultant Systems

    Institute of Scientific and Technical Information of China (English)

    干红华; 潘云鹤

    2001-01-01

    Causal reasoning is the most important feature in law consultant systems. This paper analy-ses the structure of law clauses,proposes a representation model for law knowledge in terms of causal relationships and nonmonotonic reasoning models based on it. These models are successfully applied in the implementation of NBU-CALA+ ,a law expert consultant system for case analysis and interpreta-tion.

  4. Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolor A3(2).

    Science.gov (United States)

    Ge, Ya-Dong; Cao, Zheng-Yu; Wang, Zong-Da; Chen, Lu-Lu; Zhu, You-Ming; Zhu, Guo-Ping

    2010-01-01

    We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD(+)-specific and displayed about 400-fold (k(cat)) and 1,050-fold (k(cat)/K(m)) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (K(i)=5.8 mM) and excess L-malate (K(i)=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe(2+) and Zn(2+). Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.

  5. Taguchi's experimental design for optimizing the production of novel thermostable polypeptide antibiotic from Geobacillus pallidus SAT4.

    Science.gov (United States)

    Muhammad, Syed Aun; Ahmed, Safia; Ismail, Tariq; Hameed, Abdul

    2014-01-01

    Polypeptide antimicrobials used against topical infections are reported to obtain from mesophilic bacterial species. A thermophilic Geobacillus pallidus SAT4 was isolated from hot climate of Sindh Dessert, Pakistan and found it active against Micrococcus luteus ATCC 10240, Staphylococcus aureus ATCC 6538, Bacillus subtilis NCTC 10400 and Pseudomonas aeruginosa ATCC 49189. The current experiment was designed to optimize the production of novel thermostable polypeptide by applying the Taguchi statistical approach at various conditions including the time of incubation, temperature, pH, aeration rate, nitrogen, and carbon concentrations. There were two most important factors that affect the production of antibiotic including time of incubation and nitrogen concentration and two interactions including the time of incubation/pH and time of incubation/nitrogen concentration. Activity was evaluated by well diffusion assay. The antimicrobial produced was stable and active even at 55°C. Ammonium sulphate (AS) was used for antibiotic recovery and it was desalted by dialysis techniques. The resulted protein was evaluated through SDS-PAGE. It was concluded that novel thermostable protein produced by Geobacillus pallidus SAT4 is stable at higher temperature and its production level can be improved statistically at optimum values of pH, time of incubation and nitrogen concentration the most important factors for antibiotic production.

  6. 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.

  7. Evaluation of a thermostable Newcastle disease virus strain TS09-C as an in-ovo vaccine for chickens

    Science.gov (United States)

    Yu, Qingzhong; Wang, Hongling; Luo, Qingping; Zhang, Tengfei; Zhang, Rongrong; Zhang, Wanpo; Shao, Huabin

    2017-01-01

    In-ovo vaccination is an attractive immunization approach for poultry industry. However, most of the Newcastle disease virus (NDV) vaccine strains used after hatch are unsafe, as in-ovo vaccines, due to their high pathogenicity for chicken embryos. In this study, we evaluated the safety and immunogenicity of a thermostable NDV strain TS09-C, derived from V4 strain, as in-ovo vaccine. Chickens in-ovo vaccinated with the parental V4 strain displayed greatly reduced hatchability and severe histopathological lesions in both trachea and intestine tissues, while the hatchability was not affected by in-ovo vaccination withTS09-C strain. The safe dose that infected all chicken embryos without obviously histopathological lesions was 103.0 EID50 per bird. In-ovo vaccination of chickens with TS09-C virus conferred complete protection against virulent NDV challenge. Results suggest that the thermostable NDV strain TS09-C is a safe and immunogenic in-ovo vaccine candidate that can be delivered quickly and uniformly, and induce earlier immune response. PMID:28234989

  8. High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction.

    Science.gov (United States)

    Zhang, Jun-hui; Lin, Ying; Sun, Yu-fei; Ye, Yan-rui; Zheng, Sui-ping; Han, Shuang-yan

    2012-05-10

    Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants' lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70°C for 5h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.

  9. Isolation and characterization of a novel thermostable neopullulanase-like enzyme from a hot spring in Thailand.

    Science.gov (United States)

    Tang, Kittapong; Kobayashi, Rutchadaporn Sriprang; Champreda, Verawat; Eurwilaichitr, Lily; Tanapongpipat, Sutipa

    2008-06-01

    A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60 degrees C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and gamma-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75 degrees C with K(m) and V(max) toward pullulan of 1.22+/-0.3% and 23.24+/-1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources.

  10. 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.

  11. A Thermostable α-Amylase Producing Natural Variant of Bacillus spp. Isolated From Soil in Iran

    Directory of Open Access Journals (Sweden)

    Iraj Rasooli

    2008-01-01

    Full Text Available Thermophilic processes appear more stable, rapid and less expensive and facilitate reactant activity and product recovery. Amylases have a quarter of the world enzyme market and thermostable α-amylases possess extensive commercial applications. Since little work has been done on strain isolation, growth and enzyme yield optimization, the level of thermophilic enzyme production remains relatively low. Therefore, large scale exploitation of thermophiles requires further intensive and integrated work. The present study describes isolation of an α-amylase producing bacillus from soil. The isolated bacillus was identified and named as Bacillus licheniformis Shahed-07. The strain was cultured in liquid media to produce α-amylase. The enzyme production conditions of the newly isolated bacillus revealed that the maximum enzyme production after 26 h of cultivation at pH 7.0 and 50°C. 0.5% tryptophan in production medium enhanced the enzyme productivity to two fold whereas peptone and lysin at 0.5% level showed a strong repression. Crude α-amylase characterization revealed that optimum activity was at pH 7.5 and 70°C. The crude enzyme was stable for 24 h at pH range of 6-7 at 70°C. Enzyme activity increased with temperature within the range of 40-70°C. The Bacillus licheniformis Shahed-07 strain produced thermostable α-amylase with characteristics suitable for application in starch processing and food industries.

  12. Engineering the hydrophobic residues of a GH11 xylanase impacts its adsorption onto lignin and its thermostability.

    Science.gov (United States)

    Rakotoarivonina, Harivony; Hermant, Béatrice; Aubry, Nathalie; Rémond, Caroline

    2015-12-01

    This study aimed to characterise the parameters governing the non-specific adsorption of a xylanase from Thermobacillus xylanilyticus (Tx-Xyn11) onto lignin isolated from maize stems. Such adsorption may be due to hydrophobic interactions between Tx-Xyn11 and lignin. Our strategy was to mutate hydrophobic residues present on the surface of Tx- Xyn11 into non-hydrophobic residues. Three mutants (P1, P2, and P3) with altered hydrophobic regions were produced and characterised. The thermostability of the P1 mutant was largely decreased compared with the thermostable Tx-Xyn11. The rate of adsorbed enzyme onto lignin was reduced to a similar extent for the P1 and P2 mutants, whereas the adsorption of the P3 mutant was less affected compared with that of Tx-Xyn11. When considered separately, the hydrophobic residues did not affect xylanase adsorption onto lignin. The addition of Tween 20 also led to the decreased adsorption of Tx-Xyn11 onto lignin. These results suggest that hydrophobic interactions are a key parameter in the interaction of Tx-Xyn11 with isolated lignin.

  13. A differentially conserved residue (Ile42) of GH42 β-galactosidase from Geobacillus stearothermophilus BgaB is involved in both catalysis and thermostability.

    Science.gov (United States)

    Dong, Yi-Ning; Chen, Hai-Qin; Sun, Yan-Hui; Zhang, Hao; Chen, Wei

    2015-04-01

    The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-β-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 β-galactosidases.

  14. Evaluation of an immunochromatographic assay for direct identification of thermostable direct hemolysin-producing Vibrio parahaemolyticus colonies on selective agar plates.

    Science.gov (United States)

    Kawatsu, Kentaro; Sakata, Junko; Yonekita, Taro; Kumeda, Yuko

    2015-12-01

    We evaluated the utility of an immunochromatographic assay (NH IC TDH) in identifying thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus colonies on selective agar plates. The sensitivity of the NH IC TDH assay was 100% (189 samples) and its specificity was 100% (41 samples) compared with the presence of tdh.

  15. Some Contributions to Nonmonotonic Consequence

    Institute of Scientific and Technical Information of China (English)

    ZHU Zhaohui; ZHANG Dongmo; CHEN Shifu; ZHU Wujia

    2001-01-01

    This paper introduces a non-Horn rule WRM which is a weak form of rational monotony. We explore the effects of adding this non-Horn rule to the rules for the preferential inference. In this paper, a relation |~ is said to be P + WRM iff it is a preferential inference and satisfies the rule WRM. We establish the representation theorem for P+WRM, and compare the strength of WRM with some non-Horn rules appearing in literatures. Moreover, we explore the relation between P+WRM and conditional logic, and demonstrate that P+WRM is equivalent to ‘flat' fragment of conditional logic CS4.2. Another contribution of this paper is to explore the relation between two special kinds of preferential models, i.e., PRC model and quasi-linear model. Main result reveals that the latter is a special form of the former.

  16. Structural and Functional Studies of Aspergillus oryzae Cutinase: Enhanced Thermostability and Hydrolytic Activity of Synthetic Ester and Polyester Degradation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Z.; Gosser, Y; Baker, P; Ravee, Y; Li, H; Butterfoss, G; Kong, X; Gross, R; Montclare, J; et al.

    2009-01-01

    Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.

  17. Single channel evidence for innate pore-formation by Vibrio parahaemolyticus thermostable direct haemolysin (TDH) in phospholipid bilayers.

    Science.gov (United States)

    Hardy, Simon P; Nakano, Masayuki; Iida, Tetsuya

    2004-11-01

    Vibrio parahaemolyticus thermostable direct haemolysin (TDH) is widely considered to be a pore-forming toxin. The protein has no significant homology to other known pore-forming toxins and its mechanism of action in vivo remains undefined. We demonstrate single channel pore-forming activity of V. parahaemolyticus TDH in planar lipid bilayers. Channel conductance ranged between 30-450 pS in 0.5 M KCl with a calculated cation selectivity (P(K)/P(Cl)) of 2.7. Channels were formed in NaCl and choline-Cl with and without cholesterol present and in the presence of neutral or negatively charged phospholipids. Zinc ions did not block pore formation. Whilst various techniques have previously suggested that TDH is a pore-forming toxin, the data in this study provide direct single channel evidence and indicate several features of pore formation in synthetic phospholipid membranes.

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

    DEFF Research Database (Denmark)

    Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

    2017-01-01

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

  19. Thuringiensin: A Thermostable Secondary Metabolite from Bacillus thuringiensis with Insecticidal Activity against a Wide Range of Insects

    Directory of Open Access Journals (Sweden)

    Xiaoyan Liu

    2014-07-01

    Full Text Available Thuringiensin (Thu, also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis. It has insecticidal activity against a wide range of insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species. The chemical formula of Thu is C22H32O19N5P, and it is composed of adenosine, glucose, phosphoric acid, and gluconic diacid. In contrast to the more frequently studied insecticidal crystal protein, Thu is not a protein but a small molecule oligosaccharide. In this review, a detailed and updated description of the characteristics, structure, insecticidal mechanism, separation and purification technology, and genetic determinants of Thu is provided.

  20. Improving Bread Quality with the Application of a Newly Purified Thermostable α-Amylase from Rhizopus oryzae FSIS4

    Science.gov (United States)

    Ait Kaki El-Hadef El-Okki, Amel; Gagaoua, Mohammed; Bourekoua, Hayat; Hafid, Kahina; Bennamoun, Leila; Djekrif-Dakhmouche, Shahrazed; El-Hadef El-Okki, Mohamed; Meraihi, Zahia

    2017-01-01

    A new thermostable α-amylase from Rhizopus oryzae FSIS4 was purified for first time and recovered in a single step using a three-phase partitioning (TPP) system. The fungal α-amylase, at a concentration of 1.936 U per kg of flour, was used in bread-making and compared to the commercial enzyme. The results showed a significant effect of the recovered α-amylase in the prepared bread and allowed us to improve the quality of the bread. The study indicated clearly that the recovered α-amylase is a potential candidate for future applications in the bread-making industry and in other food biotechnology applications. PMID:28231081

  1. Immobilization of Thermostable Lipase QLM on Core-Shell Structured Polydopamine-Coated Fe3O4 Nanoparticles

    Directory of Open Access Journals (Sweden)

    Chenhui Wang

    2017-02-01

    Full Text Available Here, core-shell structured polydopamine-coated Fe3O4 nanoparticles were constructed to immobilize thermostable lipase QLM from Alcaligenes sp. Systematical characterization indicated that lipase QLM was successfully immobilized on the surface of nanoparticles with an enzyme loading of 21.4 ± 1.47 mg/g immobilized enzyme. Then, the immobilized enzyme was demonstrated to possess favorable catalytic activity and stability in the ester hydrolysis, using p-nitrophenyl caprylate as the substrate. Further, it was successfully employed in the kinetic resolution of (R, S-2-octanol, and satisfactory enantioselectivity and recyclability could be obtained with an enantiomeric ratio (E of 8–15 over 10 cycle reactions. Thus, core-shell structured polydopamine-coated Fe3O4 nanoparticles can be potentially used as a carrier for enzyme immobilization to improve their activity, stability, and reusability, which is beneficial for constructing efficient catalysts for industrial biocatalysis.

  2. Thuringiensin: a thermostable secondary metabolite from Bacillus thuringiensis with insecticidal activity against a wide range of insects.

    Science.gov (United States)

    Liu, Xiaoyan; Ruan, Lifang; Peng, Donghai; Li, Lin; Sun, Ming; Yu, Ziniu

    2014-07-25

    Thuringiensin (Thu), also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis. It has insecticidal activity against a wide range of insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species. The chemical formula of Thu is C22H32O19N5P, and it is composed of adenosine, glucose, phosphoric acid, and gluconic diacid. In contrast to the more frequently studied insecticidal crystal protein, Thu is not a protein but a small molecule oligosaccharide. In this review, a detailed and updated description of the characteristics, structure, insecticidal mechanism, separation and purification technology, and genetic determinants of Thu is provided.

  3. Purification and Characterization of a Lipase with High Thermostability and Polar Organic Solvent-Tolerance from Aspergillus niger AN0512.

    Science.gov (United States)

    Liu, Guang; Hu, Songqing; Li, Lin; Hou, Yi

    2015-11-01

    An extracellular lipase (EC 3.1.1.3, AN0512Lip) from Aspergillus niger AN0512 was purified and its characteristics were investigated. After the process of ammonium sulfate precipitation followed by ion-exchange chromatography and gel filtration, the purified lipase was achieved with 203.6-fold purification and 22.1 % recovery. AN0512Lip exhibited the highest activity at 50 °C and pH 5.0. It was thermostable and pH-stable, as indicated by that more than 50 % activity retained at 60 °C for 20 h and more than 90 % activity retained at pH 3.0 for 20 h, respectively. AN0512Lip activity was stimulated by some divalent metal ions (especially Cu(2+), Ca(2+)), while greatly suppressed by EDTA, indicating that AN0512Lip was a metal-activated enzyme. Moreover, AN0512Lip exhibited high tolerance for various polar organic solvents with log P lipase activity (476 % of its original activity) was achieved after addition of 90 % (V/V) isopropanol to the reaction mixture. AN0512Lip also displayed 3-regiospecificity and great affinity for the long-chain fatty ester. The preliminary test showed that AN0512Lip was a candidate for enriching EPA and DHA in fish oil. All the unique properties, such as thermostability, Cu(2+)-dependent, 3-regiospecificity, and polar organic solvent-tolerance, indicated that AN0512Lip could have potential applications in the food industry, even in organic synthesis and the pharmaceutical industry.

  4. Characteristics of a Novel Highly Thermostable and Extremely Thermophilic Alkalitolerant Amylase from Hyperthermophilic Bacillus Strain HUTBS71

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    Hazem Akel

    2009-01-01

    Full Text Available Problem statement: This study reported the purification and characterization of a novel highly thermostable alkaline amylase from a newly isolated Bacillus strain HUTBS71. Approach: The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. Results: Maximum amylase activity (72 U mL-1 was obtained at 100°C after 10 min of incubation. The enzyme was purified 24 fold with 12.5% yield and showed a monomer band with a molecular weight of 58.8 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH and temperature, 7.8 and 100°C, respectively. It performed stability over a broad range of pH and temperature, 5.2-10.0 and 80-115°C, respectively. The half-life of the enzyme at 90 and 100°C was estimated to be 3 h. The activation energy of denaturation of purified enzyme was 2.53 kJ moL-1. The enzyme was activated by 5 mM of CoCl2, MgSO4, MnCl2, ZnSO4 and MnSO4 (relative activity was 133, 126, 133, 106.6 and 103%, respectively. It was strongly inhibited by CuSO4 and CdCl2 but less affected by NaCl, CaCl2, FeCl3, ZnCl2 and EDTA. Conclusion: The present purified amylase therefore could be defined as a highly thermostable, extremely hyperthermophilic and alkalitolerant with new properties make the present enzyme applicable for many starch processing and food industries.

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

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    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

  6. Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll Protein Complexes

    Institute of Scientific and Technical Information of China (English)

    Yunlai Tang; Mei Chen; Yinong Xu; Tingyun Kuang

    2007-01-01

    We studied the difference in thermostability of photosystem Ⅱ (PSⅡ) and leaf lipid composition between a T-DNA insertion mutant rice (Oryza sativa L.) VG28 and its wild type Zhonghua11. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSⅡ photochemistry (Fv/Fm) and oxygen-evolving activity of PSⅡ in leaves significantly decreased with increasing temperature. However, the PSⅡ activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of unsaturation of membrane lipids on the thermostability of PSll are discussed.

  7. Occurrence of urease-positive Vibrio parahaemolyticus in Kanagawa, Japan, with specific reference to presence of thermostable direct hemolysin (TDH) and the TDH-related-hemolysin genes.

    Science.gov (United States)

    Osawa, R; Okitsu, T; Morozumi, H; Yamai, S

    1996-02-01

    A total of 132 strains of V. parahaemolyticus isolated from patients and from the suspected causal food items of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were examined for the ability to hydrolyze urea, with specific reference to the presence of the thermostable direct hemolysin gene (tdh) and the gene for thermostable direct hemolysin-related hemolysin (trh). Ten strains belonging to five different O-antigen serotypes were positive for urea hydrolysis (UH+), and four of these strains did not carry tdh. A total of 106 strains carried tdh, but less than 6% of them were UH+, whereas all trh-carrying strains were UH+. The evidence suggests that urea hydrolysis is not a reliable marker for identifying tdh-carrying V. parahaemolyticus strains in Japan (the Pacific Northeast) but may be a marker for trh-carrying strains.

  8. Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases.

    Science.gov (United States)

    Kim, Jinyeong; Kim, Seungbum; Yoon, Sangyoung; Hong, Eunsoo; Ryu, Yeonwoo

    2015-08-01

    Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

  9. Improvement in thermostability of metagenomic GH11 endoxylanase (Mxyl) by site-directed mutagenesis and its applicability in paper pulp bleaching process.

    Science.gov (United States)

    Satyanarayana, Digvijay Verma T

    2013-12-01

    An attempt has been made for enhancing the thermostability of xylanase (Mxyl) retrieved from a compost-soil-based metagenomic library. The analysis of the structure of xylanase by molecular dynamics simulation revealed more structural fluctuations in β-sheets. When the surface of β-sheets was enriched with arginine residues by substituting serine/threonine by site-directed mutagenesis, the enzyme with four arginine substitutions (MxylM4) exhibited enhanced thermostability at 80 °C. The T 1/2 of MxylM4 at 80 °C, in the presence of birchwood xylan, increased from 130 to 150 min at 80 °C without any alteration in optimum pH and temperature and molecular mass. Improvement in thermostability of MxylM4 was corroborated by increase in T m by 6 °C over that of Mxyl. The K m of MxylM4, however, increased from 8.01 ± 0.56 of Mxyl to 12.5 ± 0.32 mg ml(-1), suggesting a decrease in the affinity as well as specific enzyme activity. The Mxyl as well as MxylM4 liberated chromophores and lignin-derived compounds from kraft pulp, indicating their applicability in pulp bleaching.

  10. 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.

  11. Thermo-stable carbon nanotube-TiO₂ nanocompsite as electron highways in dye-sensitized solar cell produced by bio-nano-process.

    Science.gov (United States)

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

    2015-07-17

    We produced a thermostable TiO2-(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 TiO2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO2-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 TiO2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO2 photoelectrodes.

  12. 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.

  13. Dimerization mediates thermo-adaptation, substrate affinity and transglycosylation in a highly thermostable maltogenic amylase of Geobacillus thermoleovorans.

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    Deepika Mehta

    Full Text Available BACKGROUND: Maltogenic amylases belong to a subclass of cyclodextrin-hydrolyzing enzymes and hydrolyze cyclodextrins more efficiently than starch unlike typical α-amylases. Several bacterial malto-genic amylases with temperature optima of 40-60°C have been previously characterized. The thermo-adaption, substrate preferences and transglycosylation aspects of extremely thermostable bacterial maltogenic amylases have not yet been reported. METHODOLOGY/PRINCIPAL FINDINGS: The recombinant monomeric and dimeric forms of maltogenic α-amylase (Gt-Mamy of the extremely thermophilic bacterium Geobacillus thermoleovorans are of 72.5 and 145 kDa, which are active optimally at 80°C. Extreme thermostability of this enzyme has been explained by analyzing far-UV CD spectra. Dimerization increases T1/2 of Gt-Mamy from 8.2 h to 12.63 h at 90°C and mediates its enthalpy-driven conformational thermostabilization. Furthermore, dime-rization regulates preferential substrate binding of the enzyme. The substrate preference switching of Gt-Mamy upon dimerization has been confirmed from the substrate-binding affinities of the enzyme for various high and low molecular weight substrates. There is an alteration in Km and substrate hydrolysis efficiency (Vmax/Km of the enzyme (for cyclodex-trins/starch upon dimerization. N-terminal truncation indicated the role of N-terminal 128 amino acids in the thermostabilization and modulation of substrate-binding affinity. This has been confirmed by molecular docking of β-cyclodextrin to Gt-Mamy that indicated the requirement of homodimer formation by the interaction of a few N-terminal residues of chain A with the catalytic residues of (α/β8 barrel of chain B and vice-versa for stable cyclodextrin binding. Site directed mutagenesis provided evidence for the role of N-terminal D109 at the dimeric interface in substrate affinity modulation and thermostabilization. The dimeric Gt-Mamy transglycosylates hydrolytic products of G4/G

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

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    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.

  15. Characterizing and improving the thermostability of purified xylanase from Aspergillus niger DFR-5 grown on solid-state-medium

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    Ajay Pal

    2010-12-01

    Full Text Available  The thermostability of absolutely purified xylanase from Aspergillus niger DFR-5 was improved using polyols. Supplementation of sorbitol at 2M concentration was found to increase the half-life and D-value of xylanase at elevated temperatures (45-70ºC. Thermodynamic parameters associated with the process were analyzed revealing that the stability at higher temperatures was due to the increased enthalpy (∆Hº and free energy (∆Gº change of enzyme denaturation in the presence of sorbitol. The negative values of ∆Sº (-150.093 Jmol-1K-1 at 70ºC clearly indicated that enzyme underwent a significant process of aggregation during denaturation. The enzyme required divalent cations for maximum activity and inhibited by chelator. The diminution of activity by various thiol-binding agents and enhancement by reducing agents like β-ME confirmed the essentiality of cysteine for catalysis. The enzyme had a half-life and D-value of 277 and 921 days when stored at 4 ºC.

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

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    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.

  17. 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.

  18. Molecular cloning and characterization of a thermostable lipase from deep-sea thermophile Geobacillus sp. EPT9.

    Science.gov (United States)

    Zhu, Yanbing; Li, Hebin; Ni, Hui; Xiao, Anfeng; Li, Lijun; Cai, Huinong

    2015-02-01

    A gene (1,254 bp) encoding a lipase was identified from a deep-sea hydrothermal field thermophile Geobacillus sp. EPT9. The open reading frame of this gene encoded 417 amino acid residues. The gene was cloned, overexpressed in Escherichia coli, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 44.8 kDa. When p-nitrophenyl palmitate was used as a substrate, the recombinant lipase was optimally active at 55 °C and pH 8.5. The recombinant enzyme retained 44 % residual activity after incubation at 80 °C for 1 h, which indicated that Geobacillus sp. EPT9 lipase was thermostable. Homology modeling of strain EPT9 lipase was developed with the lipase from Bacillus sp. L2 as a template. The core structure exhibits an α/β-hydrolase fold and the typical catalytic triad might consist of Ser142, Asp346, and His387. The enzymatic activity of EPT9 lipase was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays an important role in the catalytic mechanism.

  19. 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.

  20. Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko

    2009-04-01

    Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

  1. Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization.

    Science.gov (United States)

    Kundu, Nidhi; Tichkule, Swapnil; Pandit, Shashi Bhushan; Chattopadhyay, Kausik

    2017-01-15

    Pore-forming toxins (PFTs) are typically produced as water-soluble monomers, which upon interacting with target cells assemble into transmembrane oligomeric pores. Vibrio parahaemolyticus thermostable direct hemolysin (TDH) is an atypical PFT that exists as a tetramer in solution, prior to membrane binding. The TDH structure highlights a core β-sandwich domain similar to those found in the eukaryotic actinoporin family of PFTs. However, the TDH structure harbors an extended C-terminal region (CTR) that is not documented in the actinoporins. This CTR remains tethered to the β-sandwich domain through an intra-molecular disulphide bond. Part of the CTR is positioned at the inter-protomer interface in the TDH tetramer. Here we show that the truncation, as well as mutation, of the CTR compromise tetrameric assembly, and the membrane-damaging activity of TDH. Our study also reveals that intra-protomer disulphide bond formation during the folding/assembly process of TDH restrains the CTR to mediate its participation in the formation of inter-protomer contact, thus facilitating TDH oligomerization. However, once tetramerization is achieved, disruption of the disulphide bond does not affect oligomeric assembly. Our study provides critical insights regarding the regulation of the oligomerization mechanism of TDH, which has not been previously documented in the PFT family.

  2. Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus.

    Science.gov (United States)

    Ohnishi, Kiyouhisa; Nakahira, Kumiko; Unzai, Satoru; Mayanagi, Kouta; Hashimoto, Hiroshi; Shiraki, Kentaro; Honda, Takeshi; Yanagihara, Itaru

    2011-05-01

    The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.

  3. 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 Ca(2+), but inhibited by Al(3+), Mn(2+), Zn(2+), and Cu(2+) at a tested concentration of 5 mM.

  4. 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.

  5. 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.

  6. Novel thermostable lipase from Bacillus circulans IIIB153: comparison with the mesostable homologue at sequence and structure level.

    Science.gov (United States)

    Johri, S; Bhat, A; Sayed, S; Nargotra, A; Jain, A; Qazi, G N

    2012-01-01

    Thermophilic Bacillus circulans IIIB153 isolated from hot springs of North West Himalayas, India, produced an extracellular lipase, which exhibited significant biofilm disruption property on the static biofilm disruption model with a single species of Actinomyces viscosous. The gene encoding the lipase was cloned and overexpressed in Escherichia coli. Recombinant Bacillus circulans lipase (BCL), a monomer with molecular mass of 43 kDa also exhibited significant biofilm disruption activity. The enzyme was optimally active at 60°C, pH 8.5 and retained >70% of its original activity after 1 h incubation at 60°C. 3D structure of BCL developed by homology modeling showed a typical α/β hydrolase fold, a characteristic feature of lipolytic enzymes. Comparison of thermostable BCL with mesostable lipase from Chromobacterium viscosum at the sequence and structure level showed distinct variations in the structural features, with the presence of a high content of proline residues, aromatic amino acids and salt bridges. These features along with the presence of zinc-binding site observed in BCL structure could have a potential role in thermal stability of the enzyme.

  7. Characterization of a Thermostable 8-Oxoguanine DNA Glycosylase Specific for GO/N Mismatches from the Thermoacidophilic Archaeon Thermoplasma volcanium

    Directory of Open Access Journals (Sweden)

    Miki Fujii

    2016-01-01

    Full Text Available The oxidation of guanine (G to 7,8-dihydro-8-oxoguanine (GO forms one of the major DNA lesions generated by reactive oxygen species (ROS. The GO can be corrected by GO DNA glycosylases (Ogg, enzymes involved in base excision repair (BER. Unrepaired GO induces mismatched base pairing with adenine (A; as a result, the mismatch causes a point mutation, from G paired with cytosine (C to thymine (T paired with adenine (A, during DNA replication. Here, we report the characterization of a putative Ogg from the thermoacidophilic archaeon Thermoplasma volcanium. The 204-amino acid sequence of the putative Ogg (TVG_RS00315 shares significant sequence homology with the DNA glycosylases of Methanocaldococcus jannaschii (MjaOgg and Sulfolobus solfataricus (SsoOgg. The six histidine-tagged recombinant TVG_RS00315 protein gene was expressed in Escherichia coli and purified. The Ogg protein is thermostable, with optimal activity near a pH of 7.5 and a temperature of 60°C. The enzyme displays DNA glycosylase, and apurinic/apyrimidinic (AP lyase activities on GO/N (where N is A, T, G, or C mismatch; yet it cannot eliminate U from U/G or T from T/G, as mismatch glycosylase (MIG can. These results indicate that TvoOgg-encoding TVG_RS00315 is a member of the Ogg2 family of T. volcanium.

  8. The structure of a mutant enzyme of Coprinus cinereus peroxidase provides an understanding of its increased thermostability.

    Science.gov (United States)

    Houborg, Karen; Harris, Pernille; Poulsen, Jens-Christian Navarro; Schneider, Palle; Svendsen, Allan; Larsen, Sine

    2003-06-01

    Seven amino-acid substitutions introduced into the 343 amino-acid-long sequence of Coprinus cinereus peroxidase (CiP) led to a mutant enzyme (TS-rCiP) which is more stable than the native enzyme at higher temperature, pH and hydrogen peroxide concentrations. It is therefore more suitable for industrial applications. A structure determination was conducted on a deglycosylated but still active form of TS-rCiP based on X-ray diffraction data to 2.05 A resolution measured on a crystal cooled to 100 K and refined to R = 0.202 and R(free) = 0.249. The increased stability of the TS-rCiP enzyme can be understood from the structural changes of the TS-rCiP structure revealed by a comparative analysis with other known CiP structures. One of the more significant changes caused by three of the substitutions, I49S, V53A and T121A, is the conversion of a hydrophobic pocket into a hydrophilic pocket with associated changes in the water structure and the hydrogen-bonding interactions. The E239G substitution, which gives rise to increased thermostability at high pH, creates changes in the water structure and in the orientation of a phenylalanine (Phe236) in its vicinity. The three substitutions M166F, M242 and Y242F introduced to increase the oxidative stability do not introduce any structural changes.

  9. Fermentative Production and Thermostability Characterization of Amylase from Aspergillus Species and Its Application Potential Evaluation in Desizing of Cotton Cloth

    Directory of Open Access Journals (Sweden)

    Murali Krishna Chimata

    2011-01-01

    Full Text Available The production of extracellular amylase was investigated employing our laboratory isolate, Aspergillus niger sp. MK 07 and effect of process variables on enzyme production, was studied in a fermentor. It was found that amylase production was maximum when the fermentor volume was maintained at 70%, rate of agitation at 250 rpm, air supply at 2.5 vvm, inoculum concentration of 10%, and a pH of 5.0. Highest enzyme production obtained under all optimized conditions was 1734 U/mL with sucrose as carbon substrate and corn steep liquor as nitrogen source. Enzyme purification studies by ammonium sulphate precipitation and Sephadex G-100 chromatography was evaluated for obtaining purified enzyme. Thermostability of amylase were evaluated with varying concentrations from 0.2 to 0.5 M concentrations of calcium chloride and the highest activity obtained was 3115 U/mL with 0.3 M calcium chloride at 55°C. Effect of temperature and pH on the activity of purified enzyme was evaluated and the purified enzyme showed an activity till 75°C and a pH of 6.5. Application potential of partially purified alpha amylase on desizing of cotton cloth was evaluated with varying enzyme concentrations from 50 to 500 U/mL and the highest desizing activity was found to be at 300 U/mL.

  10. Production of xylooligosaccharides from the steam explosion liquor of corncobs coupled with enzymatic hydrolysis using a thermostable xylanase.

    Science.gov (United States)

    Teng, Chao; Yan, Qiaojuan; Jiang, Zhengqiang; Fan, Guangsen; Shi, Bo

    2010-10-01

    The production of xylooligosaccharides (XOs) from corncobs was studied using a two-stage process based on a steam explosion pretreatment followed by enzymatic hydrolysis. Corncobs with different chip sizes were subjected to steam explosion under different experimental conditions of temperature and time, namely 188-204 degrees C for 2.5-7.5 min. The results indicate that corncobs were optimally steam exploded at 196 degrees C for 5 min, resulting in hemicellulose recovery of 22.8%. Especially, corncobs with large chip sizes (approximately 100 mm) during steam explosion pretreatment were suitable to produce XOs. Furthermore, a thermostable xylanase from Paecilomyces themophila J18 was used to hydrolyze steam explosion liquor of corncobs (SELC) for the production of XOs. A maximum XOs yield of 28.6 g XOs/100 g xylan in corncobs was achieved and XOs syrup contained more than 90% of xylobiose and xylotriose when the hydrolysis was carried out under the optimized conditions (pH 7.0, 70 degrees C, 7.5 U mL(-1) and 2.5 h). These results suggest that the process might be effective in production of XOs for industrial applications.

  11. Engineering a high-performance, metagenomic-derived novel xylanase with improved soluble protein yield and thermostability.

    Science.gov (United States)

    Qian, Changli; Liu, Ning; Yan, Xing; Wang, Qian; Zhou, Zhihua; Wang, Qianfu

    2015-03-01

    The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340U/mg) and broad pH active range of 5.5-10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55°C, with a 10°C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60°C for 4h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.

  12. Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase produced by Moniliophthora perniciosa

    Directory of Open Access Journals (Sweden)

    Amanda R. Sena

    2011-06-01

    Full Text Available The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM was employed to evaluate the effect of variables, including inducer (yeast extract and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70% and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

  13. Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain.

    Science.gov (United States)

    Ben Ali, Mamdouh; Khemakhem, Bassem; Robert, Xavier; Haser, Richard; Bejar, Samir

    2006-02-15

    The implications of Asn315 and Val450 in the atypical starch hydrolysis profile of Bacillus stearothermophilus Amy (a-amylase) US100 have been suggested previously [Ben Ali, Mhiri, Mezghani and Bejar (2001) Enzyme Microb. Tech. 28, 537-542]. In order to confirm this hypothesis, three mutants were generated. Of these two have a single mutation, N315D or V450G, whereas the third contains both mutations. Analysis of the starch breakdown-profile of these three mutants, as well as of the wild-type, allowed us to conclude that each single mutation induces a small variation in the hydrolysis product. However, the major end product produced by the double mutant shifts from maltopentaose/maltohexaose to maltose/maltotriose, confirming the involvement of these two residues in starch hydrolysis. The superimposition of AmyUS100 model with that of Bacillus licheniformis shows in AmyUS100 an additional loop containing residues Ile214 and Gly215. Remarkably, the deletion of these two residues increases the half-life at 100 degrees C from 15 min to approx. 70 min. Moreover, this engineered amylase requires less calcium, 25 p.p.m. instead of 100 p.p.m., to reach maximal thermostability.

  14. 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.

  15. The thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus: Sequence variation and implications for detection and function.

    Science.gov (United States)

    Nilsson, William B; Turner, Jeffrey W

    2016-07-01

    Vibrio parahaemolyticus is a leading cause of bacterial food-related illness associated with the consumption of undercooked seafood. Only a small subset of strains is pathogenic. Most clinical strains encode for the thermostable direct hemolysin (TDH) and/or the TDH-related hemolysin (TRH). In this work, we amplify and sequence the trh gene from over 80 trh+strains of this bacterium and identify thirteen genetically distinct alleles, most of which have not been deposited in GenBank previously. Sequence data was used to design new primers for more reliable detection of trh by endpoint PCR. We also designed a new quantitative PCR assay to target a more conserved gene that is genetically-linked to trh. This gene, ureR, encodes the transcriptional regulator for the urease gene cluster immediately upstream of trh. We propose that this ureR assay can be a useful screening tool as a surrogate for direct detection of trh that circumvents challenges associated with trh sequence variation.

  16. Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage.

    Science.gov (United States)

    Flory, Justin D; Johnson, Trey; Simmons, Chad D; Lin, Su; Ghirlanda, Giovanna; Fromme, Petra

    2014-12-15

    PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled NA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.

  17. Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its arrhenius effect and biophysical properties.

    Science.gov (United States)

    Wang, Yu-Kuo; Huang, Sheng-Cih; Wu, Yi-Fang; Chen, Yu-Ching; Lin, Yen-Ling; Nayak, Manoswini; Lin, Yan Ren; Chen, Wen-Hung; Chiu, Yi-Rong; Li, Thomas Tien-Hsiung; Yeh, Bo-Sou; Wu, Tung-Kung

    2011-03-31

    Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 °C but is reactivated by additional heating above 80 °C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDH(WT). Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.

  18. Purification and Characterization of a New Thermostable κ-Carrageenase from the Marine Bacterium Pseudoalteromonas sp.QY203

    Institute of Scientific and Technical Information of China (English)

    LI Shangyong; JIA Panpan; WANG Linna; YU Wengong; HAN Feng

    2013-01-01

    A new extracellular κ-carrageenase,namely CgkP,34.0 kDa in molecular weight,was purified from Pseudoalteromonas sp.QY203.CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2.Sodium chloride increased its activity markedly,and KC1 increased its activity slightly.The divalent and trivalent metal ions including Cu2+,Ni2+,Zn2+,Mn2+,A13+ and Fe3+ significantly inhibited its activity,while Mg2+ did not.CgkP remained 70% of original activity after being incubated at 40℃ for 48 h,and remained 80% of the activity after being incubated at 45 ℃ for 1 h.It exhibited endo-κ-carrageenase activity,mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide.CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.

  19. Cloning, molecular modeling, and docking analysis of alkali-thermostable β-mannanase from Bacillus nealsonii PN-11.

    Science.gov (United States)

    Chauhan, Prakram Singh; Tripathi, Satya Prakash; Sangamwar, Abhays T; Puri, Neena; Sharma, Prince; Gupta, Naveen

    2015-11-01

    An alkali-thermostable β-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. The ORF encoding mannanase consisted of 1100 bp, corresponding to protein of 369 amino acids and has a catalytic domain belonging to glycoside hydrolase family 5. Cloned mannanase was smaller in size than the native mannanase by 10 kDa. This change in molecular mass could be because of difference in the glycosylation. The tertiary structure of the β-mannanase (MANPN11) was designed and it showed a classical (α/β) TIM-like barrel motif. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287. Model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive which might be responsible for the stability of the enzymes at high pH. Stability of MANPN11 at alkaline pH was further supported by the formation of a hydrophobic pocket near active site of the enzyme. To understand the ability of MANPN11 to bind with different substrates, docking studies were performed and found that mannopentose fitted properly into active site and form stable enzyme substrate complex.

  20. Extraction and purification of a highly thermostable alkaline caseinolytic protease from wastes Penaeus vannamei suitable for food and detergent industries.

    Science.gov (United States)

    Dadshahi, Zahra; Homaei, Ahmad; Zeinali, Farrokhzad; Sajedi, Reza H; Khajeh, Khosro

    2016-07-01

    A novel thermostable protease was purified from Penaeus vannamei from Persian Gulf to homogeneity level using ammonium sulfate precipitation and anion-exchange chromatography. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 24kDa on SDS-PAGE. The enzyme showed the broad highest catalytic activity for hydrolysis of the substrate with maximal activity at pH 7 and 80°C. Activity of the enzyme was inhibited by Hg(2+), Zn(2+) Co(2+) and Cu(2+), while protease activity was increased in the presence of Fe(2+) and Mn(2+) by factors of 173% and 102%, respectively. Enzyme shows a broad substrate specificity and hydrolyzes both natural and synthetic substrates. Based on the Michaelis-Menten plots, the Km with casein as substrate was 16.8μM and Vmax was 82.6μM/min. The enzyme, derived from L. vannamei, possesses unique characteristics and could be used in various industrial and biotechnological applications.

  1. 两种非单调信赖域算法的数值比较研究%A Numerical Comparison between Two Classes of Non-monotone Trust Region Algorithms

    Institute of Scientific and Technical Information of China (English)

    陈俊; 张纯

    2011-01-01

    This paper is aimed at the comparison between two typical non-monotone trust region algorithms for unconstrained optimization. In theory, they both have good convergence properties. The numerical efficiency of the two non-monotone algorithms is the focus of the comparison. Extensive numerical experiments were conducted, making use of the well-known test problems package by J.J. Mot6 et al. [ ACM Transactions on Mathematical Software, 1981,7( 1 ) : 17-41 ]. Then the two algorithms were compared by the performance profiles based on the data obtained from the numerical experiments. The analysis indicates that the numerical efficiency of the algorithm NATR is superior to that of the traditional non-monotone trust region algorithm to a certain extent.%目前求解无约束最优化问题的非单调信赖域算法根据其采用的参考函数值的不同主要有两种:一种是传统的基于最大函数值型,一种是基于函数值加权平均型.理论上该两种算法均具有较好的收敛性质,但关于这两种非单调信赖域算法在实际数值计算效率方面的比较还不充分.为此作者利用国际上广泛采用的无约束优化测试函数包(J.J.More et al.ACM Transactions on Mathematical Software,1981,7(1):17—41)对这两种方法进行大量的数值试验,并采用剖面分析方法对试验数据进行较全面的分析比较,结果表明基于函数值平均权重的非单调信赖域算法其数值效率在一定程度上优于传统的非单调信赖域算法.

  2. 基于投影数据非单调性全变分恢复的低剂量CT重建%Nonmonotone Total Variation Minimization Based Projection Restoration for Low-Dose CT Reconstruction

    Institute of Scientific and Technical Information of China (English)

    钱姗姗; 黄静; 马建华; 张华; 刘楠; 张喜乐; 冯前进; 陈武凡

    2011-01-01

    为获取低剂量CT图像的优质重建,本文提出一种基于投影数据非单调性全变分恢复的低剂量CT重建方法.新方法首先通过非线性Anscombe变换将满足Poisson分布的投影数据转化为近似Gaussian分布,其后对变换后的Gaussian型数据进行非单调性全变分最小化算法(Nonmonotone Total Variation Minimization,NTVM)滤波,最后对Anscombe逆变换数据实现传统的滤波反投影(Filtered Back Projection,FBP) CT重建.仿真和临床低剂量CT重建实验表明,本文方法在噪声清除、伪影抑制和缩短重建时间等方面均有上佳表现.%In order to improve the reconstruction quality of low-dose CT image,a new approach is proposed based on low-dose CT projection restoration in this paper. First, projection data is transformed from Poisson distribution to Gaussian distribution using nonlinear Anscombe transform. Then, the Anscombe transformed data is filtered by an efficient nonmonotone total variation minimization denoising algorithm. Last, the reconstruction is achieved by inverse Anscombe transform and filtered back projection (FBP) method. Simulated and clinical low-dose CT data experimental results demonstrate that a high-quality CT image can be reconstructed.

  3. 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.

  4. Screening the thermophilic and hyperthermophilic bacterial population of three Iranian hot-springs to detect the thermostable α- amylase producing strain

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    A Sajjadian

    2010-06-01

    Full Text Available Background: Screening is a routine procedure for isolation of microorganisms which are able to produce special metabolites. Purified thermostable α-amylase from bacterial sources is widely used in different industries. In this study we analyzed samples collected from three different hot springs in Iran to detect any strains capable of producing thermostable α-amylase."nMaterials and Methods: Hot water samples from Larijan (67°C, pH 6.5, Mahallat (46°C, pH 7, and Meshkinshahr (82°C, pH 6, were cultivated in screening starch agar plates and incubated at 65°C for 24 hours. Thereafter, the plates were stained with Gram's iodine solution."nResults and Discussion: The bacterial colonies from the Meshkinshahr hot-spring produced the largest haloforming zone. Based on the phenotypic tests, the strain was identified as Bacillus sp. The culture condition was optimized for biosynthesis of α-amylase. The enzyme was produced at maximum level when it was incubated at 70 °C in the presence of soluble starch (1% at pH 6. The addition of calcium (10 mM and peptone (1% to the mineral medium, shortened the lag period and improved the growth and α-amylase synthesis. The addition of glucose (1% to the culture greatly diminished the syntheses of α -amylase. Importantly, the enzyme extract retained 100% activity when incubated for 45 minutes at 100°C."nConclusion: The Meshkinshahr hot-spring is rich in the Bacillus spp thermostable α-amylase producing strain of the thermophilic bacterial population. Iranian hot-springs like Meshkinshahr, have large microbial storages and can be used as sources of different biological products like enzymes. The enzyme which was produced with Bacillus sp. could hydrolyse polymers like starch and was used at laboratory scale successfully.

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

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    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.

  6. Higher thermostability of l-lactate dehydrogenases is a key factor in decreasing the optical purity of d-lactic acid produced from Lactobacillus coryniformis.

    Science.gov (United States)

    Gu, Sol-A; Jun, Chanha; Joo, Jeong Chan; Kim, Seil; Lee, Seung Hwan; Kim, Yong Hwan

    2014-05-10

    Lactobacillus coryniformis is known to produce d-lactic acid as a dominant fermentation product at a cultivation temperature of approximately 30°C. However, the considerable production of l-lactic acid is observed when the fermentation temperature is greater than 40°C. Because optically pure lactates are synthesized from pyruvate by the catalysis of chiral-specific d- or l-lactate dehydrogenase, the higher thermostability of l-LDHs is assumed to be one of the key factors decreasing the optical purity of d-lactic acid produced from L. coryniformis at high temperature. To verify this hypothesis, two types of d-ldh genes and six types of l-ldh genes based on the genomic information of L. coryniformis were synthesized and expressed in Escherichia coli. Among the LDHs tested, five LDHs showed activity and were used to construct polyclonal antibodies. d-LDH1, l-LDH2, and l-LDH3 were found to be expressed in L. coryniformis by Western blotting analysis. The half-life values (t1/2) of the LDHs at 40°C were estimated to be 10.50, 41.76, and 2311min, and the T50(10) values were 39.50, 39.90, and 58.60°C, respectively. In addition, the Tm values were 36.0, 41.0, and 62.4°C, respectively, which indicates that l-LDH has greater thermostability than d-LDH. The higher thermostability of l-LDHs compared with that of d-LDH1 may be a major reason why the enantiopurity of d-lactic acid is decreased at high fermentation temperatures. The key enzymes characterized will suggest a direction for the design of genetically modified lactic acid bacteria to produce optically pure d-lactic acid. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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    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.

  8. Properties of thermostable hemicellulolytic enzymes from Thermomonospora strain 29 grown in solid state fermentation on coffee processing solid waste.

    Science.gov (United States)

    Srivastava, K C

    1993-01-01

    During decaffeination of Coffee Processing Plant Solid Wastes (CPSW) by actinomycetes, Thermomonospora, Strain 29 exhibited high titers of cellulase and xylanase. This organism, originally isolated on soybean seed coat was grown in solid state fermentation on CPSW supplemented with mineral salts. Enzymes recovered were arabinosidase, xylanase, and beta-D-xylosidase. Higher activity of the former two enzymes was in the extracellular broth, whereas the beta-D-xylosidase activity was highest in the cell fraction. The enzymes were characterized after precipitation with (NH(4))(2)SO(4), dialysis, and gel filtration. Production of all three enzymes was inhibited by monomeric sugars and sugar alcohols but not by arabinoxylan, xylans, or xylan containing water insoluble carbohydrates. The optimum pH for the activity was 6.5, 7.0, and 7.5 for beta-xylosidase, xylanase and arabinosidase (alpha-L-arabinofuranosidase, alpha-arabinosidase, alpha-L-arabinosidase) respectively. These enzymes were stable in the pH range of 6.5 to 8.0. All three enzymes were thermostable up to 80 degrees C. At 55 degrees C, arabinosidase had the longest half life of 120 h. However, at 40 degrees C, xylanase had the longest half life (504 h). At either temperature, beta-D-xylosidase had the shortest half life. The molecular weights (kDa), and Kms (mM) were estimated to be 95, 0.27; 45, 12.4; and 106, 0.67 for arbinosidase, xylanase, and beta-xylosidase respectively. Step wise addition of the three enzymes showed higher saccharification of lignocellulosics.

  9. A halotolerant thermostable lipase from the marine bacterium Oceanobacillus sp. PUMB02 with an ability to disrupt bacterial biofilms

    Science.gov (United States)

    Seghal Kiran, George; Nishanth Lipton, Anuj; Kennedy, Jonathan; Dobson, Alan DW; Selvin, Joseph

    2014-01-01

    A halotolerant thermostable lipase was purified and characterized from the marine bacterium Oceanobacillus sp. PUMB02. This lipase displayed a high degree of stability over a wide range of conditions including pH, salinity, and temperature. It was optimally active at 30 °C and pH 8.0 respectively and was stable at higher temperatures (50–70 °C) and alkaline pH. The molecular mass of the lipase was approximately 31 kDa based on SDS-PAGE and MALDI-ToF fingerprint analysis. Conditions for enhanced production of lipase by Oceanobacillus sp. PUMB02 were attained in response surface method-guided optimization with factors such as olive oil, sucrose, potassium chromate, and NaCl being evaluated, resulting in levels of 58.84 U/ml being achieved. The biofilm disruption potential of the PUMB02 lipase was evaluated and compared with a marine sponge metagenome derived halotolerant lipase Lpc53E1. Good biofilm disruption activity was observed with both lipases against potential food pathogens such as Bacillus cereus MTCC1272, Listeria sp. MTCC1143, Serratia sp. MTCC4822, Escherichia coli MTCC443, Pseudomonas fluorescens MTCC1748, and Vibrio parahemolyticus MTCC459. Phase contrast microscopy, scanning electron microscopy, and confocal laser scanning microscopy showed very effective disruption of pathogenic biofilms. This study reveals that marine derived hydrolytic enzymes such as lipases may have potential utility in inhibiting biofilm formation in a food processing environment and is the first report of the potential application of lipases from the genus Oceanobacillus in biofilm disruption strategies. PMID:25482232

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

    Science.gov (United States)

    Gururaj, P; Ramalingam, Subramanian; Nandhini Devi, Ganesan; Gautam, Pennathur

    2016-01-01

    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 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/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 45kDa 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°C 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 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  11. 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.

  12. Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus.

    Science.gov (United States)

    Kamruzzaman, Muhammad; Bhoopong, Phuangthip; Vuddhakul, Varaporn; Nishibuchi, Mitsuaki

    2008-09-15

    The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh+ strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh+ strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh+ sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh+ strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.

  13. Association of Vibrio parahaemolyticus thermostable direct hemolysin with lipid rafts is essential for cytotoxicity but not hemolytic activity.

    Science.gov (United States)

    Matsuda, Shigeaki; Kodama, Toshio; Okada, Natsumi; Okayama, Kanna; Honda, Takeshi; Iida, Tetsuya

    2010-02-01

    Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, induces cytotoxicity in cultured cells. However, the mechanism of TDH's cytotoxic effect including its target molecules on the plasma membrane of eukaryotic cells remains unclear. In this study, we identified the role of lipid rafts, cholesterol- and sphingolipid-enriched microdomains, in TDH cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (MbetaCD), a raft-disrupting agent, inhibited TDH cytotoxicity. TDH was associated with detergent-resistant membranes (DRMs), and MbetaCD eliminated this association. In contrast, there was no such association between a nontoxic TDH mutant and DRMs. The disruption of lipid rafts neither affected hemolysis nor inhibited Ca(2+) influx into HeLa cells induced by TDH. These findings indicate that the cytotoxicity but not the hemolytic activity of TDH is dependent on lipid rafts. The exogenous and endogenous depletion of cellular sphingomyelin also prevented TDH cytotoxicity, but a direct interaction between TDH and sphingomyelin was not detected with either a lipid overlay assay or a liposome absorption test. Treatment with sphingomyelinase (SMase) at 100 mU/ml disrupted the association of TDH with DRMs but did not affect the localization of lipid raft marker proteins (caveolin-1 and flotillin-1) with DRMs. These results suggest that sphingomyelin is important for the association of TDH with lipid rafts but is not a molecular target of TDH. We hypothesize that TDH may target a certain group of rafts that are sensitive to SMase at a certain concentration, which does not affect other types of rafts.

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

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    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.

  15. Swapping of pro-sequences between keratinases of Bacillus licheniformis and Bacillus pumilus: altered substrate specificity and thermostability.

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    Rajput, Rinky; Tiwary, Ekta; Sharma, Richa; Gupta, Rani

    2012-08-10

    Pro-sequences were swapped in cis between keratinases from Bacillus licheniformis (Ker BL) and Bacillus pumilus (Ker BP) to construct Ker ProBP-BL and Ker ProBL-BP, respectively. Expression of these keratinases was carried out constitutively by E. coli HB101-pEZZ18 system. They were characterized with respect to their parent enzymes, Ker BL and Ker BP, respectively. Ker ProBP-BL became more thermostable with a t(1/2) of 45 min at 80°C contrary to Ker BL which was not stable beyond 60°C. Similarly, the activity of Ker ProBP-BL on keratin and casein substrate, i.e. K:C ratio increased to 1.2 in comparison to 0.1 for Ker BL. Hydrolysis of insulin B-chain revealed that the cleavage sites increased to six from four in case of Ker ProBP-BL in comparison to Ker BL. However, cleavage sites decreased from seven to four in case of Ker ProBL-BP in comparison to the parent keratinase, Ker BP. Likewise, Ker ProBL-BP revealed altered pH and temperature kinetics with optima at pH 10 and 60°C in comparison to Ker BP which had optima at pH 9 and 70°C. It also cleaved soluble substrates with better efficiency in comparison to Ker BP with K:C ratio of 1.6. Pro-sequence mediated conformational changes were also observed in trans and were almost similar to the features acquired by the chimeras constructed in cis by swapping the pro-sequence region.

  16. [Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks].

    Science.gov (United States)

    Obata, Hiromi; Shimojima, Yukako; Konishi, Noriko; Monma, Chie; Yano, Kazuyoshi; Kai, Akemi; Morozumi, Satoshi; Fukuyama, Masafumi

    2006-07-01

    The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.

  17. Reduction of acrylamide level through blanching with treatment by an extremely thermostable L-asparaginase during French fries processing.

    Science.gov (United States)

    Zuo, Shaohua; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-07-01

    Bacterial L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid. It is normally used as an antineoplastic drug applied in lymphoblastic leukemia chemotherapy and as a food processing aid in baked or fried food industry to inhibit the formation of acrylamide. The present study demonstrates cloning, expression, and characterization of a thermostable L-asparaginase from Thermococcus zilligii AN1 TziAN1_1 and also evaluates the potential for enzymatic acrylamide mitigation in French fries using this enzyme. The recombinant L-asparaginase was purified to homogeneity by nickel-affinity chromatography. The purified enzyme displayed the maximum activity at pH 8.5 and 90 °C, and the optimum temperature was the highest ever reported. The K m, k cat, and k cat/K m values toward L-asparagine were measured to be 6.08 mM, 3267 s(-1), and 537.3 mM(-1) s(-1), respectively. The enzyme retained 70 % of its original activity after 2 h of incubation at 85 °C. When potato samples were treated with 10 U/mL of L-asparaginase at 80 °C for only 4 min, the acrylamide content in final French fries was reduced by 80.5 % compared with the untreated control. Results of this study revealed that the enzyme was highly active at elevated temperatures, reflecting the potential of the T. zilligii L-asparaginase in the food processing industry.

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

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    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.

  19. Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana

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    Mickiewicz Małgorzata

    2010-10-01

    Full Text Available Abstract Background In recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods. Results We report the characterization of single-stranded DNA binding proteins (SSBs from the thermophilic bacteria Thermotoga maritima (TmaSSB and Thermotoga neapolitana (TneSSB. They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino acid residues with a calculated molecular mass of 16.30 and 16.58 kDa, respectively. The similarity between amino acid sequences of these proteins is very high: 90% identity and 95% similarity. Surprisingly, both TmaSSB and TneSSB possess a quite low sequence similarity to Escherichia coli SSB (36 and 35% identity, 55 and 56% similarity, respectively. They are functional as homotetramers containing one single-stranded DNA binding domain (OB-fold in each monomer. Agarose mobility assays indicated that the ssDNA-binding site for both proteins is salt independent, and fluorescence spectroscopy resulted in a size of 68 ± 2 nucleotides. The half-lives of TmaSSB and TneSSB were 10 h and 12 h at 100°C, respectively. When analysed by differential scanning microcalorimetry (DSC the melting temperature (Tm was 109.3°C and 112.5°C for TmaSSB and TneSSB, respectively. Conclusion The results showed that TmaSSB and TneSSB are the most thermostable SSB proteins identified to date, offering an attractive alternative to TaqSSB and TthSSB in molecular biology applications, especially with using high temperature e. g. polymerase chain reaction (PCR.

  20. 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.

  1. Enzymatic activity of Lecithin:retinol acyltransferase: a thermostable and highly active enzyme with a likely mode of interfacial activation.

    Science.gov (United States)

    Horchani, Habib; Bussières, Sylvain; Cantin, Line; Lhor, Mustapha; Laliberté-Gemme, Jean-Sébastien; Breton, Rock; Salesse, Christian

    2014-06-01

    Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100°C. The results of this study highly improved our understanding of this enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Purification and partial characterization of a non-O1 Vibrio cholerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaemolyticus.

    OpenAIRE

    Yoh, M; Honda, T.; Miwatani, T

    1986-01-01

    A newly identified hemolysin (NAG-rTDH), which is related to the thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus produced by non-O1 Vibrio cholerae, was studied. NAG-rTDH was purified by successive column chromatographies on DEAE-cellulose and an immunoaffinity column coupled with anti-Vp-TDH immunoglobulin. The molecular weight of NAG-rTDH was estimated as 18,500, similar to that of Vp-TDH, as judged by sodium dodecyl sulfate slab gel electrophoresis, but its charge or mole...

  3. Development and Evaluation of a Rapid, Simple, and Sensitive Immunochromatographic Assay To Detect Thermostable Direct Hemolysin Produced by Vibrio parahaemolyticus in Enrichment Cultures of Stool Specimens

    OpenAIRE

    Kawatsu, Kentaro; Ishibashi, Masanori; Tsukamoto,Teizo

    2006-01-01

    Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numb...

  4. The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity.

    Science.gov (United States)

    García-Huante, Yolanda; Cayetano-Cruz, Maribel; Santiago-Hernández, Alejandro; Cano-Ramírez, Claudia; Marsch-Moreno, Rodolfo; Campos, Jorge E; Aguilar-Osorio, Guillermo; Benitez-Cardoza, Claudia G; Trejo-Estrada, Sergio; Hidalgo-Lara, María Eugenia

    2017-01-01

    A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

  5. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    Science.gov (United States)

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  6. A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp. – A robust, thermostable alternative to mezophilic prototype BbvI

    Indian Academy of Sciences (India)

    Joanna Zebrowska; Olga Zołnierkiewicz; Marta A Skowron; Agnieszka Zylicz-Stachula; Joanna Jezewska-Frackowiak; Piotr M Skowron

    2016-03-01

    Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers’ biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5′-GCAGC(N8/12)-3′ DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase-MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.

  7. ISOLATION OF A NEW PENICILLIUM CHRYSOGENUM STRAIN BF02 FROM AGRICULTURAL SOIL OF RURAL INDIA PRODUCING A THERMOSTABLE LOW KM CELLOBIASE

    Directory of Open Access Journals (Sweden)

    Samudra Prosad Banik

    2014-02-01

    Full Text Available A new strain of Penicillium chrysogenum producing a low Km, thermostable cellobiase was isolated from agricultural soil of rural West Bengal, India. The culture filtrate showed strong antibacterial activity against gram positive organisms, gave two cellobiase activity bands on native PAGE zymography and two peaks on sephacryl S200 gel chromatography. The higher molecular weight peak had a higher specific activity and was characterised further. It gave three bands corresponding to 130 kDa, 65 kDa and 55 kDa in SDS-PAGE and showed a Km of 0.196 mM against p- nitrophenyl β-D glucopyranoside. It was highly thermostable and retained full activity after one hour incubation at 55°C with temperature optima of 75°C. Its pH stability ranged between 4.5 and 8.5 with optimal activity at pH 5 and it retained more than 65% activity in presence of 100 mM guanidium hydrochloride and 2 M urea. Apart from Zn+2 and Hg+2, enzyme activity was not affected by other metal ions and in presence of Mg+2 and Mn+2, activity was boosted. It also retained more than 80% activity in presence of 0.5% glucose. This is one of the very few reports of an efficient cellobiase from the Penicillium genus which can be utilised for biotechnological applications.

  8. Nanotechnology enabled enhancement of enzyme activity and thermostability: study on impaired pectate lyase from attenuated Macrophomina phaseolina in presence of hydroxyapatite nanoparticle.

    Directory of Open Access Journals (Sweden)

    Nalok Dutta

    Full Text Available In this paper we show that hydroxyapatite nanoparticles (NP can not only act as a chaperon (by imparting thermostability but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP, APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy-enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time.

  9. Nanotechnology enabled enhancement of enzyme activity and thermostability: study on impaired pectate lyase from attenuated Macrophomina phaseolina in presence of hydroxyapatite nanoparticle.

    Science.gov (United States)

    Dutta, Nalok; Mukhopadhyay, Arka; Dasgupta, Anjan Kr; Chakrabarti, Krishanu

    2013-01-01

    In this paper we show that hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina) showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy-enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time.

  10. Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1.

    Science.gov (United States)

    Yu, Shuhuai; Wang, Xiao; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2016-08-10

    Inulin fructotransferase (IFTase) is an important enzyme that produces di-d-fructofuranose 1,2':2,3' dianhydride (DAF III), which is beneficial for human health. Present investigations mainly focus on screening and characterizing IFTase, including catalytic efficiency and thermostability, which are two important factors for enzymatic industrial applications. However, few reports aimed to improve these two characteristics based on the structure of IFTase. In this work, a structural model of IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed through homology modeling. Analysis of this model reveals that two residues, Ser-309 and Ser-333, may play key roles in the structural stability. Therefore, the functions of the two residues were probed by site-directed mutagenesis combined with the Nano-DSC method and assays for residual activity. In contrast to other mutations, single mutation of serine 309 (or serine 333) to threonine did not decrease the enzymatic stability, whereas double mutation (serine 309 and serine 333 to threonine) can enhance thermostability (by approximately 5 °C). The probable mechanisms for this enhancement were investigated.

  11. A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp.--A robust, thermostable alternative to mezophilic prototype BbvI.

    Science.gov (United States)

    Zebrowska, Joanna; Zolnierkiewicz, Olga; Skowron, Marta A; Zylicz-Stachula, Agnieszka; Jezewska-Frackowiak, Joanna; Skowron, Piotr M

    2016-03-01

    Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers' biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5'-GCAGC(N8/12)-3'DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (Λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of Λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase- MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.

  12. Geometry optimization and excited state properties of the new symmetric (E)-Stilbene derivative for application in thermostable polarizing PVA-films: A combined experimental and DFT approach

    Science.gov (United States)

    Shahab, Siyamak; Almodarresiyeh, Hora Alhosseini; Filippovich, Liudmila; Hajikolaee, Fatemeh Haji; Kumar, Rakesh; Darroudi, Mahdieh; Mashayekhi, Mahsa

    2016-09-01

    In the present work, quantum-chemical calculations used for the structural analysis of the new symmetric (E)-Stilbene derivative: (Potassium 2,2‧-((ethane-1,2-diylbis(4,1-phenylene))bis(azanediyl))diacetate) (P) (Trans isomer) using DFT/B3LYP/LanL2MB level of theory. The FT-IR and 1H NMR spectra of the compound P are presented. The electronic absorption spectrum of the P in aqueous medium was calculated using TDB3LYP/LanL2MB method. The excitation energies, electronic transitions and oscillator strengths for studied structure have also been calculated. The experimental, calculated UV/Vis and emission spectra of the P are presented and discussed. The geometry optimization of the molecule P in excited state and its electronic spectrum was carried out by the same method. In excited state the molecule P loses its symmetry and becomes unstable form. First time on the basis of polyvinyl alcohol (PVA) and the new designed structure P thermostable polarizing film absorbing at λmax = 410 nm was created. Polarizing Efficiency (PE) of obtained film is 96% at Stretching Degree (Rs) 3.0. In oriented PVA-films is the phenomenon of anisotropy of thermal conductivity (λ||/λ⊥) which is very important for development of thermostable polarizing films.

  13. Introduction of Pea DNA Helicase 45 Into Sugarcane (Saccharum spp. Hybrid) Enhances Cell Membrane Thermostability And Upregulation Of Stress-responsive Genes Leads To Abiotic Stress Tolerance.

    Science.gov (United States)

    Augustine, Sruthy Maria; Ashwin Narayan, J; Syamaladevi, Divya P; Appunu, C; Chakravarthi, M; Ravichandran, V; Tuteja, Narendra; Subramonian, N

    2015-05-01

    DNA helicases are motor proteins that play an essential role in nucleic acid metabolism, by providing a duplex-unwinding function. To improve the drought and salinity tolerance of sugarcane, a DEAD-box helicase gene isolated from pea with a constitutive promoter, Port Ubi 2.3 was transformed into the commercial sugarcane variety Co 86032 through Agrobacterium-mediated transformation, and the transgenics were screened for tolerance to soil moisture stress and salinity. The transgene integration was confirmed through polymerase chain reaction, and the V 0 transgenic events showed significantly higher cell membrane thermostability under normal irrigated conditions. The V 1 transgenic events were screened for tolerance to soil moisture stress and exhibited significantly higher cell membrane thermostability, transgene expression, relative water content, gas exchange parameters, chlorophyll content, and photosynthetic efficiency under soil moisture stress compared to wild-type (WT). The overexpression of PDH45 transgenic sugarcane also led to the upregulation of DREB2-induced downstream stress-related genes. The transgenic events demonstrated higher germination ability and better chlorophyll retention than WT under salinity stress. Our results suggest the possibility for development of increased abiotic stress tolerant sugarcane cultivars through overexpression of PDH45 gene. Perhaps this is the first report, which provides evidence for increased drought and salinity tolerance in sugarcane through overexpression of PDH45.

  14. Enhanced Thermostability of Lipoxygenase from Anabaena sp. PCC 7120 by Site-Directed Mutagenesis Based on Computer-Aided Rational Design.

    Science.gov (United States)

    Diao, Hanwen; Zhang, Chong; Wang, Shuicheng; Lu, Fengxia; Lu, Zhaoxin

    2016-04-01

    Lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX) was thermally unstable. So, improving the thermostability of the enzyme was quite essential. The target site of Ana-LOX selected for site-directed mutagenesis was based on computer-aided rational design. The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40. Compared to the wild-type enzyme which has a half-life (T 1/2) of inactivation of 3.8 min at 50 °C, the T 1/2 of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0 min, respectively. The double mutant V421A/V40A showed a synergistic effect with a T 1/2 value of 8.3 min, resulting in a 1.18-fold improvement compared to the original Ana-LOX. V421A, V40A, and V421A/V40A also obtained 4.83, 41.58, and 80.07 % increase in specific activity, respectively. This study provides useful theoretical reference for enzyme molecular modification and computer-aided rational design.

  15. Bioprocess exploration for thermostable α-amylase production of a deep-sea thermophile Geobacillus sp. in high-temperature bioreactor.

    Science.gov (United States)

    Jiang, Tao; Huang, Mengmeng; He, Hao; Lu, Jian; Zhou, Xiangshan; Cai, Menghao; Zhang, Yuanxing

    2016-08-17

    Geobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40 U/ml. Then, a high-temperature batch culture of the thermophile in a 15 l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600 rpm and 15 l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79 U/ml and productivity of 19804 U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.

  16. Rapid biodegradation of aflatoxin B1 by metabolites of Fusarium sp. WCQ3361 with broad working temperature range and excellent thermostability.

    Science.gov (United States)

    Wang, Cuiqiong; Li, Zhongyuan; Wang, Hui; Qiu, Haiyan; Zhang, Minghui; Li, Shuang; Luo, Xuegang; Song, Yajian; Zhou, Hao; Ma, Wenjian; Zhang, Tongcun

    2017-03-01

    Contamination of food and feed by aflatoxin B1 (AFB1) poses serious economic and health problems worldwide, so the development of biological methods for effective AFB1 degradation is strongly required. Among three AFB1-degrading microorganisms isolated from moldy peanut, Fusarium sp. WCQ3361 could remove AFB1 extremely effectively, with a degradation ratio of 70.20% after 1 min and 95.38% after 24 h. Its degradation ratio was not much affected by temperature change (0-90 °C) and it also displayed excellent thermostability, maintaining 99.40% residual activity after boiling for 10 min. Since protease K could reduce the AFB1 degradation ratio by 55.15%, it is proposed that the effective component for AFB1 degradation is a protein. The AFB1 degradation ability of Fusarium sp. WCQ3361 was further verified by feed stock detoxification and the MTT test with HepG2 cells. In addition, no degradation products were detected by preliminary liquid chromatography/mass spectrometry, suggesting that AFB1 might be metabolized to products with different chemical characteristics from AFB1. Fusarium sp. WCQ3361 is the first reported AFB1 degradation fungus belonging to the genus Fusarium with broad working temperature range, excellent thermostability and high activity, which provides a potential highly useful solution for dealing with AFB1 contamination in the human diet and animal feed. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  17. Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

    Science.gov (United States)

    Saito, Shioko; Iwade, Yoshito; Tokuoka, Eisuke; Nishio, Tomohiro; Otomo, Yoshimitsu; Araki, Emiko; Konuma, Hirotaka; Nakagawa, Hiroshi; Tanaka, Hiroyuki; Sugiyama, Kanji; Hasegawa, Akio; Sugita-Konishi, Yoshiko; Hara-Kudo, Yukiko

    2015-02-01

    Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

  18. Mechanism Analyses of Pressure Non-Monotonic Effect on Flammability Limits%压力对可燃极限非单调作用的机理分析

    Institute of Scientific and Technical Information of China (English)

    田雪沁; 陶志强; 张海

    2011-01-01

    1D, laminar premixed CH4/Air flames at elevated pressures were numerically studied to assess pressure effect on flammability limits, with attention primarily paid to maximum flame temperature loss caused by radiation, dominant chain branching and termination reactions and species profiles at different pressures. It was found that radiation heat loss effect increases with decreasing equivalence ratio, especially at the turning point showing the non-monotonic pressure effect. As pressure increases, sensitivity coefficient of main chain termination reaction H+O2+M=HO2+M changes non-monotonously, firstly increases then decreases and this chain termination reaction is gradually substituted by reaction HO2+CH3=OH+CH3O. The main reason of such change is that the relative concentration of HO2 species becomes higher than that of other species as pressure increases.%采用CHEMKIN的PREMIX模块对非常压下贫燃侧的一维、层流CH4/Air预混火焰进行数值模拟,分析了不同的压力下辐射引起的最高火焰温度损失、主要反应的敏感性系数和主要自由基摩尔分数的变化。结果表明,辐射热损失随着当量比的下降而加强,在非单调变化的拐点附近,热辐射损失对最高火焰温度的相对变化作用明显加强。随着压力增大,近极限火焰的主要链终止反应H+O2+M=HO2+M的敏感性呈现先增大后减小的非单调变化,并逐渐被HO2+CH3=OH+CH30取代;引起以上变化的原因是,随着压力增大,HO2对其它主要基团(OH、O、H)的相对浓度明显增大。

  19. Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16.

    Science.gov (United States)

    Sheu, Der-Shyan; Chen, Wen-Ming; Lai, Yung-Wei; Chang, Rey-Chang

    2012-05-01

    The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

  20. Partially thermostated Kac model

    Science.gov (United States)

    Tossounian, Hagop; Vaidyanathan, Ranjini

    2015-08-01

    We study a system of N particles interacting through the Kac collision, with m of them interacting, in addition, with a Maxwellian thermostat at temperature /1 β . We use two indicators to understand the approach to the equilibrium Gaussian state. We prove that (i) the spectral gap of the evolution operator behaves as /m N for large N and (ii) the relative entropy approaches its equilibrium value (at least) at an eventually exponential rate ˜ /m N 2 for large N. The question of having non-zero entropy production at time 0 remains open. A relationship between the Maxwellian thermostat and the thermostat used in the work of Bonetto et al. [J. Stat. Phys. 156(4), 647-667 (2014)] is established through a Van Hove limit.

  1. 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.

  2. Thermostability of montmorillonitic clays

    OpenAIRE

    Petr Jelínek; Dobosz, Stanisław M.; Jaroslav Beňo

    2014-01-01

    Bentonite is one of the most widespread used clays connected with various applications. In the case of foundry technology, bentonite is primarily used as a binder for mold manufacture. Thermal stability of bentonites is a natural property of clay minerals and it depends on the genesis, source and chemical composition of the clay. This property is also closely connected to bentonite structure. According to DTA analysis if only one peak of dehydroxylation is observed (about 600 ºC), the cis- is...

  3. Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase produced by Moniliophthora perniciosa

    Directory of Open Access Journals (Sweden)

    Amanda R. Sena

    2011-06-01

    Full Text Available The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM was employed to evaluate the effect of variables, including inducer (yeast extract and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70% and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70% e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram

  4. 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.

  5. Alicyclobacillus sp. strain CC2, a thermo-acidophilic bacterium isolated from Deception Island (Antarctica) containing a thermostable superoxide dismutase enzyme

    Institute of Scientific and Technical Information of China (English)

    Daniela N. Correa-Llantén; Maximiliano J. Amenábar; Patricio A. Muñoz; María T. Monsalves; Miguel E. Castro; Jenny M.Blamey

    2014-01-01

    A gram-positive, rod-shaped, aerobic, thermo-acidophilic bacterium CC2 (optimal temperature 55℃and pH 4.0), belonging to the genus Alicyclobacillus was isolated from geothermal soil collected from“Cerro Caliente”, Deception Island, Antarctica. Owing to the harsh environmental conditions found in this territory, microorganisms are exposed to conditions that trigger the generation of reactive oxygen species (ROS). They must have an effective antioxidant defense system to deal with this oxidative stress. We focused on one of the most important enzymes: superoxide dismutase, which was partially purified and characterized. This study presents the ifrst report of a thermo-acidophilic bacterium isolated from Deception Island with a thermostable superoxide dismutase (SOD).

  6. 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

  7. 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...

  8. The urease gene cluster of Vibrio parahaemolyticus does not influence the expression of the thermostable direct hemolysin (TDH) gene or the TDH-related hemolysin gene.

    Science.gov (United States)

    Nakaguchi, Yoshitsugu; Okuda, Jun; Iida, Tetsuya; Nishibuchi, Mitsuaki

    2003-01-01

    In order to investigate why the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) of Vibrio parahaemolyticus are produced at low levels from urease-positive strains, the effect of the functional urease gene cluster of V. parahaemolyticus on the expression of the tdh and trh genes was examined. Transcriptional lacZ fusions with the tdh1, tdh2, trh1 and trh2 genes representing variants of the tdh and trh genes were integrated into the chromosome of an Escherichia coli strain and a urease-negative V. parahaemolyticus strain. The plasmid-borne urease gene cluster introduced and expressed in these constructs did not affect expression of any of the fusion genes. The amount of TDH produced from a Kanagawa phenomenon-positive V. parahaemolyticus did not change by introduction of the urease gene cluster either. It was concluded therefore that the urease gene cluster is not involved in the regulation of tdh and trh expression.

  9. Urea hydrolysis and suppressed production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus associated with presence of TDH-related hemolysin genes.

    Science.gov (United States)

    Okitsu, T; Osawa, R; Pornruangwong, S; Yamai, S

    1997-05-01

    A total of 18 strains of V. parahaemolyticus isolated from patients of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were assayed for presence of the thermostable direct hemolysin (TDH) gene and the TDH-related hemolysin (TRH) genes (trh 1 and trh 2) with specific reference to their ability to hydrolyze urea and TDH production. A polymerase chain reaction assay revealed that all urea-hydrolyzing strains (9 strains) carried either trh 1 gene or trh 2 gene. The strains carrying the trh genes as well as the tdh gene produced TDH less by a factor of 4 to 16 than those carrying only the tdh gene, suggesting the expression of the tdh gene was suppressed by the presence of trh gene through a mechanism yet to be defined.

  10. Evaluation of two assay kits for thermostable direct hemolysin (TDH) as an indicator of TDH-related hemolysin (TRH) produced by Vibrio parahaemolyticus.

    Science.gov (United States)

    Yoh, M; Kawakami, N; Funakoshi, Y; Okada, K; Honda, T

    1995-01-01

    Reversed passive latex agglutination (RPLA) or enzyme-linked immunosorbent assay kits with beads (Bead-ELISA) are commercially available in Japan to detect the thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus isolates. We evaluated whether these kits can be used to assay the pathogenic toxin, TDH-related hemolysin (TRH), produced by some so-called Kanagawa phenomenon-negative V. parahaemolyticus strains isolated from patients with diarrhea. Our results showed that the two kits, RPLA and Bead-ELISA, can detect TRH, although they were originally developed for detection of TDH. This may be due to the use of polyclonal anti-TDH antisera that cross react with TRH. Although the sensitivity for TDH detection by RPLA and Bead-ELISA differed tenfold, that for TRH detection was essentially equal. The minimum concentration of TRH required for detection by the two assay kits was about 10 ng/ml.

  11. An amperometric D-amino acid biosensor prepared with a thermostable D-proline dehydrogenase and a carbon nanotube-ionic liquid gel.

    Science.gov (United States)

    Tani, Yuji; Itoyama, Yukiko; Nishi, Kenichi; Wada, Chikahiro; Shoda, Yoshio; Satomura, Takenori; Sakuraba, Haruhiko; Ohshima, Toshihisa; Hayashi, Yukako; Yabutani, Tomoki; Motonaka, Junko

    2009-07-01

    Carbon nanotube (CNT) gel, which is composed of a mixture of single-wall CNT, an ionic liquid, and a thermostable D-proline dehydrogenase (D-Pro DH) immobilized electrode was utilized for the determination of D-amino acids (DAAs) in food samples. When a critical comparison with CNT, Ketjen Black (KB), and carbon powder (CP) was also carried out, the CNT/D-Pro DH immobilized electrode showed the highest sensitivity and the lowest detection limit of D-proline. In addition, the CNT/D-Pro DH immobilized electrode was applied to detection of DAAs in rice wine and vinegar samples. The concentrations of DAAs in rice wine and vinegar samples were 0.0210 +/- 0.0001 and 0.55 +/- 0.05 mmol L(-1), respectively.

  12. In situ synthesized 3D heterometallic metal-organic framework (MOF) as a high-energy-density material shows high heat of detonation, good thermostability and insensitivity.

    Science.gov (United States)

    Feng, Yaya; Liu, Xiangyu; Duan, Linqiang; Yang, Qi; Wei, Qing; Xie, Gang; Chen, Sanping; Yang, Xuwu; Gao, Shengli

    2015-02-07

    A reticular 3D heterometallic metal-organic framework (MOF), [Cu4Na(Mtta)5(CH3CN)]n () (N% = 40.08%), has been synthesized, using a 5-methyl tetrazole (Mtta) ligand formed from acetonitrile and azide, through in situ synthesis and structurally characterized by X-ray single crystal diffraction. The fluorescence spectra demonstrate that undergoes an interesting structural transformation in aqueous solution, yielding the compound [Cu4Na(Mtta)5H2O]n () as confirmed by (1)H NMR, IR and PXRD. Thermoanalysis showed that possesses excellent thermostability up to 335 °C. The calculated detonation properties and the sensitivity test illustrate that compound could be used as a potential explosive. In addition, the non-isothermal kinetics for were studied using the Kissinger and Ozawa-Doyle methods. The enthalpy of formation was obtained from the determination of the constant-volume combustion energy.

  13. Continuous Production of Thermostable beta-Amylase with Clostridium thermosulfurogenes: Effect of Culture Conditions and Metabolite Levels on Enzyme Synthesis and Activity.

    Science.gov (United States)

    Nipkow, A; Shen, G J; Zeikus, J G

    1989-03-01

    A beta-amylase-overproducing mutant of Clostridium thermosulfurogenes was grown in continuous culture on soluble starch to produce thermostable beta-amylase. Enzyme productivity was reasonably stable over periods of weeks to months. The pH and temperature optima for beta-amylase production were pH 6.0 and 60 degrees C, respectively. Enzyme concentration was maximized by increasing biomass concentration by using high substrate concentrations and by maintaining a low growth rate. beta-Amylase concentration reached 90 U ml at a dilution rate of 0.07 h in a 3% starch medium. A further increase in enzyme activity levels was limited by acetic acid inhibition of growth and low beta-amylase productivity at low growth rates.

  14. A thermostable glucoamylase from Bispora sp. MEY-1 with stability over a broad pH range and significant starch hydrolysis capacity.

    Directory of Open Access Journals (Sweden)

    Huifang Hua

    Full Text Available Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.A glucoamylase gene (gla15 was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2-11.0 and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.GLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.

  15. Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766.

    OpenAIRE

    Xu, M.; Iida, T; Yamamoto, K.; Takarada, Y; Miwatani, T; Honda, T.

    1994-01-01

    Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide g...

  16. Improved Production of Thermostable Cellulase from Thermoascus aurantiacus RCKK by Fermentation Bioprocessing and Its Application in the Hydrolysis of Office Waste Paper, Algal Pulp, and Biologically Treated Wheat Straw.

    Science.gov (United States)

    Jain, Kavish Kumar; Kumar, Sandeep; Deswal, Deepa; Kuhad, Ramesh Chander

    2017-02-01

    Thermostable cellulases have wide variety of applications and distinctive advantages, but their low titer becomes the hurdle in their commercialization. In the present work, an assessment of optimum levels of significant factors (temperature, moisture ratio, inoculum size, and ammonium sulfate) and the effect of their interactions on production of thermostable CMCase, FPase, and β-glucosidase by Thermoascus aurantiacus RCKK under solid-state fermentation (SSF) was carried out using central composite design (CCD) of response surface methodology (RSM). The study revealed 33, 13, and 8 % improvement in FPase, CMCase, and β-glucosidase production, respectively. Moreover, crude cellulase from T. aurantiacus RCKK efficiently hydrolyzed office waste paper, algal pulp (Gracillaria verulosa), and biologically treated wheat straw at 60 °C with sugar release of about 830 mg/ml, 285 mg/g, and 260 mg/g of the substrate, respectively. The thermostable enzyme from T. aurantiacus RCKK holds potential to be used in biofuel industry.

  17. Immunization with a thermostable newcastle disease virus K148/08 strain originated from wild mallard duck confers protection against lethal viscerotropic velogenic newcastle disease virus infection in chickens.

    Science.gov (United States)

    Jeong, Seung-Hwan; Lee, Dong-Hun; Kim, Byoung-Yoon; Choi, Soo-Won; Lee, Joong-Bok; Park, Seung-Yong; Choi, In-Soo; Song, Chang-Seon

    2013-01-01

    Newcastle disease (ND) is one of the most devastating poultry infections because of its worldwide distribution and accompanying economical threat. In the present study, we characterized the ND virus (NDV) K148/08 strain from wild mallard duck, with regard to safety, thermostability, immunogenicity, and protective efficacy against velogenic ND viral infection. The NDV K148/08 strain offered enhanced immunogenicity and safety relative to commercially available vaccine strains. The NDV K148/08 strain was safe in 1-day-old SPF chicks after vaccination using a coarse or cabinet-type fine sprayer. We demonstrated that the NDV K148/08 strain elicited high levels of antibody responses and provided protective efficacy against lethal NDV challenge. In addition, the thermostability of the NDV K148/08 strain was as high as that of the thermostable V4 strain. Therefore, the NDV K148/08 strain may be useful to ensure NDV vaccine performance and effectiveness in developing countries, especially in remote areas without cold chains.

  18. 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

  19. Glucose-based PD solution, but not icodextrin-based PD solution, induces plasminogen activator inhibitor-1 and tissue-type plasminogen activator in human peritoneal mesothelial cells via ERK1/2.

    Science.gov (United States)

    Katsutani, Masahira; Ito, Takafumi; Masaki, Takao; Kohno, Nobuoki; Yorioka, Noriaki

    2007-04-01

    Peritoneal dialysis (PD) solutions containing glucose are considered to cause peritoneal fibrosis. Plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) participate in fibrogenesis of various organs, and human peritoneal mesothelial cells (HPMC) can produce PAI-1 and t-PA following glucose stimulation. Icodextrin has been widely used as an alternative osmotic agent. In this study, we investigated whether icodextrin-based PD solution reduced the production of PAI-1 and t-PA by HPMC. We also examined the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2). Glucose-based PD solutions increased the production of PAI-1 and t-PA by HPMC, whereas icodextrin-based PD solution exerted lesser effects. Glucose-based PD solutions activated ERK1/2, and PD98059 inhibited the production of PAI-1 and t-PA-responses not observed with icodextrin-based PD solution. In conclusion, glucose-based PD solutions, unlike icodextrin-based PD solution, induce overproduction of PAI-1 and t-PA via the ERK1/2 pathway.

  20. Improvement of Catalytic Efficiency, Thermo-stability and Dye Decolorization Capability of Pleurotus ostreatus IBL-02 laccase by Hydrophobic Sol Gel Entrapment

    Science.gov (United States)

    2012-01-01

    Background In serious consideration of the worldwide environmental issues associated with the extensive use of the textile dyes and effluents generated thereof, the scientists across the world are in search for potential treatment technologies for their treatment. In such scenario the ligninolytic enzymes provide a potential alternative because they are cost effective, eco-friendly and can be applied to wide range of dye containing industrial effluents. Results Laccase produced from Pleurotus ostreatus IBL-02 during decolorization of the reactive textile dye Drimarene brilliant red K-4BL (DBR K-4BL) was purified and immobilized by hydrophobic gel entrapment. The crude laccase was 4.2-fold purified with specific activity of 573.52 U/mg after passing through the DEAE-Sepharose ion exchange and Sephadex-G-100 chromatography columns. P. ostreatus IBL-02 laccase was found to be a homogenous monomeric protein as evident by single band corresponding to 67 kDa on native and sodium dodesylsulfate polyacrylamide gel electrophoresis (PAGE). The laccase was immobilized by entrapment in Sol–gel matrix of trimethoxysilane (T) and proplytetramethoxysilane (P) prepared using different T:P molar ratios. The free and immobilized laccases were compared to investigate the effect of immobilization on catalytic efficiency and thermo-stability features. Laccase immobilized in the Sol–gel of 1:5 T:P ratio was optimally active and thermo-stable fraction at pH 5, 60°C with half-life of 3 h and 50 min. Laccases immobilized in 1:2 and 1:5 T:P ratio gels had significantly higher Km (83 and100mM) and Vmax (1000 and 1111 mM/mg) values as compared to free laccase. After 5 h reaction time varying decolorization percentages with a maximum of 100% were achieved for different dyes and effluents. Conclusions In summary, P. ostreatus IBL-02 laccase was immobilized by entrapping in a Sol–gel matrix with an objective to enhance its catalytic and stability properties. Sol–gel entrapped laccase