Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution.

Science.gov (United States)

Varrot, A; Hastrup, S; Schülein, M; Davies, G J

1999-01-15

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-05-01

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

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.

Thermostable enzymes as biocatalysts in the biofuel industry.

Science.gov (United States)

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

2010-01-01

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

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

Science.gov (United States)

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

2012-01-01

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

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,

Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

Science.gov (United States)

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

2018-03-01

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

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

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.

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.

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.

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

Science.gov (United States)

He, Xiaoming

2011-01-01

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

Purification and Characterization of Thermostable Cellulase from ...

African Journals Online (AJOL)

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

Thermostability in rubredoxin and its relationship to mechanical rigidity

Science.gov (United States)

Rader, A. J.

2010-03-01

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

Thermostability in rubredoxin and its relationship to mechanical rigidity

International Nuclear Information System (INIS)

Rader, A J

2010-01-01

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

Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

Directory of Open Access Journals (Sweden)

Siika-aho Matti

2011-01-01

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

Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

Science.gov (United States)

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

2015-12-01

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

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....

Optimization of medium composition for thermostable protease ...

African Journals Online (AJOL)

SERVER

2008-04-17

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1986-01-01

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

Evaluation of thermostable enzymes for bioethanol processing

DEFF Research Database (Denmark)

Skovgaard, Pernille Anastasia

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

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.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

Science.gov (United States)

Xue, Xianli; Wu, Yilan; Qin, Xing; Ma, Rui; Luo, Huiying; Su, Xiaoyun; Yao, Bin

2016-07-11

The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N

Thermostable crude endoglucanase produced by Aspergillus ...

African Journals Online (AJOL)

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

Comparison of the thermostability of cellulases from various thermophilic fungi

Energy Technology Data Exchange (ETDEWEB)

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

1987-10-01

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

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

NARCIS (Netherlands)

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

1992-01-01

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

Thermostability in endoglucanases is fold-specific

Science.gov (United States)

2011-01-01

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

Thermostability in endoglucanases is fold-specific

Directory of Open Access Journals (Sweden)

Wolt Jeffrey D

2011-02-01

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

Purification and Characterization of Thermostable Cellulase from ...

African Journals Online (AJOL)

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

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

Science.gov (United States)

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

2015-07-09

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

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.

Ruthenium(II)- bipyridyl with extended π-system: Improved thermo ...

Indian Academy of Sciences (India)

aInorganic and Physical Chemistry Division, Indian Institute of Chemical Technology, Uppal Road, Tarnaka, ... A new extended thermo-stable high molar extinction coefficient bipyridyl ruthenium(II) complex ... cyanines and metal free organic sensitizers have been ..... Iodide-based ionic liquids are more viscous than.

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

African Journals Online (AJOL)

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

Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

Science.gov (United States)

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

2016-11-01

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

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

Science.gov (United States)

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

2016-01-01

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

Highly thermostable fluorescent proteins

Science.gov (United States)

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

2011-03-22

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

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.

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.

Approaches for improving thermostability characteristics in cellulases.

Science.gov (United States)

Anbar, Michael; Bayer, Edward A

2012-01-01

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

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...

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

Science.gov (United States)

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

2007-10-01

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

Expression of Acidothermus cellulolyticus thermostable cellulases in tobacco and rice plants

Directory of Open Access Journals (Sweden)

Xiran Jiang

2017-01-01

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

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

Science.gov (United States)

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

2015-09-01

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

Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

Science.gov (United States)

Malau, N. D.; Sianturi, M.

2017-03-01

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

Development of a thermostable microneedle patch for influenza vaccination

Science.gov (United States)

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

2017-01-01

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

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

Science.gov (United States)

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

2016-10-01

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

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

Science.gov (United States)

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

2007-01-01

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

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

International Nuclear Information System (INIS)

Ji, Hong-Fang; Zhang, Hong-Yu

2007-01-01

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

Thermostable cellulase from a thermomonospora gene

Science.gov (United States)

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

1997-10-14

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

FireProt: web server for automated design of thermostable proteins

Science.gov (United States)

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

2017-01-01

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

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

International Nuclear Information System (INIS)

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

1983-01-01

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

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.

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.

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

Directory of Open Access Journals (Sweden)

Meng eWang

2015-05-01

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

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

Directory of Open Access Journals (Sweden)

Chengtuo Niu

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-06-15

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

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Perioxidases play important roles in abscisic acid (ABA)-simulating photosystem II (PSII) thermostabilty of apple tree rootstock leaves

Czech Academy of Sciences Publication Activity Database

Brestic, M.; Shao, H. B.; Ferus, P.; Malbeck, Jiří

2011-01-01

Roč. 10, č. 71 (2011), s. 15891-15900 ISSN 1684-5315 Institutional research plan: CEZ:AV0Z50380511 Keywords : Photosystem II thermostability * antioxidant activity * phytohormones Subject RIV: EF - Botanics Impact factor: 0.573, year: 2010

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

Science.gov (United States)

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

2012-03-13

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

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

Science.gov (United States)

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

2012-01-01

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

SUSTAINABLE PAPER - Biotechnical modification of mechanical pulp. Final report; KESTAeVAe PAPERI - Mekaanisen massan biotekninen muokkaus. Loppuraportti

Energy Technology Data Exchange (ETDEWEB)

Pere, J.; Liukkonen, S.; Gullichsen, J.; Viikari, L.

1997-12-31

In this work the application of purified enzymes in mechanical pulping was studied. The aim was to gain energy savings in secondary refining of mechanical pulp by modifying pulp fractions with enzymes. One special objective was to increase the flexibility and bonding ability of long fibre fraction. The main interest was in Trichoderma reesei cellulases (CBH I, CBH II, EG I) and hemicellulases (xylanase, mannanase), but a few commercial enzyme preparations (pectinase, cellulase) were also tested. Coarse mechanical pulp (CSF 350-600 ml) or functioned reject (CSF 550-700 ml) were treated with enzymes (45 deg C. pH 5,2-16 h). After enzymatic treatment the pulps were subjected to secondary refining either with a laboratory scale disk refiner (Sprout-Waldron) or a wing defibrator. Some of the results obtained in laboratory scale were further verified in pilot scale. The accessibility of mechanical pulp to enzymatic hydrolysis was limited and therefore yield losses of raw material due to the enzymatic treatments were usually very low, < 0,6 % of original dry weight. The liberation of soluble oligosaccharides was affected by the enzyme used and also depended on the freeness level and metal composition of the pulp. Endoglucanase (EG I) and mannanase solubilized reducing sugars more efficiently than cellobiohydrolases (CBH I, CBH II). If secondary refining was performed with the atmospheric disk refiner no energy savings or improvement in pulp properties were gained with any of the enzymes tested as compared with the untreated reference. But energy savings up to 20-30 % were obtained when the pulp was pretreated with CBH I prior to secondary refining with the wing defibrator. Pretreatment of the pulp with mannanase gave small energy savings (10-20 %), too. Boosting of secondary refining with CBH I and mannanase was attained while retaining good handsheet properties of the pulp. The positive effects of CBH I on secondary refining were further verified in pilot scale. In a two

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

Energy Technology Data Exchange (ETDEWEB)

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

2003-01-01

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

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

Science.gov (United States)

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

2015-10-01

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

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.

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

Science.gov (United States)

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

2018-05-18

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

Thermostability promotes the cooperative function of split adenylate kinases.

Science.gov (United States)

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

2008-05-01

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

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

Science.gov (United States)

Shivhare, Devendra

2017-01-01

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

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

Directory of Open Access Journals (Sweden)

Eleni Gomes

2007-02-01

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

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

Science.gov (United States)

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

2017-12-01

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

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

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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.

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

Directory of Open Access Journals (Sweden)

Fabrizio Pucci

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

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

Science.gov (United States)

Skouri-Gargouri, Houda; Gargouri, Ali

2008-11-01

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

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...

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

Science.gov (United States)

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

2017-06-19

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

Conduction Mechanism by Using CBH Model in Fe3+ and Mn3+ Ion Modified Pb(Zr0.65−xAxTi0.35O3 (A = Mn3+/Fe3+ Ceramics

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Niranjan Sahu

2013-01-01

Full Text Available Polycrystalline samples of manganese and iron substituted lead zirconium titanate (PZT with general formula Pb(Zr0.65−xAxTi0.35O3 (A = Mn3+ and Fe3+ ceramics have been synthesized by high temperature solid state reaction technique. X-ray diffraction (XRD patterns were recorded at room temperature to study the crystal structure. All the patterns could be refined by employing the Rietveld method to R3c space group with rhombohedral symmetry. Microstructural properties of the materials were analyzed by scanning electron microscope (SEM, and compositional analysis was carried out by energy dispersive spectrum (EDS measurements. All the materials exhibit ferroelectric to paraelectric transition. The variation of dielectric constant and loss tangent with temperature and frequency is investigated. The decrease of activation energy and increases of AC conductivity with the Fe3+ or Mn3+ ion concentration have been observed. The AC conductivity has been analyzed by the power law. The frequency exponent with the function of temperature has been analyzed by assuming that the AC conduction mechanism is the correlated barrier hopping (CBH model. The conduction in the present sample is found to be of bipolaron type for Mn3+ ion-doped sample. However, the conduction mechanism could not be explained by CBH model for Fe3+ ion-doped sample.

Purification and properties of a thermostable pullulanase from Clostridium thermosulfurgenes EM1 which hydrolyses both. alpha. -1,6 and. alpha. -1,4-glycosidic linkages

Energy Technology Data Exchange (ETDEWEB)

Spreinat, A [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

1990-08-01

A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the {alpha}-1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an {alpha}-1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K{sub m} values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60deg-65deg C and a pH optimum at 5.5-6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. (orig.).

Enzymatic conversion of pretreated biomass into fermentable sugars for biorefinery operation

Science.gov (United States)

Gao, Dahai

2011-12-01

Depleting petroleum reserves and potential climate change caused by fossil fuel consumption have attracted significant attention towards the use of alternative renewable resources for production of fuels and chemicals. Lignocellulosic biomass provides a plentiful resource for the sustainable production of biofuels and biochemicals and could serve as an important contributor to the world energy portfolio in the near future. Successful biological conversion of lignocellulosic biomass requires an efficient and economical pretreatment method, high glucose/xylose yields during enzymatic hydrolysis and fermentation of both hexose and pentose to ethanol. High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. Core glycosyl hydrolases were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I), cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and beta-Glucosidase (betaG). The two core hemicellulases were an endoxylanase (EX) and a beta-xylosidase (betaX). A diverse set of accessory hemicellulases from bacterial sources was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (˜20 mg protein/g glucan) using an in-house developed enzyme cocktail and this cocktail was compared to commercial enzyme. Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products

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

Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense

NARCIS (Netherlands)

Gusakov, A.V.; Sinitsyn, A.P.; Salanovich, T.N.; Bukhtojarov, F.E.; Markov, A.V.; Ustinov, B.B.; Zeijl, C.V.; Punt, P.; Burlingame, R.

2005-01-01

Two forms of cellobiohydrolase I (CBH I, Cel7A) were purified from the culture ultrafiltrate of a mutant strain of the fungus Chrysosporium lucknowense, an industrial producer of cellulases and hemicellulases. The enzymes had different molecular masses (52 and 65 kDa, SDS-PAGE data) but the same pI

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

KAUST Repository

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

2015-01-01

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

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

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

Science.gov (United States)

Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

2011-05-01

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

DEFF Research Database (Denmark)

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

2014-01-01

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

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

Science.gov (United States)

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

2018-06-01

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

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

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.

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.

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

Science.gov (United States)

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

2016-11-01

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

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.

Engineering of pectinolytic enzymes for enhanced thermostability

DEFF Research Database (Denmark)

Larsen, Dorte Møller

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

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

Science.gov (United States)

2015-12-23

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

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

Science.gov (United States)

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

2018-05-09

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

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

DEFF Research Database (Denmark)

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

2006-01-01

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

Understanding thermostability and pH dependent properties of proteins

DEFF Research Database (Denmark)

Galberg, Pernille

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

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

Directory of Open Access Journals (Sweden)

Salleh Abu

2009-04-01

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

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

Science.gov (United States)

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

2018-01-01

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

Crystallization and preliminary crystallographic analysis of maganese(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from Bacillus sp. JF8

International Nuclear Information System (INIS)

Senda, Miki; Hatta, Takashi; Kimbara, Kazuhide; Senda, Toshiya

2010-01-01

A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized in two forms using the sitting-drop vapour-diffusion method. Both crystals diffracted to approximately 1.3 Å resolution. A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized. The initial screening for crystallization was performed by the sitting-drop vapour-diffusion method using a crystallization robot, resulting in the growth of two crystal forms. The first crystal belonged to space group P1, with unit-cell parameters a = 62.7, b = 71.4, c = 93.6 Å, α = 71.2, β = 81.0, γ = 64.0°, and diffracted to 1.3 Å resolution. The second crystal belonged to space group I222, with unit-cell parameters a = 74.2, b = 90.8, c = 104.3 Å, and diffracted to 1.3 Å resolution. Molecular-replacement trials using homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (28% amino-acid sequence identity) as a search model provided a satisfactory solution for both crystal forms

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.

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

Science.gov (United States)

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

2016-01-01

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

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

International Nuclear Information System (INIS)

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

2011-01-01

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

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

Science.gov (United States)

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

2017-05-01

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

Thermostating extended Lagrangian Born-Oppenheimer molecular dynamics.

Science.gov (United States)

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

2015-04-21

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

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

Science.gov (United States)

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

2015-03-01

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

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

Science.gov (United States)

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

2014-09-01

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

Protein features as determinants of wild-type glycoside hydrolase thermostability

DEFF Research Database (Denmark)

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

2017-01-01

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

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

NARCIS (Netherlands)

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

1992-01-01

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

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

NARCIS (Netherlands)

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

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

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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.

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

Directory of Open Access Journals (Sweden)

Hongde An

2015-11-01

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

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.

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

Science.gov (United States)

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

2016-12-01

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

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

Science.gov (United States)

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

2018-03-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Science.gov (United States)

2012-08-01

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

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

NARCIS (Netherlands)

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

2012-01-01

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

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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.

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

International Nuclear Information System (INIS)

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

1983-01-01

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

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

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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

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

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Chi-Ho Chan

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

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

Science.gov (United States)

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

2014-04-10

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

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

Science.gov (United States)

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

2015-09-01

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

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

DEFF Research Database (Denmark)

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

2007-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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

2018-01-01

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

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.

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.

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

International Nuclear Information System (INIS)

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

2012-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

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

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Yolanda Bautista

2017-12-01

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

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

Science.gov (United States)

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

2011-07-01

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

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

Science.gov (United States)

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

2016-01-22

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

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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

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Jiivittha Veno

2017-11-01

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

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.

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.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

Energy Technology Data Exchange (ETDEWEB)

Manikandan, K. [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bhardwaj, Amit [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ghosh, Amit [Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036 (India); Reddy, V. S. [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ramakumar, S., E-mail: ramak@physics.iisc.ernet.in [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012 (India)

2005-08-01

A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

International Nuclear Information System (INIS)

Manikandan, K.; Bhardwaj, Amit; Ghosh, Amit; Reddy, V. S.; Ramakumar, S.

2005-01-01

A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution

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

Czech Academy of Sciences Publication Activity Database

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

2004-01-01

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

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

Science.gov (United States)

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

2015-06-28

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

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.

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.

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

Science.gov (United States)

2013-01-01

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

Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2

International Nuclear Information System (INIS)

Shariff, Fairolniza Mohd; Rahman, Raja Noor Zaliha Raja Abd.; Ali, Mohd Shukuri Mohamad; Chor, Adam Leow Thean; Basri, Mahiran; Salleh, Abu Bakar

2010-01-01

Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P2 1 2 1 2 1 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V M ) of 2.85 Å 3 Da −1 and a solvent content of 57%

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

Science.gov (United States)

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

2017-12-01

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

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

Science.gov (United States)

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

2016-01-01

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

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.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

Science.gov (United States)

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The

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

Science.gov (United States)

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

2003-01-01

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

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

OpenAIRE

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

2014-01-01

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

Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup.

Science.gov (United States)

Jia, Dong-Xu; Zhou, Lin; Zheng, Yu-Guo

2017-04-01

Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn 2+ at 85°C for 48h. The K m and k cat /K m values for ToGI were 81.46mM and 21.77min -1 mM -1 , respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2016-10-15

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

Thermostable cross-protective subunit vaccine against Brucella species.

Science.gov (United States)

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

2014-12-01

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

Study of the leaching of heavy metals from waste water sludge and incinerator's ash, using coupled thermostated columns and DTPA as complex agent

International Nuclear Information System (INIS)

Vite T, J.; Vite T, M.; Guerrero D, J.; Carreno de Leon, M.C.

2000-01-01

We studied the metallic composition from waste water sludge and incinerators ashes of an incinerator located in Toluca, Mexico, the qualitative studies were made using the Activation Analysis technique, and fluorescence X-ray techniques. The quantitative analysis of heavy metals in the wastes were made using Inductively coupled plasma atomic emission spectrometry (Icp-Aes). For leaching the samples, we used four coupled thermostated columns, each one had a p H of 2,5, 7 and 10. The flux of the air was of 1600 cc/min. The temperature was maintain constant in 60 Centigrade using a thermostated system. For this study we used 100 g of wastes mixed with mineral acid or sodium hydroxide to reach p H 2,5,7 and 10. We added a reducing and tensoactive agents and finally DTPA as complex agent. With this method, we obtain a better leaching efficiency using a complex agent. However the high DTPA cost, make this process expansive that is why we recommend to work with another classes of complex agents, that be cheaper to leach metals of different chemistry matrix. (Author)

Simple method for direct crown base height estimation of individual conifer trees using airborne LiDAR data.

Science.gov (United States)

Luo, Laiping; Zhai, Qiuping; Su, Yanjun; Ma, Qin; Kelly, Maggi; Guo, Qinghua

2018-05-14

Crown base height (CBH) is an essential tree biophysical parameter for many applications in forest management, forest fuel treatment, wildfire modeling, ecosystem modeling and global climate change studies. Accurate and automatic estimation of CBH for individual trees is still a challenging task. Airborne light detection and ranging (LiDAR) provides reliable and promising data for estimating CBH. Various methods have been developed to calculate CBH indirectly using regression-based means from airborne LiDAR data and field measurements. However, little attention has been paid to directly calculate CBH at the individual tree scale in mixed-species forests without field measurements. In this study, we propose a new method for directly estimating individual-tree CBH from airborne LiDAR data. Our method involves two main strategies: 1) removing noise and understory vegetation for each tree; and 2) estimating CBH by generating percentile ranking profile for each tree and using a spline curve to identify its inflection points. These two strategies lend our method the advantages of no requirement of field measurements and being efficient and effective in mixed-species forests. The proposed method was applied to a mixed conifer forest in the Sierra Nevada, California and was validated by field measurements. The results showed that our method can directly estimate CBH at individual tree level with a root-mean-squared error of 1.62 m, a coefficient of determination of 0.88 and a relative bias of 3.36%. Furthermore, we systematically analyzed the accuracies among different height groups and tree species by comparing with field measurements. Our results implied that taller trees had relatively higher uncertainties than shorter trees. Our findings also show that the accuracy for CBH estimation was the highest for black oak trees, with an RMSE of 0.52 m. The conifer species results were also good with uniformly high R 2 ranging from 0.82 to 0.93. In general, our method has

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

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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.

Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics

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Jäger Gernot

2010-08-01

Full Text Available Abstract Background It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH and endoglucanases (EG. Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose. Results First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir and kinetics (pseudo-first-order. Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured. It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose. Conclusions α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation

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

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Myat, Lin; Ryu, Gi-Hyung

2014-01-30

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

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

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Ahlgren Simon

2011-09-01

Full Text Available Abstract Background The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP. Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. Results We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™ to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. Conclusions Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

The Cerebellar-Cerebral Microstructure Is Disrupted at Multiple Sites in Very Preterm Infants with Cerebellar Haemorrhage.

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Neubauer, Vera; Djurdjevic, Tanja; Griesmaier, Elke; Biermayr, Marlene; Gizewski, Elke Ruth; Kiechl-Kohlendorfer, Ursula

2018-01-01

Recent advances in magnetic resonance imaging (MRI) techniques have prompted reconsideration of the anatomical correlates of adverse outcomes in preterm infants. The importance of the contribution made by the cerebellum is now increasingly appreciated. The effect of cerebellar haemorrhage (CBH) on the microstructure of the cerebellar-cerebral circuit is largely unexplored. To investigate the effect of CBH on the microstructure of cerebellar-cerebral connections in preterm infants aged microstructure (fractional anisotropy [FA] and apparent diffusion coefficient) were quantified in 5 vulnerable regions (the centrum semiovale, posterior limb of the internal capsule, corpus callosum, and superior and middle cerebellar peduncles). Group differences between infants with CBH and infants without CBH were assessed. There were 267 infants included in the study. Infants with CBH (isolated and combined) had significantly lower FA values in all regions investigated. Infants with isolated CBH showed lower FA in the middle and superior cerebellar peduncles and in the posterior limb of the internal capsule. This study provides evidence that CBH causes alterations in localised and remote WM pathways in the developing brain. The disruption of the cerebellar-cerebral microstructure at multiple sites adds further support for the concept of developmental diaschisis, which is propagated as an explanation for the consequences of early cerebellar injury on cognitive and affective domains. © 2017 S. Karger AG, Basel.

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

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Jørgensen, F; Hansen, O C; Stougaard, P

2004-06-01

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

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

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

2018-03-01

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

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

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

2018-03-01

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

Observed Correlation between Aerosol and Cloud Base Height for Low Clouds at Baltimore and New York, United States

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Sium Gebremariam

2018-04-01

Full Text Available The correlation between aerosol particulate matter with aerodynamic diameter ≤2.5 μ m (PM2.5 and cloud base height (CBH of low clouds (CBH lower than 1.5 km a.g.l. at Baltimore and New York, United States, for an 8 year period (2007–2014 was investigated using information from the Automated Surface Observing System (ASOS observations and collocated U.S. Environmental Protection Agency (EPA observations. The lifting condensation level (LCL heights were calculated and compared with the CBH. The monthly average observations show that PM2.5 decreases from 2007 to 2014 while there is no significant trend found for CBH and LCL. The variability of the LCL height agrees well with CBH but LCL height is systematically lower than CBH (~180 m lower. There was a significant negative correlation found between CBH–LCL and PM2.5. All of the cloud cases were separated into polluted and clean conditions based on the distribution of PM2.5 values. The distributions of CBH–LCL in the two groups show more cloud cases with smaller CBH–LCL in polluted conditions than in clean conditions.

Improved retrieval of cloud base heights from ceilometer using a non-standard instrument method

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Wang, Yang; Zhao, Chuanfeng; Dong, Zipeng; Li, Zhanqing; Hu, Shuzhen; Chen, Tianmeng; Tao, Fa; Wang, Yuzhao

2018-04-01

Cloud-base height (CBH) is a basic cloud parameter but has not been measured accurately, especially under polluted conditions due to the interference of aerosol. Taking advantage of a comprehensive field experiment in northern China in which a variety of advanced cloud probing instruments were operated, different methods of detecting CBH are assessed. The Micro-Pulse Lidar (MPL) and the Vaisala ceilometer (CL51) provided two types of backscattered profiles. The latter has been employed widely as a standard means of measuring CBH using the manufacturer's operational algorithm to generate standard CBH products (CL51 MAN) whose quality is rigorously assessed here, in comparison with a research algorithm that we developed named value distribution equalization (VDE) algorithm. It was applied to both the profiles of lidar backscattering data from the two instruments. The VDE algorithm is found to produce more accurate estimates of CBH for both instruments and can cope with heavy aerosol loading conditions well. By contrast, CL51 MAN overestimates CBH by 400 m and misses many low level clouds under such conditions. These findings are important given that CL51 has been adopted operationally by many meteorological stations in China.

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

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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.

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...

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

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Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

2009-11-13

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

Temperature and pH influence adsorption of cellobiohydrolase onto lignin by changing the protein properties.

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Lu, Xianqin; Wang, Can; Li, Xuezhi; Zhao, Jian

2017-12-01

Non-productive adsorption of cellulase onto lignin restricted the movement of cellulase and also hindered the cellulase recycling in bioconversion of lignocellulose. In this study, effect of temperature and pH on adsorption and desorption of cellobiohydrolase (CBH) on lignin and its possible mechanism were discussed. It found that pH value and temperature influenced the adsorption and desorption behaviors of CBH on lignin. Different thermodynamic models suggested that the action between lignin and CBH was physical action. More CBH was adsorbed onto lignin, but lower initial adsorption velocity was detected at 50°C comparing with 4°C. Elevating pH value could improve desorption of cellulase from lignin. The changes of hydrophobicity and electric potential on protein surface may partially explain the impact of environmental conditions on the adsorption and desorption behaviors of CBH on lignin, and comparing to electrical interaction, the hydrophobicity may be the dominating factor influencing the behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characteristics of Lignin Fractions from Dilute Acid Pretreated Switchgrass and Their Effect on Cellobiohydrolase from Trichoderma longibrachiatum

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Lan Yao

2018-02-01

Full Text Available To investigate the interactions between acid pretreated switchgrass lignin and cellobiohydrolase (CBH, three different lignin fractions were isolated from dilute acid pretreated switchgrass by (i ethanol extraction, followed by (ii dioxane/H2O extraction, and (iii cellulase treatment, respectively. Structural properties of each lignin fraction were elucidated by GPC, 13C-NMR, and 2D-HSQC NMR analyses. The adsorptions of CBH to the isolated lignin fractions were also studied by Langmuir adsorption isotherms. Ethanol-extractable lignin fraction, mainly composed of syringyl (S and guaiacyl (G units, had the lowest molecular weight, while dioxane/H2O-extracted lignin fraction had the lowest S/G ratio with higher content of p-coumaric acid (pCA unit. The residual lignin fraction after enzymatic treatment had the highest S/G ratio without hydroxyphenyl (H unit. Strong associations were found between lignin properties such as lignin composition and S/G ratio and its non-productive enzyme adsorption factors including the maximum adsorption capacity and binding strength.

Dielectric and electrical conductivity studies of bulk lead (II) oxide (PbO)

Energy Technology Data Exchange (ETDEWEB)

Darwish, A.A.A., E-mail: aaadarwish@gmail.com [Department of Physics, Faculty of Education at Al-Mahweet, Sana’a University, Al-Mahwit (Yemen); Department of Physics, Faculty of Science, University of Tabuk, P.O. Box 741, Tabuk 71491, Tabuk (Saudi Arabia); El-Zaidia, E.F.M.; El-Nahass, M.M. [Department of Physics, Faculty of Education, Ain Shams University, Rorxy, Cairo 11757 (Egypt); Hanafy, T.A. [Department of Physics, Faculty of Science, University of Tabuk, P.O. Box 741, Tabuk 71491, Tabuk (Saudi Arabia); Department of Physics, Faculty of Science, Fayoum University, 63514 El Fayoum (Egypt); Al-Zubaidi, A.A. [Department of Physics, Faculty of Science, University of Tabuk, P.O. Box 741, Tabuk 71491, Tabuk (Saudi Arabia)

2014-03-15

Highlights: • The AC measurements of PbO were measured at temperature range 313–523 K. • The dielectric constants increased with temperature. • The mechanism responsible for AC conduction is electronic hopping. -- Abstract: The dielectric properties, the impedance spectroscopy and AC conductivity of bulk PbO have been investigated as a function of frequency and temperature. The measurements were carried out in the frequency range from 40 to 5 × 10{sup 6} Hz and in temperature range from 313 to 523 K. The frequency response of dielectric constant, ε{sub 1}, and dielectric loss index, ε{sub 2}, as a function of temperature were studied. The values of ε{sub 1} and ε{sub 2} were found to decrease with the increase in frequency. However, they increase with the increase in temperature. The presence of a single arc in the complex modulus spectrum at different temperatures confirms the single-phase character of the PbO. The AC conductivity exhibited a universal dynamic response: σ{sub AC} = Aω{sup s}. The AC conductivity was also found to increase with increasing temperature and frequency. The correlation barrier hopping (CBH) model was found to apply to the AC conductivity data. The calculated values of s were decreased with temperature. This behavior reveals that the conduction mechanism for PbO samples is CBH. The activation energy for AC conductivity decreases with increasing frequency. This confirms that the hopping conduction to the dominant mechanism for PbO samples.

Structurally stabilized organosilane-templated thermostable mesoporous titania.

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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.

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

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

2013-04-15

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

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

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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.

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

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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.

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

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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.

Production of extremely alkaliphilic, halotolerent, detergent, and thermostable mannanase by the free and immobilized cells of Bacillus halodurans PPKS-2. Purification and characterization.

Science.gov (United States)

Vijayalaxmi, S; Prakash, P; Jayalakshmi, S K; Mulimani, V H; Sreeramulu, K

2013-09-01

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0-16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.

Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry.

Science.gov (United States)

Niu, Chengtuo; Zhu, Linjiang; Hill, Annie; Alex Speers, R; Li, Qi

2017-01-01

To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing. A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg -1 and 189 s -1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml -1 , which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively. The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

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

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

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Tosatto Silvio CE

2005-12-01

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

Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

Energy Technology Data Exchange (ETDEWEB)

Byun, Jung-Sue [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of); Rhee, Jin-Kyu [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Oh, Jong-Won [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

2006-02-01

Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method. EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4–C6 at a temperature of 303–363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est{sub pc}-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 Å resolution from an EstE1 crystal; the crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 73.71, c = 234.23 Å. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient V{sub M} is calculated to be 2.2 Å{sup 3} Da{sup −1} and the solvent content is 44.1%.

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

Science.gov (United States)

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

2018-05-04

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

Construction and engineering of a thermostable self-sufficient cytochrome P450

Energy Technology Data Exchange (ETDEWEB)

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

2009-06-19

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

Construction and engineering of a thermostable self-sufficient cytochrome P450

International Nuclear Information System (INIS)

Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

2009-01-01

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

Thermostable Bacteriocin BL8 from Bacillus licheniformis isolated from marine sediment.

Science.gov (United States)

Smitha, S; Bhat, S G

2013-03-01

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

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.

Reconstitution of β-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

International Nuclear Information System (INIS)

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

2006-01-01

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

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

Science.gov (United States)

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

2018-04-01

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

Ultrasonically detectable cerebellar haemorrhage in preterm infants.

LENUS (Irish Health Repository)

McCarthy, Lisa Kenyon

2011-07-01

To determine the frequency and pattern of cerebellar haemorrhage (CBH) on routine cranial ultrasound (cUS) imaging in infants of ≤32 weeks gestation, and to investigate how extremely preterm infants with CBH differ from those with severe intraventricular haemorrhage (IVH).

Effects of Calcium Ions on the Thermostability and Spectroscopic Properties of the LH1-RC Complex from a New Thermophilic Purple Bacterium Allochromatium tepidum.

Science.gov (United States)

Kimura, Yukihiro; Lyu, Shuwen; Okoshi, Akira; Okazaki, Koudai; Nakamura, Natsuki; Ohashi, Akira; Ohno, Takashi; Kobayashi, Manami; Imanishi, Michie; Takaichi, Shinichi; Madigan, Michael T; Wang-Otomo, Zheng-Yu

2017-05-18

The light harvesting-reaction center (LH1-RC) complex from a new thermophilic purple sulfur bacterium Allochromatium (Alc.) tepidum was isolated and characterized by spectroscopic and thermodynamic analyses. The purified Alc. tepidum LH1-RC complex showed a high thermostability comparable to that of another thermophilic purple sulfur bacterium Thermochromatium tepidum, and spectroscopic characteristics similar to those of a mesophilic bacterium Alc. vinosum. Approximately 4-5 Ca 2+ per LH1-RC were detected by inductively coupled plasma atomic emission spectroscopy and isothermal titration calorimetry. Upon removal of Ca 2+ , the denaturing temperature of the Alc. tepidum LH1-RC complex dropped accompanied by a blue-shift of the LH1 Q y absorption band. The effect of Ca 2+ was also observed in the resonance Raman shift of the C3-acetyl νC═O band of bacteriochlorophyll-a, indicating changes in the hydrogen-bonding interactions between the pigment and LH1 polypeptides. Thermodynamic parameters for the Ca 2+ -binding to the Alc. tepidum LH1-RC complex indicated that this reaction is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. Our data support a hypothesis that Alc. tepidum may be a transitional organism between mesophilic and thermophilic purple bacteria and that Ca 2+ is one of the major keys to the thermostability of LH1-RC complexes in purple bacteria.

Perioxidases play important roles in abscisic acid (ABA)-simulating ...

African Journals Online (AJOL)

Leaf photosynthetic activity limited by summer heat stress represents large constraint to production process of fruit trees. To cope with this problem, we tested photosystem II (PS II) thermostability in clonal apple tree rootstocks with different growth intensity - semi-vigorous-MM106 and dwarfing-J-TE-F experiencing summer ...

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.

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

Science.gov (United States)

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

2015-11-01

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

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

Directory of Open Access Journals (Sweden)

David Bednar

2015-11-01

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

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.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

Science.gov (United States)

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

2017-07-01

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the

A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

Directory of Open Access Journals (Sweden)

Hugo Oliveira

Full Text Available Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68 and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively. Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermostability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

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.

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

Science.gov (United States)

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

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

Science.gov (United States)

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

2006-06-01

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

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

International Nuclear Information System (INIS)

Hassanpour, S.; Khoylou, F.

2003-01-01

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

Ethanol from Agriculture for Arkansas and America

Energy Technology Data Exchange (ETDEWEB)

Hood, Elizabeth E [Arkansas State Univ., Jonesboro, AR (United States)

2017-03-27

The purpose of this project was to develop technology that would facilitate production of sugars from agricultural residues to enable biofuels and biobased product manufacturing. Our primary technology is to use genetic engineering to put bacterial and fungal cellulase genes into corn kernels, using the grain as the production system for the enzymes. At the beginning of this DoE funded program, we were producing two cellulases—E1 endocellulase from a bacterium found in a hot spring at Yellowstone National Park, and CBH I exocellulase from a wood rot fungus. Our team developed several new regulatory sequences (promoters) that increased enzyme protein accumulation in two kernel compartments (embryo and endosperm). We were also able to capitalize on the diverse genetics of corn to increase protein accumulation. High oil germplasm in particular was instrumental in this increase. A second task in the program was to produce enzymes and proteins that enhanced the activity of the E1 and CBH I enzymes. Our team produced CBH II, from the same wood rot fungus at a level that enabled highly enhanced deconstruction activity of E1 and CBH I in a synergistic manner. We analyzed an additional protein, expansin from cucumber that was expressed in the maize grain expression system. This protein had been previously shown to enhance cellulase activity (D. Cosgrove, Penn State University), and required a large-scale production platform. Our team showed that the corn production system allows industrial amounts of active expansin to be harvested from the grain. One of the challenges of any new production system is to maximize recovery of active ingredient from the raw materials at a cost compatible with its final use. Our team showed that low pH extraction of grain solubilized the enzymes without contamination of native corn protein and active product could be concentrated through ultrafiltration. The final outcomes of this project were the following: 3 cellulase enzymes and the

Enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c(552) through introduction of an extra methylene group into its hydrophobic protein interior.

Science.gov (United States)

Tai, Hulin; Irie, Kiyofumi; Mikami, Shin-ichi; Yamamoto, Yasuhiko

2011-04-19

Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c(552) (HT) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729-25734] indicated that a void space, which is large enough to accommodate a methyl group, exists in the hydrophobic protein interior near the heme. We tried to reduce the void space through the replacement of a Val by Ile or Leu (Val/Ile or Val/Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The Val/Ile mutation was found to cause further enhancement of the thermostability of the oxidized HT, as reflected in the increase of the denaturation temperature (T(m)) value by ∼ 3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E(m)) of the Val/Ile mutant exhibited a negative shift of ∼ 50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E(m) value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650-13651]. In contrast, the Val/Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T(m) values of the oxidized and reduced proteins by ∼ 3 and ∼ 5 deg, respectively, and the E(m) value of the Val/Leu mutant happened to be similar to that of the Val/Ile one. The E

Dipeptidyl peptidase IV is involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes in Aspergillus aculeatus.

Science.gov (United States)

Tani, Shuji; Yuki, Shota; Kunitake, Emi; Sumitani, Jun-Ichi; Kawaguchi, Takashi

2017-06-01

We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 Aspergillus aculeatus T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (cbhI) promoter and the orotidine 5'-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and cbhI expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (dppIV) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and cbhI expression. The deletion of dppIV resulted in a significant reduction in the cellulose-responsive expression of both cbhI as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in A. aculeatus. In contrast, the dppIV deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in A. aculeatus.

Structural analysis of sumoylated proteins in Schizosaccharomyces pombe

DEFF Research Database (Denmark)

Jørgensen, Maria Louise Mønster

or Sap1-DNA interactions. In addition, the Sap1 function relationship was investigated in vivo by repeating a search for suppressors of the slow growth phenotype of abp1Δ cbh1Δ mutants. Autonomously replicating sequence binding protein 1 (Abp1) and cenp-B homologue 1 (Cbh1) co-localise with Sap1 in some...

Accurate and computationally efficient prediction of thermochemical properties of biomolecules using the generalized connectivity-based hierarchy.

Science.gov (United States)

Sengupta, Arkajyoti; Ramabhadran, Raghunath O; Raghavachari, Krishnan

2014-08-14

In this study we have used the connectivity-based hierarchy (CBH) method to derive accurate heats of formation of a range of biomolecules, 18 amino acids and 10 barbituric acid/uracil derivatives. The hierarchy is based on the connectivity of the different atoms in a large molecule. It results in error-cancellation reaction schemes that are automated, general, and can be readily used for a broad range of organic molecules and biomolecules. Herein, we first locate stable conformational and tautomeric forms of these biomolecules using an accurate level of theory (viz. CCSD(T)/6-311++G(3df,2p)). Subsequently, the heats of formation of the amino acids are evaluated using the CBH-1 and CBH-2 schemes and routinely employed density functionals or wave function-based methods. The calculated heats of formation obtained herein using modest levels of theory and are in very good agreement with those obtained using more expensive W1-F12 and W2-F12 methods on amino acids and G3 results on barbituric acid derivatives. Overall, the present study (a) highlights the small effect of including multiple conformers in determining the heats of formation of biomolecules and (b) in concurrence with previous CBH studies, proves that use of the more effective error-cancelling isoatomic scheme (CBH-2) results in more accurate heats of formation with modestly sized basis sets along with common density functionals or wave function-based methods.

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

DEFF Research Database (Denmark)

Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

2017-01-01

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

Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

Directory of Open Access Journals (Sweden)

Pichapak Sriyapai

2015-06-01

Full Text Available A thermostable esterase gene (hydS14 was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG and catalytic triad (Ser88-Asp208-His235 of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases, has three conserved regions, and contains the novel motif (GY(FSLG, which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14 was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6, displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1. In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

Characterization of a thermostable recombinant l-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 and its application for the production of l-fructose and l-rhamnulose.

Science.gov (United States)

Chen, Ziwei; Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2018-04-01

l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized. The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co 2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co 2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg -1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L -1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L -1 l-mannose, respectively. The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

Directory of Open Access Journals (Sweden)

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.

A two-dimensional zinc(II) coordination polymer based on mixed dimethyl succinate and bipyridine ligands: synthesis, structure, thermostability and luminescence properties.

Science.gov (United States)

Liu, Yang; Feng, Yong Lan; Fu, Wei Wei

2016-04-01

From the viewpoint of crystal engineering, the construction of crystalline polymeric materials requires a rational choice of organic bridging ligands for the self-assembly process. Multicarboxylate ligands are of particular interest due to their strong coordination activity towards metal ions, as well as their various coordination modes and versatile conformations. The structural chemistry of dicarboxylate-based coordination polymers of transition metals has been developed through the grafting of N-containing organic linkers into carboxylate-bridged transition metal networks. A new luminescent two-dimensional zinc(II) coordination polymer containing bridging 2,2-dimethylsuccinate and 4,4'-bipyridine ligands, namely poly[[aqua(μ2-4,4'-bipyridine-κ(2)N:N')bis(μ3-2,2-dimethylbutanedioato)-κ(4)O(1),O(1'):O(4):O(4');κ(5)O(1):O(1),O(4):O(4),O(4')-dizinc(II)] dihydrate], {[Zn2(C6H8O4)2(C10H8N2)(H2O)]·2H2O}n, has been synthesized under hydrothermal conditions and characterized by single-crystal X-ray diffraction and elemental, IR and thermogravimetric analyses. In the structure, the 2,2-dimethylsuccinate ligands link linear tetranuclear Zn(II) subunits into one-dimensional chains along the c axis. 4,4'-Bipyridine acts as a tethering ligand expanding these one-dimensional chains into a two-dimensional layered structure. Hydrogen-bonding interactions between the water molecules (both coordinated and free) and carboxylate O atoms strengthen the packing of the layers. Furthermore, the luminescence properties of the complex were investigated. The compound exhibits a blue photoluminescence in the solid state at room temperature and may be a good candidate for potential hybrid inorganic-organic photoactive materials.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

OpenAIRE

Sigoillot Jean-Claude; Kim-Anh To; Haltrich Dietmar; Berrin Jean-Guy; Thi-Thu Dang; Bien-Cuong Do; Yamabhai Montarop

2009-01-01

Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pret...

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

Science.gov (United States)

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

2017-11-01

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

Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering.

Science.gov (United States)

Nakanishi, Akihito; Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

2012-10-23

To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes.

Science.gov (United States)

Mohammad, Balsam T; Al Daghistani, Hala I; Jaouani, Atef; Abdel-Latif, Saleh; Kennes, Christian

2017-01-01

The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis . This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes

Directory of Open Access Journals (Sweden)

Balsam T. Mohammad

2017-01-01

Full Text Available The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis. This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

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...

SU-E-J-236: Audiovisual Biofeedback Improves Breath-Hold Lung Tumor Position Reproducibility Measured with 4D MRI

International Nuclear Information System (INIS)

Lee, D; Pollock, S; Keall, P; Greer, P; Lapuz, C; Ludbrook, J; Kim, T

2015-01-01

Purpose: Audiovisual biofeedback breath-hold (AVBH) was employed to reproduce tumor position on inhale and exhale breath-holds for 4D tumor information. We hypothesize that lung tumor position will be more consistent using AVBH compared with conventional breath-hold (CBH). Methods: Lung tumor positions were determined for seven lung cancer patients (age: 25 – 74) during to two separate 3T MRI sessions. A breathhold training session was performed prior to the MRI sessions to allow patients to become comfortable with AVBH and their exhale and inhale target positions. CBH and AVBH 4D image datasets were obtained in the first MRI session (pre-treatment) and the second MRI session (midtreatment) within six weeks of the first session. Audio-instruction (MRI: Siemens Skyra) in CBH and verbal-instruction (radiographer) in AVBH were used. A radiation oncologist contoured the lung tumor using Eclipse (Varian Medical Systems); tumor position was quantified as the centroid of the contoured tumor after rigid registration based on vertebral anatomy across two MRI sessions. CBH and AVBH were compared in terms of the reproducibility assessed via (1) the difference between the two exhale positions for the two sessions and the two inhale positions for the sessions. (2) The difference in amplitude (exhale to inhale) between the two sessions. Results: Compared to CBH, AVBH improved the reproducibility of two exhale (or inhale) lung tumor positions relative to each other by 33%, from 6.4±5.3 mm to 4.3±3.0 mm (p=0.005). Compared to CBH, AVBH improved the reproducibility of exhale and inhale amplitude by 66%, from 5.6±5.9 mm to 1.9±1.4 mm (p=0.005). Conclusions: This study demonstrated that audiovisual biofeedback can be utilized for improving the reproducibility of breath-hold lung tumor position. These results are advantageous towards achieving more accurate emerging radiation treatment planning methods, in addition to imaging and treatment modalities utilizing breath

SU-E-J-236: Audiovisual Biofeedback Improves Breath-Hold Lung Tumor Position Reproducibility Measured with 4D MRI

Energy Technology Data Exchange (ETDEWEB)

Lee, D; Pollock, S; Keall, P [Radiation Physics Laboratory, Sydney Medical School, The University of Sydney, NSW (Australia); Greer, P [School of Mathematical and Physical Sciences, The University of Newcastle, Newcastle, NSW (Australia); Department of Radiation Oncology, Calvary Mater Newcastle, Newcastle, NSW (Australia); Lapuz, C; Ludbrook, J [Department of Radiation Oncology, Calvary Mater Newcastle, Newcastle, NSW (Australia); Kim, T [Radiation Physics Laboratory, Sydney Medical School, The University of Sydney, NSW (Australia); Department of Radiation Oncology, University of Virginia Health System, Charlottesville, VA (United States)

2015-06-15

Purpose: Audiovisual biofeedback breath-hold (AVBH) was employed to reproduce tumor position on inhale and exhale breath-holds for 4D tumor information. We hypothesize that lung tumor position will be more consistent using AVBH compared with conventional breath-hold (CBH). Methods: Lung tumor positions were determined for seven lung cancer patients (age: 25 – 74) during to two separate 3T MRI sessions. A breathhold training session was performed prior to the MRI sessions to allow patients to become comfortable with AVBH and their exhale and inhale target positions. CBH and AVBH 4D image datasets were obtained in the first MRI session (pre-treatment) and the second MRI session (midtreatment) within six weeks of the first session. Audio-instruction (MRI: Siemens Skyra) in CBH and verbal-instruction (radiographer) in AVBH were used. A radiation oncologist contoured the lung tumor using Eclipse (Varian Medical Systems); tumor position was quantified as the centroid of the contoured tumor after rigid registration based on vertebral anatomy across two MRI sessions. CBH and AVBH were compared in terms of the reproducibility assessed via (1) the difference between the two exhale positions for the two sessions and the two inhale positions for the sessions. (2) The difference in amplitude (exhale to inhale) between the two sessions. Results: Compared to CBH, AVBH improved the reproducibility of two exhale (or inhale) lung tumor positions relative to each other by 33%, from 6.4±5.3 mm to 4.3±3.0 mm (p=0.005). Compared to CBH, AVBH improved the reproducibility of exhale and inhale amplitude by 66%, from 5.6±5.9 mm to 1.9±1.4 mm (p=0.005). Conclusions: This study demonstrated that audiovisual biofeedback can be utilized for improving the reproducibility of breath-hold lung tumor position. These results are advantageous towards achieving more accurate emerging radiation treatment planning methods, in addition to imaging and treatment modalities utilizing breath

Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

Directory of Open Access Journals (Sweden)

Leontina GURGU

2012-12-01

Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

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

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

2009-11-01

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

Allergy to fish collagen: Thermostability of collagen and IgE reactivity of patients' sera with extracts of 11 species of bony and cartilaginous fish.

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Kobayashi, Yukihiro; Kuriyama, Takuma; Nakagawara, Ryoko; Aihara, Michiko; Hamada-Sato, Naoko

2016-10-01

Parvalbumin was identified as a major fish allergen, and has been well investigated. Collagen was identified as a second allergen; however, its allergenic properties remain uncharacterized. Although fish is an important staple in coastal countries, its thermostability is unknown. Therefore, we aimed to determine the thermostability of fish collagen as an allergen. Meat of seven bony and four cartilaginous fishes was heated at various temperatures and times, and extracts were analyzed using SDS-PAGE, IgE-ELISA, and SPTs. Collagen was dissolved from heated meat of Pacific mackerel into a crude extract. Collagen in the extracts was degraded at a high heating load-140 °C (10 min) or 100 °C (320 min). However, ELISA revealed the IgE reactivities of patients' sera with the extracts were unchanged even after heating the samples. Patients strongly reacted to extract proteins of other bony fish, which were detected by patients' IgE even after heating at 100 °C (320 min). In contrast, reactivities of the extracts of cartilaginous fish were lower than those of bony fish. SPTs in one patient revealed that all bony and cartilaginous fish extracts prepared from heated meat elicited allergic reactions. The IgE reactivity of patients' sera to fish collagen in extracts was retained even when fish meat was treated by a high heating load. As for the fish collagen, the IgE reactivities to cartilaginous fish were lower than that to bony fish. Reducing IgE reactivity to fish meat using heat is difficult, and other modalities will be required to produce hypoallergenic fish meat. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

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

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Bhattacharya, Abhishek; Shrivastava, Ankita; Sharma, Anjana

2013-06-01

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

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

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

2015-03-02

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

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

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

2017-01-01

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

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

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

2016-02-01

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

Compensator design for hysteresis of a stacked PZT actuator using a congruency-based hysteresis model

International Nuclear Information System (INIS)

Nguyen, Phuong-Bac; Choi, Seung-Bok

2012-01-01

This paper proposes a rate-independent hysteresis compensator for a stacked PZT (lead zirconate titanate) actuator. From a congruency-based hysteresis (CBH) model which is derived from the inherent properties of this actuator, especially the congruency, a feedforward compensator associated with it is developed. The formulation of the proposed compensator is based on an assumption that the inverse operator also possesses the same properties as the CBH model does. This implies that the compensator also possesses properties such as the wiped-out loop closing between the consecutive control points and congruency. Consequently, the expressions for the compensator can be conducted by exploiting the equations for the CBH model in two cases of monotonic increase and monotonic decrease of input excitation. In order to assess the performance of the compensator, several experiments in both open-loop and closed-loop controls are undertaken. In the open-loop control experiment, the performance of the feedforward compensator using the CBH model is compared with the classical Preisach model-based one in three cases of reference waveforms. In the closed-loop control experiment, the proposed compensator is incorporated into a PID (proportional-integral-derivative) control system and the performance of this integrated system is then evaluated and compared to that of the PID with and without compensator. (paper)

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.

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

International Nuclear Information System (INIS)

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

2010-01-01

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

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

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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.

Development of thermophilic tailor-made enzyme mixtures for the bioconversion of agricultural and forest residues

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Anthi eKarnaouri

2016-02-01

Full Text Available Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5 and MtEG7, in the presence of other four accessory enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a and β-glucosidase MtBGL3, were tested as a 9-component cocktail against one model substrate (phosphoric acid swollen cellulose and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch and spruce biomass, as forest residues. Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC, high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch. Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that

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.

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

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

2013-01-01

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

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

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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.

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

Energy Technology Data Exchange (ETDEWEB)

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

1990-08-01

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

Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization.

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Huiping Liu

Full Text Available Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid (ABTS, syringaldazine (SGZ, guaiacol, and 2,6-dimethoxyphenol (2,6-DMP as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0-11.0 and thermostable at 40°C-90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

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Ueno, Tomohiro; Niimi, Hideki; Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

2015-01-01

Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and

Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity.

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Shrinivas, Dengeti; Kumar, Raghwendra; Naik, G R

2012-01-01

The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.

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

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Ashwini Naganthran

2017-09-01

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

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

International Nuclear Information System (INIS)

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

2003-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2003-10-01

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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.

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

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M. MARINOVIC-CINCOVIC

2004-02-01

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

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.

Effect of enzymatic (thermostable α-amylase) treatment on the physicochemical and antioxidant properties of extruded rice incorporated with soybean flour.

Science.gov (United States)

Xu, Enbo; Wu, Zhengzong; Pan, Xiaowei; Long, Jie; Wang, Fang; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

2016-04-15

In order to determine the effect of enzymatic extrusion on the physicochemical and antioxidant properties of rice/soybean mixture, different mass ratios (100/0, 95/5, 85/15, 70/30, 50/50 and 25/75%, w/w) were treated with thermostable α-amylase. The reduced special mechanical energy and the enhanced product temperature were closely and regularly linked with the increase of soybean content. The bulk density and water solubility index increased, and the water absorption index and viscosities decreased remarkably after enzymatic extrusion, however, the modification caused by α-amylase were dramatically eliminated with the increase of soybean content to ∼50%. Moreover, the addition of enzyme exhibited an improvement of the total phenolic/flavonoid content (TPC/TFC) and antioxidant capacities compared to traditional extrusion. The TPC/TFC retention of extrudate (ratios of 85/15 and 70/30%) attained over 90%, but dramatically decreased (72.91 and 67.81%, respectively) with soybean added to 75%, probably due to the great reduction of starch substrate for enzymatic hydrolysis. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Synthesis and characterisation of Cu(II), Ni(II), Mn(II), Zn(II) and VO(II ...

Indian Academy of Sciences (India)

Unknown

Synthesis and characterisation of Cu(II), Ni(II), Mn(II), Zn(II) and VO(II) Schiff base complexes derived from o-phenylenediamine and acetoacetanilide. N RAMAN*, Y PITCHAIKANI RAJA and A KULANDAISAMY. Department of Chemistry, VHNSN College, Virudhunagar 626 001, India e-mail: ra_man@123india.com.

Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride

International Nuclear Information System (INIS)

Li, Conghu; Tian, Zhenhua; Liu, Wentao; Li, Guoying

2015-01-01

The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen. - Highlights: • Acylated collagen retained the unique triple helix conformation. • Acylated collagen had stronger thermostability than native collagen. • Amide I was the most sensitive band to the temperature for acylated collagen. • Amide II was the most sensitive band to the temperature for native collagen. • Auto-peak at 1680 cm −1 for acylated collagen disappeared at higher temperature

Biologically active new Fe(II, Co(II, Ni(II, Cu(II, Zn(II and Cd(II complexes of N-(2-thienylmethylenemethanamine

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C. SPÎNU

2008-04-01

Full Text Available Iron(II, cobalt(II, nickel (II, copper (II, zinc(II and cadmium(II complexes of the type ML2Cl2, where M is a metal and L is the Schiff base N-(2-thienylmethylenemethanamine (TNAM formed by the condensation of 2-thiophenecarboxaldehyde and methylamine, were prepared and characterized by elemental analysis as well as magnetic and spectroscopic measurements. The elemental analyses suggest the stoichiometry to be 1:2 (metal:ligand. Magnetic susceptibility data coupled with electronic, ESR and Mössbauer spectra suggest a distorted octahedral structure for the Fe(II, Co(II and Ni(II complexes, a square-planar geometry for the Cu(II compound and a tetrahedral geometry for the Zn(II and Cd(II complexes. The infrared and NMR spectra of the complexes agree with co-ordination to the central metal atom through nitrogen and sulphur atoms. Conductance measurements suggest the non-electrolytic nature of the complexes, except for the Cu(II, Zn(II and Cd(II complexes, which are 1:2 electrolytes. The Schiff base and its metal chelates were screened for their biological activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and the metal chelates were found to possess better antibacterial activity than that of the uncomplexed Schiff base.

Characteristics of cloud occurrence using ceilometer measurements and its relationship to precipitation over Seoul

Science.gov (United States)

Lee, Sanghee; Hwang, Seung-On; Kim, Jhoon; Ahn, Myoung-Hwan

2018-03-01

Clouds are an important component of the atmosphere that affects both climate and weather, however, their contributions can be very difficult to determine. Ceilometer measurements can provide high resolution information on atmospheric conditions such as cloud base height (CBH) and vertical frequency of cloud occurrence (CVF). This study presents the first comprehensive analysis of CBH and CVF derived using Vaisala CL51 ceilometers at two urban stations in Seoul, Korea, during a three-year period from January 2014 to December 2016. The average frequency of cloud occurrence detected by the ceilometers is 54.3%. It is found that the CL51 is better able to capture CBH as compared to another ceilometer CL31 at a nearby meteorological station because it could detect high clouds more accurately. Frequency distributions for CBH up to 13,000 m providing detailed vertical features with 500-m interval show 55% of CBHs below 2 km for aggregated CBHs. A bimodal frequency distribution was observed for three-layers CBHs. A monthly variation of CVF reveals that frequency concentration of lower clouds is found in summer and winter, and higher clouds more often detected in spring and autumn. Monthly distribution features of cloud occurrence and precipitation are depending on seasons and it might be easy to define their relationship due to higher degree of variability of precipitation than cloud occurrence. However, a fluctuation of cloud occurrence frequency in summer is similar to precipitation in trend, whereas clouds in winter are relatively frequent but precipitation is not accompanied. In addition, recent decrease of summer precipitation could be mostly explained by a decrease of cloud occurrence. Anomalous precipitation recorded sometimes is considerably related to corresponding cloud occurrence. The diurnal and daily variations of CBH and CVF from ceilometer observations and the analysis of microwave radiometer measurements for two typical cloudiness cases are also reviewed

包覆赤磷制备工艺及其热安定性研究%Preparation technique of microcapsulated red phosphorusand its thermostability

Institute of Scientific and Technical Information of China (English)

刘杰; 关华; 宋东明

2017-01-01

为了降低赤磷的吸湿性,提高其热安定性,该文采用氢氧化铝-密胺树脂包覆赤磷(MRP).通过单因素实验优化了制备包覆赤磷的工艺,采用傅里叶变换红外(FTIR)光谱仪和扫描电子显微镜(SEM)对包覆层进行了表征,用热重-差热分析法(TG-DTA)分析了包覆前后赤磷样品的热安定性.结果表明,制备包覆赤磷的最佳工艺参数为,九水合硝酸铝的质量为0.10 g,甲醛和三聚氰胺总质量为0.40 g、摩尔比为3∶1,反应温度为80 ℃,反应时间为1 h.在此工艺条件下,包覆赤磷(MRP)的吸湿率降幅最大,达到96.88%,且包覆物质量分数小于5%,包覆材料在赤磷表面的包覆状况良好.相比于未包覆赤磷,包覆后赤磷的外推起始温度、峰值温度和外推终点温度分别提高42.8 ℃、51.7 ℃和48.4 ℃;自发火温度提高58.8 ℃,表明包覆赤磷的热安定性大幅提高.%In order to reduce the hygroscopicity of red phosphorus and improve its thermostability,the aluminium hydroxide/melamine-formaldehyde resin microcapsulated red phosphorus(MRP)is successfully prepared.The process of preparing the coated red phosphorus is optimized by the single factor test.The coating layer of the red phosphorus is characterized by the Fourier transform infrared(FTIR)spectrometer and the scanning electron microscopy(SEM).The thermostability of the red phosphorus(RP)and the MRP is investigated by the thermogravimetric analysis-differential thermal analysis(TG-DTA)in air atmosphere.Results show that the optimal process parameters for preparing the coated red phosphorus are,the mass of the aluminum nitrate nonahydrate 0.10 g,the total mass of the formaldehyde and melamine 0.40 g,the molar ratio of the formaldehyde and the melamine 3∶1,the reaction temperature 80 ℃,the reaction time 1 h.Under these optimal process parameters,the moisture absorption ratio of MRP reduces 96.88%(the greatest reduction),the mass fraction of coating materials is below 5

Complexes of cobalt(II), nickel(II), copper(II), zinc(II), cadmium(II) and dioxouranium(II) with thiophene-2-aldehydethiosemicarbazone

International Nuclear Information System (INIS)

Singh, Balwan; Misra, Harihar

1986-01-01

Metal complexes of thiosemicarbazides have been known for their pharmacological applications. Significant antitubercular, fungicidal and antiviral activities have been reported for thiosemicarbazides and their derivatives. The present study describes the systhesis and characterisation of complexes of Co II , Cu II , Zn II ,Cd II and UO II with thiosemicarbazone obtained by condensing thiophene-2-aldehyde with thiosemicarbazide. 17 refs., 2 tables. (author)

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

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A. L. Rumyantseva

2018-01-01

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

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

Science.gov (United States)

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

1992-07-01

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

Mn(II), Zn(II) and VO(II) Schiff

Indian Academy of Sciences (India)

Home; Journals; Journal of Chemical Sciences; Volume 113; Issue 3. Synthesis and characterisation of Cu(II), Ni(II), Mn(II), Zn(II) and VO(II) Schiff base complexes derived from o-phenylenediamine and acetoacetanilide. N Raman Y Pitchaikani Raja A Kulandaisamy. Inorganic Volume 113 Issue 3 June 2001 pp 183-189 ...

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.

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.

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

Science.gov (United States)

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

2003-04-01

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

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

Science.gov (United States)

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

2015-01-01

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

Study of the leaching of heavy metals from waste water sludge and incinerator's ash, using coupled thermostated columns and DTPA as complex agent; Estudio de la extraccion de metales pesados de lodos y cenizas de aguas residuales usando columnas termostatizadas acopladas y DTPA como agente complejante

Energy Technology Data Exchange (ETDEWEB)

Vite T, J.; Vite T, M.; Guerrero D, J.; Carreno de Leon, M.C. [Departamento de Estudios del Ambiente, Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)

2000-07-01

We studied the metallic composition from waste water sludge and incinerators ashes of an incinerator located in Toluca, Mexico, the qualitative studies were made using the Activation Analysis technique, and fluorescence X-ray techniques. The quantitative analysis of heavy metals in the wastes were made using Inductively coupled plasma atomic emission spectrometry (Icp-Aes). For leaching the samples, we used four coupled thermostated columns, each one had a p H of 2,5, 7 and 10. The flux of the air was of 1600 cc/min. The temperature was maintain constant in 60 Centigrade using a thermostated system. For this study we used 100 g of wastes mixed with mineral acid or sodium hydroxide to reach p H 2,5,7 and 10. We added a reducing and tensoactive agents and finally DTPA as complex agent. With this method, we obtain a better leaching efficiency using a complex agent. However the high DTPA cost, make this process expansive that is why we recommend to work with another classes of complex agents, that be cheaper to leach metals of different chemistry matrix. (Author)

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.

Adsorption of Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Fe(II), and As(V) on bacterially produced metal sulfides.

Science.gov (United States)

Jong, Tony; Parry, David L

2004-07-01

The adsorption of Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Fe(II) and As(V) onto bacterially produced metal sulfide (BPMS) material was investigated using a batch equilibrium method. It was found that the sulfide material had adsorptive properties comparable with those of other adsorbents with respect to the specific uptake of a range of metals and, the levels to which dissolved metal concentrations in solution can be reduced. The percentage of adsorption increased with increasing pH and adsorbent dose, but decreased with increasing initial dissolved metal concentration. The pH of the solution was the most important parameter controlling adsorption of Cd(II), Cu(II), Fe(II), Ni(II), Pb(II), Zn(II), and As(V) by BPMS. The adsorption data were successfully modeled using the Langmuir adsorption isotherm. Desorption experiments showed that the reversibility of adsorption was low, suggesting high-affinity adsorption governed by chemisorption. The mechanism of adsorption for the divalent metals was thought to be the formation of strong, inner-sphere complexes involving surface hydroxyl groups. However, the mechanism for the adsorption of As(V) by BPMS appears to be distinct from that of surface hydroxyl exchange. These results have important implications to the management of metal sulfide sludge produced by bacterial sulfate reduction.

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.

Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches.

Science.gov (United States)

Woon, James Sy-Keen; Mackeen, Mukram M; Illias, Rosli M; Mahadi, Nor M; Broughton, William J; Murad, Abdul Munir Abdul; Abu Bakar, Farah Diba

2017-01-01

Aspergillus niger , along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger . In this study, the gene encoding a cellobiohydrolase B ( cbh B) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic ® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment. Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N -glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p -nitrophenyl-cellobioside ( p NPC) and p -nitrophenyl-cellobiotrioside ( p NPG3) but was not active towards crystalline substrates like Avicel ® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum catalysis occurred at 50 °C and

Synthesis and spectroscopic studies of biologically active tetraazamacrocyclic complexes of Mn(II, Co(II, Ni(II, Pd(II and Pt(II

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Monika Tyagi

2014-01-01

Full Text Available Complexes of Mn(II, Co(II, Ni(II, Pd(II and Pt(II were synthesized with the macrocyclic ligand, i.e., 2,3,9,10-tetraketo-1,4,8,11-tetraazacycoletradecane. The ligand was prepared by the [2 + 2] condensation of diethyloxalate and 1,3-diamino propane and characterized by elemental analysis, mass, IR and 1H NMR spectral studies. All the complexes were characterized by elemental analysis, molar conductance, magnetic susceptibility measurements, IR, electronic and electron paramagnetic resonance spectral studies. The molar conductance measurements of Mn(II, Co(II and Ni(II complexes in DMF correspond to non electrolyte nature, whereas Pd(II and Pt(II complexes are 1:2 electrolyte. On the basis of spectral studies an octahedral geometry has been assigned for Mn(II, Co(II and Ni(II complexes, whereas square planar geometry assigned for Pd(II and Pt(II. In vitro the ligand and its metal complexes were evaluated against plant pathogenic fungi (Fusarium odum, Aspergillus niger and Rhizoctonia bataticola and some compounds found to be more active as commercially available fungicide like Chlorothalonil.

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

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Sandeep Kaur Saggu

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

Inflation of polymer melts into elliptic and circular cylinders

DEFF Research Database (Denmark)

Rasmussen, Henrik Koblitz; Christensen, Jens Horslund; Gøttsche, Søren

2000-01-01

A thin sheet (membrane) of the polymeric material is clamped between a Teflon-coated thermostated plate and a thermostated aluminium cylinder. By applying thermostated air through the plate, the polymer membrane deforms into an elliptic or a circular cylinder. The position of the top of the infla......A thin sheet (membrane) of the polymeric material is clamped between a Teflon-coated thermostated plate and a thermostated aluminium cylinder. By applying thermostated air through the plate, the polymer membrane deforms into an elliptic or a circular cylinder. The position of the top...

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

2013-07-01

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

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

Science.gov (United States)

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

2013-03-01

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

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

Science.gov (United States)

Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

2010-06-01

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

Effect of Cu(II), Cd(II) and Zn(II) on Pb(II) biosorption by algae Gelidium-derived materials.

Science.gov (United States)

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2008-06-15

Biosorption of Pb(II), Cu(II), Cd(II) and Zn(II) from binary metal solutions onto the algae Gelidium sesquipedale, an algal industrial waste and a waste-based composite material was investigated at pH 5.3, in a batch system. Binary Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II) solutions have been tested. For the same equilibrium concentrations of both metal ions (1 mmol l(-1)), approximately 66, 85 and 86% of the total uptake capacity of the biosorbents is taken by lead ions in the systems Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II), respectively. Two-metal results were fitted to a discrete and a continuous model, showing the inhibition of the primary metal biosorption by the co-cation. The model parameters suggest that Cd(II) and Zn(II) have the same decreasing effect on the Pb(II) uptake capacity. The uptake of Pb(II) was highly sensitive to the presence of Cu(II). From the discrete model it was possible to obtain the Langmuir affinity constant for Pb(II) biosorption. The presence of the co-cations decreases the apparent affinity of Pb(II). The experimental results were successfully fitted by the continuous model, at different pH values, for each biosorbent. The following sequence for the equilibrium affinity constants was found: Pb>Cu>Cd approximately Zn.

Systemic inflammation combined with neonatal cerebellar haemorrhage aggravates long-term structural and functional outcomes in a mouse model.

Science.gov (United States)

Tremblay, Sophie; Pai, Alex; Richter, Lindsay; Vafaei, Rod; Potluri, Praneetha; Ellegood, Jacob; Lerch, Jason P; Goldowitz, Daniel

2017-11-01

Despite the increased recognition of cerebellar injury in survivors of preterm birth, the neurodevelopmental consequences of isolated cerebellar injury have been largely unexplored and our current understanding of the functional deficits requires further attention in order to translate knowledge to best practices. Preterm infants are exposed to multiple stressors during their postnatal development including perinatal cerebellar haemorrhage (CBH) and postnatal infection, two major risk factors for neurodevelopmental impairments. We developed a translational mouse model of CBH and/or inflammation to measure the short- and long-term outcomes in cerebellar structure and function. Mice exposed to early combined insults of CBH and early inflammatory state (EIS) have a delay in grasping acquisition, neonatal motor deficits and deficient long-term memory. CBH combined with late inflammatory state (LIS) does not induce neonatal motor problems but leads to poor fine motor function and long-term memory deficits at adulthood. Early combined insults result in poor cerebellar growth from postnatal day 15 until adulthood shown by MRI, which are reflected in diminished volumes of cerebellar structures. There are also decreases in volumes of gray matter and hippocampus. Cerebellar microgliosis appears 24h after the combined insults and persists until postnatal day 15 in the cerebellar molecular layer and cerebellar nuclei in association with a disrupted patterning of myelin deposition, a delay of oligodendrocyte maturation and reduced white matter cerebellar volume. Together, these findings reveal poor outcomes in developing brains exposed to combined cerebellar perinatal insults in association with cerebellar hypoplasia, persistence of microgliosis and alterations of cerebellar white matter maturation and growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Regional Brain Shrinkage over Two Years: Individual Differences and Effects of Pro-Inflammatory Genetic Polymorphisms

Science.gov (United States)

Persson, N.; Ghisletta, P.; Dahle, C.L.; Bender, A.R.; Yang, Y.; Yuan, P.; Daugherty, A.M.; Raz, N.

2014-01-01

We examined regional changes in brain volume in healthy adults (N = 167, age 19-79 years at baseline; N = 90 at follow-up) over approximately two years. With latent change score models, we evaluated mean change and individual differences in rates of change in 10 anatomically-defined and manually-traced regions of interest (ROIs): lateral prefrontal cortex (LPFC), orbital frontal cortex (OF), prefrontal white matter (PFw), hippocampus (HC), parahippocampal gyrus (PhG), caudate nucleus (Cd), putamen (Pt), insula (In), cerebellar hemispheres (CbH), and primary visual cortex (VC). Significant mean shrinkage was observed in the HC, CbH, In, OF, and the PhG, and individual differences in change were noted in all regions, except the OF. Pro-inflammatory genetic variants mediated shrinkage in PhG and CbH. Carriers of two T alleles of interleukin-1β (IL-1βC-511T, rs16944) and a T allele of methylenetetrahydrofolate reductase (MTHFRC677T, rs1801133) polymorphisms showed increased PhG shrinkage. No effects of a pro-inflammatory polymorphism for C-reactive protein (CRP-286C>A>T, rs3091244) or apolipoprotein (APOE) ε4 allele were noted. These results replicate the pattern of brain shrinkage observed in previous studies, with a notable exception of the LPFC thus casting doubt on the unique importance of prefrontal cortex in aging. Larger baseline volumes of CbH and In were associated with increased shrinkage, in conflict with the brain reserve hypothesis. Contrary to previous reports, we observed no significant linear effects of age and hypertension on regional brain shrinkage. Our findings warrant further investigation of the effects of neuroinflammation on structural brain change throughout the lifespan. PMID:25264227

Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure.

Science.gov (United States)

Ishak, Siti Nor Hasmah; Aris, Sayangku Nor Ariati Mohamad; Halim, Khairul Bariyyah Abd; Ali, Mohd Shukuri Mohamad; Leow, Thean Chor; Kamarudin, Nor Hafizah Ahmad; Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd

2017-09-25

Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

Science.gov (United States)

Yildirim, Vildan; Baltaci, Mustafa Ozkan; Ozgencli, Ilknur; Sisecioglu, Melda; Adiguzel, Ahmet; Adiguzel, Gulsah

2017-12-01

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K M and V max values were calculated as 0.197 mg/mL and 7.29 μmol.mL - 1 .min - 1 , respectively.

Effect of chromone-substituted benzothiazolium halides on photosynthetic processes

International Nuclear Information System (INIS)

Kralova, K.; Sersen, F.; Gasparova, R.; Lacova, M.

1998-01-01

The effects of 3-R 2 -2[2-(6-R 1 -chromone-3-yl)ethenyl]benzothiazolium halides (CBH) on photosynthetic electron transport in spinach chloroplasts and in the legal suspension of Chlorella vulgaris were investigated. Using EPR spectroscopy it was confirmed that these compounds containing in their molecules two heterocyclic skeletons, namely benzothiazole and chromone, interact with the intermediate D + , corresponding to the tyrosine radical Tyr D situated in D 2 protein on the donor side of photosystem 2. Consequently, higher concentrations of CBH inhibited oxygen evolution rate in Chlorella vulgaris and the inhibitory effectiveness depended on the lipophilicity of the of the compound. (authors)

Physicochemical properties of 3,4,5-trimethoxybenzoates of Mn(II, Co(II, Ni(II and Zn(II

Directory of Open Access Journals (Sweden)

W. FERENC

2005-09-01

Full Text Available The complexes of Mn(II, Co(II, Ni(II, Cu(II and Zn(II with 3,4,5-trimethoxybenzoic acid anion of the formula: M(C10H11O52·nH2O, where n = 6 for Ni(II, n = 1 for Mn(II, Co(II, Cu(II, and n = 0 for Zn, have been synthesized and characterized by elemental analysis, IR spectroscopy, X–ray diffraction measurements, thermogravimetry and magnetic studies. They are crystalline compounds characterized by various symmetry. They decompose in various ways when heated in air to 1273 K. At first, they dehydrate in one step and form anhydrous salts. The final products of decomposition are oxides of the respective metals (Mn2O3, Co3O4, NiO, CuO, ZnO. The solubilities of the analysed complexes in water at 293 K are in the orders of 10-2 – 10-4 mol dm-3. The magnetic susceptibilities of the Mn(II, Co(II, Ni(II and Cu(II complexes were measured over the range of 76–303 K and the magnetic moments were calculated. The results show that the 3,4,5-trimethoxybenzoates of Mn(II, Co(II and Ni(II are high-spin complexes but that of Cu(II forms a dimer [Cu2(C10H11O54(H2O2]. The carboxylate groups bind as monodentate or bidentate chelating or bridging ligands.

Synthesis and spectral studies of manganese(II), cobalt(II), nickel(II), copper(II), zinc(II), cadmium(II) and mercury(II) complexes of 4-oxo-4H-1-benzopyran-3-carboxaldehyde hydrazone derivatives

International Nuclear Information System (INIS)

Nawar, N.; Khattab, M.A.; Bekheit, M.M.; El-Kaddah, A.H.

1996-01-01

A few complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Hg(II) with 4-oxo-4H-1-benzopyran-3-(carboxaldehyde-4-chlorobenzylhydrazone) (BCBH) and 4-oxo-4H-1-benzopyran-3-(carboxaldehyde-4-methylbenzylhydrazone) (BMBH) have been synthesised and characterized by elemental analysis, molar conductivities, magnetic measurements and infrared (IR) and visible spectral studies. The IR spectra show that BCBH and BMBH behave as bidentate ligands either in the keto or enol form. (author). 24 refs., 2 tabs

Synthesis, Characterization, and Biological Activity of Mn(II, Fe(II, Co(II, Ni(II, Cu(II, Zn(II, and Cd(II Complexes of N-Thiophenoyl-N′-Phenylthiocarbohydrazide

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M. Yadav

2013-01-01

Full Text Available Mn(II, Fe(II, Co(II, Ni(II, Cu(II, Zn(II, and Cd(II complex of N-thiophenoyl -N′-phenylthiocarbohydrazide (H2 TPTH have been synthesized and characterized by elemental analysis, magnetic susceptibility measurements, infrared, NMR, electronic, and ESR spectral studies. The complexes were found to have compositions [Mn(H TPTH2], [Co(TPTH (H2O2], [Ni(TPTH (H2O2], [Cu(TPTH], [Zn(H TPTH], [Cd(H TPTH2], and [Fe(H TPTH2(EtOH]. The magnetic and electronic spectral studies suggest square planar geometry for [Cu(TPTH], tetrahedral geometry for [Zn(TPTH] and [Cd(H TPTH2], and octahedral geometry for rest of the complexes. The infrared spectral studies of the 1 : 1 deprotonated complexes suggest bonding through enolic oxygen, thiolato sulfur, and both the hydrazinic nitrogens. Thus, H2TPTH acts as a binegative tetradentate ligand. H2 TPTH and its metal complexes have been screened against several bacteria and fungi.

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5.

Science.gov (United States)

Rachadech, W; Navacharoen, A; Ruangsit, W; Pongtharangkul, T; Vangnai, A S

2010-01-01

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.

Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

Directory of Open Access Journals (Sweden)

James Sy-Keen Woon

2017-10-01

Full Text Available Background Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. Methods In this study, the gene encoding a cellobiohydrolase B (cbhB from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercial cellulase preparation (Cellic® CTec2 and was used to hydrolyse oil palm empty fruit bunch (OPEFB, one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR to screen for any compositional changes upon enzymatic treatment. Results Recombinant CBHB was over-expressed as a hyperglycosylated protein attached to N-glycans. CBHB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC, p-nitrophenyl-cellobioside (pNPC and p-nitrophenyl-cellobiotrioside (pNPG3 but was not active towards crystalline substrates like Avicel® and Sigmacell cellulose. Characterisation of purified CBHB using MUC as the model substrate revealed that optimum

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

Directory of Open Access Journals (Sweden)

Yasser R. Abdel-Fattah

2013-01-01

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

Chemical speciation of Pb(II, Cd(II, Hg(II, Co(II, Ni(II, Cu(II and Zn(II binary complexes of l-methionine in 1,2-propanediol-water mixtures

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M. Padma Latha

2007-04-01

Full Text Available Chemical speciation of Pb(II, Cd(II, Hg(II, Co(II, Ni(II, Cu(II and Zn(II complexes of L-methionine in 0.0-60 % v/v 1,2-propanediol-water mixtures maintaining an ionic strength of 0.16 M at 303 K has been studied pH metrically. The active forms of ligand are LH2+, LH and L-. The predominant species detected are ML, MLH, ML2, ML2H, ML2H2 and MLOH. Models containing different numbers of species were refined by using the computer program MINIQUAD 75. The best-fit chemical models were arrived at based on statistical parameters. The trend in variation of complex stability constants with change in the dielectric constant of the medium is explained on the basis of electrostatic and non-electrostatic forces.

Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans

International Nuclear Information System (INIS)

Doan, Thi-Ngoc-Thanh; Prabhu, Ponnandy; Kim, Jin-Kwang; Ahn, Yeh-Jin; Natarajan, Sampath; Kang, Lin-Woo; Park, Geon Tae; Lim, Sang-Boem; Lee, Jung-Kul

2010-01-01

l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution. l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å 3 Da −1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method

cobalt (ii), nickel (ii)

African Journals Online (AJOL)

DR. AMINU

Department of Chemistry Bayero University, P. M. B. 3011, Kano, Nigeria. E-mail: hnuhu2000@yahoo.com. ABSTRACT. The manganese (II), cobalt (II), nickel (II) and .... water and common organic solvents, but are readily soluble in acetone. The molar conductance measurement [Table 3] of the complex compounds in.

Amobilisasi Sel Bacillus licheniformis KA-08 dalam Menghasilkan Keratinase Termostabil

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Anthoni Agustien

2012-10-01

Full Text Available Isolate local Bacillus licheniformis KA-08 known extracellular thermostable keratinase producers. Scale up of thermostable keratinase production can be with cells immobilized. The objective of the research is to thermostable keratinase production of B. licheniformis KA-08 cells immobilization. Thermostable keratinase activities were determined with modification of Brandelli and Riffel method. Protein concentration of enzyme determined with Lowry method. Immobilization of cells by Ca-alginate matrix with Adinarayana method, alginate concentration and amount of alginate bead effects with Beshay method. The result extracellular thermostable keratinase of B. licheniformis KA-08 cells immobilized was maximum produced at 12 times incubation with activity as 9.25 U/mg. Three percent alginate has optimum activity. Three hundred alginate beads has optimum activity. Cells immobilized ofB. licheniformis KA-08 has scale up of thermostable keratinase activity at 2 times than free cells. Thermostable keratinase produced by cell immobilized was nine cycles.

Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw

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Billard Hélène

2012-02-01

Full Text Available Abstract Background An efficient hydrolysis of lignocellulosic substrates to soluble sugars for biofuel production necessitates the interplay and synergistic interaction of multiple enzymes. An optimized enzyme mixture is crucial for reduced cost of the enzymatic hydrolysis step in a bioethanol production process and its composition will depend on the substrate and type of pretreatment used. In the present study, an experimental design was used to determine the optimal composition of a Trichoderma reesei enzyme mixture, comprising the main cellulase and hemicellulase activities, for the hydrolysis of steam-exploded wheat straw. Methods Six enzymes, CBH1 (Cel7a, CBH2 (Cel6a, EG1 (Cel7b, EG2 (Cel5a, as well as the xyloglucanase Cel74a and the xylanase XYN1 (Xyl11a were purified from a T. reesei culture under lactose/xylose-induced conditions. Sugar release was followed in milliliter-scale hydrolysis assays for 48 hours and the influence of the mixture on initial conversion rates and final yields is assessed. Results The developed model could show that both responses were strongly correlated. Model predictions suggest that optimal hydrolysis yields can be obtained over a wide range of CBH1 to CBH2 ratios, but necessitates a high proportion of EG1 (13% to 25% which cannot be replaced by EG2. Whereas 5% to 10% of the latter enzyme and a xylanase content above 6% are required for highest yields, these enzymes are predicted to be less important in the initial stage of hydrolysis. Conclusions The developed model could reliably predict hydrolysis yields of enzyme mixtures in the studied domain and highlighted the importance of the respective enzyme components in both the initial and the final hydrolysis phase of steam-exploded wheat straw.

The thermal stability of yellow fever vaccines

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Ricardo Ishak

1990-09-01

Full Text Available The assessment of yellow fever vaccine thermostability both in lyophilized form and after reconstitution were analyzed. Two commercial yellow fever vaccines were assayed for their thermal stability. Vaccines were exposed to test temperatures in the range of 8 (graus C to 45 (graus C. Residual infectivity was measured by a plaque assay using Vero cells. The titre values were used in an accelerated degradation test that follows the Arrhenius equation and the minimum immunizing dose was assumed to be 10 (ao cubo particles forming unit (pfu/dose. Some of the most relevant results include that (i regular culture medium show the same degradation pattern of a reconstituted 17D-204 vaccine; (ii reconstituted YF-17D-204 showed a predictable half life of more than six days if kept at 0 (graus C; (iii there are differences in thermostability between different products that are probably due to both presence of stabilizers in the preparation and the modernization in the vaccine production; (iv it is important to establish a proper correlation between the mouse infectivity test and the plaque assay since the last appears to be more simple, economical, and practical for small laboratories to assess the potency of the vaccine, and (v the accelerated degradation test appears to be the best procedure to quantify the thermostability of biological products.

Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry.

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Niu, Canfang; Yang, Peilong; Luo, Huiying; Huang, Huoqing; Wang, Yaru; Yao, Bin

2017-08-30

Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

Cr(III,Mn(II,Fe(III,Co(II,Ni(II,Cu(II and Zn(II Complexes with Diisobutyldithiocarbamato Ligand

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Mohammad Tarique

2011-01-01

Full Text Available The synthesis of sulphur and nitrogen containing dithiocarbamato ligand derived from diisobutylamine as well as its coordination compounds with 3d series transition metals is presented. These synthesized compounds were characterized on the basis of elemental analysis, conductometric measurements and IR spectral studies. The analytical data showed the stoichiometry 1:2 and 1:3 for the compounds of the types ML2 {M=Mn(II, Co(II, Ni(II, Cu(II and Zn(II} and M'L3{M'=Cr(III and Fe(III} respectively. The conductometric measurements proved the non-electrolytic behaviour of all the compounds. The bidentate nature of dithiocarbamato moiety was confirmed on the basis of IR spectral data.

Computer augumented modelling studies of Pb(II, Cd(II, Hg(II, Co(II, Ni(II, Cu(II and Zn(II complexes of L-glutamic acid in 1,2-propanediol–water mixtures

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MAHESWARA RAO VEGI

2008-12-01

Full Text Available Chemical speciation of Pb(II, Cd(II, Hg(II, Co(II, Ni(II, Cu(II and Zn(II complexes of L-glutamic acid was studied at 303 K in 0–60 vol. % 1,2-propanediol–water mixtures, whereby the ionic strength was maintained at 0.16 mol dm-3. The active forms of the ligand are LH3+, LH2 and LH–. The predominant detected species were ML, ML2, MLH, ML2H and ML2H2. The trend of the variation in the stability constants with changing dielectric constant of the medium is explained based on the cation stabilizing nature of the co-solvents, specific solvent–water interactions, charge dispersion and specific interactions of the co-solvent with the solute. The effect of systematic errors in the concentrations of the substances on the stability constants is in the order alkali > > acid > ligand > metal. The bioavailability and transportation of metals are explained based on distribution diagrams and stability constants.

Synthesis and characterization of heterobimetallic complexes of the type [Cu(pn2][MCl4] where M = Co(II, Ni(II, Cu(II, Zn(II, Cd(II, and Hg(II

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Seema Yadav

2016-11-01

Full Text Available A series of new bimetallic transition metal complexes of the type [Cu(pn2] [MCl4] have been synthesized (where M = Co(II, Ni(II, Cu(II, Zn(II, Cd(II and Hg(II, pn = 1,3-diaminopropane and characterized by elemental analysis, molar conductance, TGA, IR and electronic spectra. All the compounds are 1:1 electrolyte in DMF. The Cu(II ion is square-planar while metal ions in the anionic moiety acquire their usual tetrahedral arrangement. On the basis of these studies it is concluded that anionic moiety is electrically stabilized by its cationic counterpart.

Development of a D-amino acids electrochemical sensor based on immobilization of thermostable D-Proline dehydrogenase within agar gel membrane

International Nuclear Information System (INIS)

Tani, Yuji; Tanaka, Katsuhito; Yabutani, Tomoki; Mishima, Yuji; Sakuraba, Haruhiko; Ohshima, Toshihisa; Motonaka, Junko

2008-01-01

A novel biosensor for determination of D-amino acids (DAAs) in biological samples by using an electrode based on immobilization of a thermostable D-Proline dehydrogenase (D-Pro DH) within an agar gel membrane was developed. The electrode was simply prepared by spin-coating the agar solution with the D-Pro DH on a glassy carbon (GC) electrode. An electrocatalytic oxidation current of 2,6-dichloroindophenol (DCIP) was observed at -100 mV vs. Ag/AgCl with the addition of 5 and 20 mmol L -1 D-proline. The current response and its relative standard deviation were 0.15 μA and 7.6% (n = 3), respectively, when it was measured in a pH 8.0 phosphate buffer solution containing 10 mmol L -1 D-proline and 0.5 mmol L -1 DCIP at 50 deg. C. The current response of D-proline increased with increase of the temperature of the sample solution up to 70 deg. C. The electrocatalytic response at the D-Pro DH/agar immobilized electrode subsequently maintained for 80 days. Finally, the D-Pro DH/agar immobilized electrode was applied to determination of DAAs in a human urine sample. The determined value of DAAs in the human urine and its R.S.D. were 1.39 ± 0.12 mmol L -1 and 8.9% (n = 3), respectively

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

Science.gov (United States)

Kim, Hye-Jung; Ryu, Se-Ah; Kim, Pil; Oh, Deok-Kun

2003-01-01

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.

A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

Science.gov (United States)

Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

2017-12-11

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

Small Diameter Bomb Increment II (SDB II)

Science.gov (United States)

2015-12-01

Selected Acquisition Report (SAR) RCS: DD-A&T(Q&A)823-439 Small Diameter Bomb Increment II (SDB II) As of FY 2017 President’s Budget Defense... Bomb Increment II (SDB II) DoD Component Air Force Joint Participants Department of the Navy Responsible Office References SAR Baseline (Production...Mission and Description Small Diameter Bomb Increment II (SDB II) is a joint interest United States Air Force (USAF) and Department of the Navy

Synthesis and structural characterization of nickel(II), cobalt(II), Zinc(II), manganese(II), cadmium(II) and uranium(VI) complexes of α-oximinoacetoacet-o/p-anisidide thiosemicarbazone

International Nuclear Information System (INIS)

Patel, P.S.; Patel, M.M.; Ray, R.M.

1993-01-01

A few metal complexes of α-oximinoacetoacet-o/p-anisidide thiosemicarbazones (OAOATS)/(OAPATS) with Ni(II), Co(II), Zn(II), Mn(II), Hg(II), Cd(II) and UO 2 (II) have been prepared and characterized by elemental analyses, conductivity, differential scanning calorimetry study, thermogravimetric analyses and infrared and electronic spectral measurements in conjunction with magnetic susceptibility measurements at room temperature. They have also been tested for their antimicrobial activities. (author). 24 refs., 2 tabs

Competitive adsorption of copper(II), cadmium(II), lead(II) and zinc(II) onto basic oxygen furnace slag

International Nuclear Information System (INIS)

Xue Yongjie; Hou Haobo; Zhu Shujing

2009-01-01

Polluted and contaminated water can often contain more than one heavy metal species. It is possible that the behavior of a particular metal species in a solution system will be affected by the presence of other metals. In this study, we have investigated the adsorption of Cd(II), Cu(II), Pb(II), and Zn(II) onto basic oxygen furnace slag (BOF slag) in single- and multi-element solution systems as a function of pH and concentration, in a background solution of 0.01 M NaNO 3 . In adsorption edge experiments, the pH was varied from 2.0 to 13.0 with total metal concentration 0.84 mM in the single element system and 0.21 mM each of Cd(II), Cu(II), Pb(II), and Zn(II) in the multi-element system. The value of pH 50 (the pH at which 50% adsorption occurs) was found to follow the sequence Zn > Cu > Pb > Cd in single-element systems, but Pb > Cu > Zn > Cd in the multi-element system. Adsorption isotherms at pH 6.0 in the multi-element systems showed that there is competition among various metals for adsorption sites on BOF slag. The adsorption and potentiometric titrations data for various slag-metal systems were modeled using an extended constant-capacitance surface complexation model that assumed an ion-exchange process below pH 6.5 and the formation of inner-sphere surface complexes at higher pH. Inner-sphere complexation was more dominant for the Cu(II), Pb(II) and Zn(II) systems

Competitive adsorption of copper(II), cadmium(II), lead(II) and zinc(II) onto basic oxygen furnace slag

Energy Technology Data Exchange (ETDEWEB)

Xue Yongjie [School of Resource and Environment Science, Wuhan University, Hubei, Wuhan (China); Wuhan Kaidi Electric Power Environmental Protection Co. Ltd., Hubei, Wuhan (China)], E-mail: xueyj@mail.whut.edu.cn; Hou Haobo; Zhu Shujing [School of Resource and Environment Science, Wuhan University, Hubei, Wuhan (China)

2009-02-15

Polluted and contaminated water can often contain more than one heavy metal species. It is possible that the behavior of a particular metal species in a solution system will be affected by the presence of other metals. In this study, we have investigated the adsorption of Cd(II), Cu(II), Pb(II), and Zn(II) onto basic oxygen furnace slag (BOF slag) in single- and multi-element solution systems as a function of pH and concentration, in a background solution of 0.01 M NaNO{sub 3}. In adsorption edge experiments, the pH was varied from 2.0 to 13.0 with total metal concentration 0.84 mM in the single element system and 0.21 mM each of Cd(II), Cu(II), Pb(II), and Zn(II) in the multi-element system. The value of pH{sub 50} (the pH at which 50% adsorption occurs) was found to follow the sequence Zn > Cu > Pb > Cd in single-element systems, but Pb > Cu > Zn > Cd in the multi-element system. Adsorption isotherms at pH 6.0 in the multi-element systems showed that there is competition among various metals for adsorption sites on BOF slag. The adsorption and potentiometric titrations data for various slag-metal systems were modeled using an extended constant-capacitance surface complexation model that assumed an ion-exchange process below pH 6.5 and the formation of inner-sphere surface complexes at higher pH. Inner-sphere complexation was more dominant for the Cu(II), Pb(II) and Zn(II) systems.

Competitive adsorption of copper(II), cadmium(II), lead(II) and zinc(II) onto basic oxygen furnace slag.

Science.gov (United States)

Xue, Yongjie; Hou, Haobo; Zhu, Shujing

2009-02-15

Polluted and contaminated water can often contain more than one heavy metal species. It is possible that the behavior of a particular metal species in a solution system will be affected by the presence of other metals. In this study, we have investigated the adsorption of Cd(II), Cu(II), Pb(II), and Zn(II) onto basic oxygen furnace slag (BOF slag) in single- and multi-element solution systems as a function of pH and concentration, in a background solution of 0.01M NaNO(3). In adsorption edge experiments, the pH was varied from 2.0 to 13.0 with total metal concentration 0.84mM in the single element system and 0.21mM each of Cd(II), Cu(II), Pb(II), and Zn(II) in the multi-element system. The value of pH(50) (the pH at which 50% adsorption occurs) was found to follow the sequence Zn>Cu>Pb>Cd in single-element systems, but Pb>Cu>Zn>Cd in the multi-element system. Adsorption isotherms at pH 6.0 in the multi-element systems showed that there is competition among various metals for adsorption sites on BOF slag. The adsorption and potentiometric titrations data for various slag-metal systems were modeled using an extended constant-capacitance surface complexation model that assumed an ion-exchange process below pH 6.5 and the formation of inner-sphere surface complexes at higher pH. Inner-sphere complexation was more dominant for the Cu(II), Pb(II) and Zn(II) systems.

Preparation of Schiff s base complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) and their spectroscopic, magnetic, thermal, and antifungal studies

International Nuclear Information System (INIS)

Parekh, H.M.; Patel, M.N.

2006-01-01

The potassium salt of salicylidene-DL-alanine (KHL), bis(benzylidene)ethylenediamine (A 1 ), thiophene-o-carboxaldene-p-toluidine (A 2 ), and its metal complexes of the formula [(M II (L)(A)(H 2 O)] (M=Mn(II), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II); A = A 1 or A 2 ) are prepared. They are characterized by elemental analysis, magnetic susceptibility measurements, thermogravimetric analysis, and infrared and electronic spectral studies. The electronic spectral and magnetic moment data suggest an octahedral geometry for the complexes. All of these complexes, metal nitrates, fungicides (bavistin and emcarb), and ligands are screened for their antifungal activity against Aspergillus niger, Fusarium oxysporum, and Aspergillus flavus using a plate poison technique. The complexes show higher activity than those of the free ligands, metal nitrate, and the control (DMSO) and moderate activity against bavistin and emcarb [ru

Lactate dehydrogenase of Mugil sp. (Mugilidae, Perciformes. Lack of electrokinetic, thermostability and kinetic differences among individuals with different number of scales

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Marcelo dos Santos

2000-03-01

Full Text Available The scale number in lateral sets (SNS of Mugil sp. (Mugilidae, Perciformes collected in the lagoon-estuarine region of Cananéia, State of São Paulo ranges from 33 to 39. Electrokinetic, kinetic and thermostability properties of lactate dehydrogenase (LDH were tested to determine if individuals with different SNS correspond to different species or populations of mullet. As in many other teleosts, LDH-A*, LDH-B*, and LDH-C* loci were detected. Through a two-fold serial dilution method applied to 10 different tissues of Mugil sp., a bidirectionally divergent expression of these loci was suggested. No association among LDH electrophoretic pattern, thermal inactivation, kinetic responses and different SNS was observed. The apparent Km (pyr values obtained here were similar to Km values obtained by other authors for muscle and heart LDH or their purified isoforms. The effect of NaCl on Km and Vmax values of Mugil sp. (35 and 39 SNS individuals indicates that this salt behaves as a competitive inhibitor, since it decreases enzyme-substrate affinity. Thus, electrokinetic and thermostability behavior, Km and Vmax values and the effect of NaCl do not permit us to consider these mullets, with SNS ranging from 33 to 39, as belonging to different populations or species.O número de escamas em séries laterais (SNS de exemplares de Mugil sp. (Mugilidae, Perciformes coletados na região estuarino-lagunar de Cananéia, Estado de São Paulo, varia de 33 a 39. A fim de tentar determinar se exemplares com diferentes SNS corresponderiam a diferentes espécies ou populações de tainhas, foram analisadas as propriedades eletrocinéticas, cinéticas e de termoestabilidade da sua lactato desidrogenase (LDH. A exemplo de muitos teleósteos, a LDH de Mugil sp. mostrou-se codificada por 3 locos gênicos: LDH-A*, LDH-B* e LDH-C*. Método de diluições seriadas aplicado a 10 diferentes tecidos dessa espécie sugeriu um padrão bidirecionalmente divergente de express

thermo-stable alpha-amylase(S) from irradiated microbial origin utilizing agricultural and environmental wastes under solid state fermentation conditions

International Nuclear Information System (INIS)

Moustafa, O.E.A.

2002-01-01

an investigation concerning the production of thermo-stable α-amylases by thermophilic bacterial and fungal isolates has been undertaken. nine thermophilic bacteria and five teen fungi were isolated from different localities viz. phyllosphere of water hyacinth, different desert plants leaves, fermented dough, oven dust, garbage , and soil. their amylolytic activities were tested by dinitrosalicylic acid color reagent (Dns) method when grown on some environmental pollutants (garbage and water hyacinth) as well as industrial wastes (Bagasse, biscuit, corn flex and dough residues ) as the sole carbon source at 65 o C for bacterial and at 50 o C for fungal isolates . isolates No. B 1 ,B 2 ,B 5 ,B 6 ,B 7 ,B 8 ,B 9 , and F 4 ,F 6 ,F 8 ,F 1 2,F 1 3 and F 1 5, exhibited the highest α -amylase production when grown on water hyacinth, while B 4 ,F 3 ,F 1 1 and F 1 3, on dough ; (B 3 ,F 9 and F 1 0 ) on bagasse and ( F 1 ,F 2 ,F 5 ,F 7 ,F 1 1 and F 1 4) on garbage. Out of the nine identified bacterial isolated, only two isolates viz; actinobacillus actinomycetemcomitans, B1 and strepto bacillus moniliformis, B 7 , exhibited the ability to produce high percentages of α amylases at 55 o C (while still able to produce the enzyme within 45-70 o C)

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

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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.

Cu(II) AND Zn(II)

African Journals Online (AJOL)

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SYNTHESIS OF 2,2-DIMETHYL-4-PHENYL-[1,3]-DIOXOLANE USING ZEOLITE. ENCAPSULATED Co(II), Cu(II) AND Zn(II) COMPLEXES. B.P. Nethravathi1, K. Rama Krishna Reddy2 and K.N. Mahendra1*. 1Department of Chemistry, Bangalore University, Bangalore-560001, India. 2Department of Chemistry, Government ...

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

Science.gov (United States)

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

2015-01-01

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

Spectroscopic and thermal degradation behavior of Mg(II, Ca(II, Ba(II and Sr(II complexes with paracetamol drug

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Moamen S. Refat

2017-05-01

Full Text Available Complexes of Mg(II, Ca(II, Ba(II and Sr(II with paracetamol drug were synthesized and characterized by elemental analysis, conductivity, UV–Vis, IR, and 1H NMR spectroscopy and thermal analysis, as well as screened for antimicrobial activity. The IR spectral data suggested that the ligand behaves as paracetamol behaves as a neutral bidentate ligand coordinated to the metal ions via the lone pair of electrons of nitrogen and carbonyl-O atoms of the amide group. From the microanalytical data, the stoichiometry of the complexes reacts with Mg(II, Ca(II, Ba(II and Sr(II by molar ratios (2:1 (paracetamol:metal ion. The thermal behavior (TG/DTG of the complexes was studied. The ligand and their metal complexes were screened against both of antibacterial and fungicidal activities.

AC electrical conductivity and dielectric relaxation studies on n-type organic thin films of N, N‧-Dimethyl-3,4,9,10-perylenedicarboximide (DMPDC)

Science.gov (United States)

Qashou, Saleem I.; Darwish, A. A. A.; Rashad, M.; Khattari, Z.

2017-11-01

Both Alternating current (AC) conductivity and dielectric behavior of n-type organic thin films of N, N‧-Dimethyl-3,4,9,10-perylenedicarboximide (DMPDC) have been investigated. Fourier transformation infrared (FTIR) spectroscopy is used for identifying both powder and film bonds which confirm that there are no observed changes in the bonds between the DMPDC powder and evaporated films. The dependence of AC conductivity on the temperature for DMPDC evaporated films was explained by the correlated barrier hopping (CBH) model. The calculated barrier height using CBH model shows a decreasing behavior with increasing temperature. The mechanism of dielectric relaxation was interpreted on the basis of the modulus of the complex dielectric. The calculated activation energy of the relaxation process was found to be 0.055 eV.

Solar photocatalytic removal of Cu(II), Ni(II), Zn(II) and Pb(II): Speciation modeling of metal-citric acid complexes

International Nuclear Information System (INIS)

Kabra, Kavita; Chaudhary, Rubina; Sawhney, R.L.

2008-01-01

The present study is targeted on solar photocatalytic removal of metal ions from wastewater. Photoreductive deposition and dark adsorption of metal ions Cu(II), Ni(II), Pb(II) and Zn(II), using solar energy irradiated TiO 2 , has been investigated. Citric acid has been used as a hole scavenger. Modeling of metal species has been performed and speciation is used as a tool for discussing the photodeposition trends. Ninety-seven percent reductive deposition was obtained for copper. The deposition values of other metals were significantly low [nickel (36.4%), zinc (22.2%) and lead (41.4%)], indicating that the photocatalytic treatment process, using solar energy, was more suitable for wastewater containing Cu(II) ions. In absence of citric acid, the decreasing order deposition was Cu(II) > Ni(II) > Pb(II) > Zn(II), which proves the theoretical thermodynamic predictions about the metals

Cd(II), Cu(II)

African Journals Online (AJOL)

user

Depending on the way goethite was pretreated with oxalic acid, affinity for Cd(II) varied ...... Effects and mechanisms of oxalate on Cd(II) adsorption on goethite at different ... precipitation, surfactant mediation, hydrothermal and micro-emulsion.

Sequestration of Cu(II), Ni(II), and Co(II) by ethyleneimine immobilized on silica

International Nuclear Information System (INIS)

Arakaki, Luiza N.H.; Alves, Ana Paula M.; Silva Filho, Edson C. da; Fonseca, Maria G.; Oliveira, Severino F.; Espinola, Jose Geraldo P.; Airoldi, Claudio

2007-01-01

Thermodynamic data on interaction of Cu(II), Ni(II), and Co(II) with silica modified with ethyleneimine are obtained by calorimetric titration. The amount of ethyleneimine anchored on silica surface was estimated to be 0.70 mmol g -1 . The enthalpies of binding Ni(II), Cu(II) and Co(II), are -3.59 ± 0.001, -4.88 ± 0.001, and -7.75 ± 0.003 kJ mol -1 , respectively

Solid-phase extraction of Mn(II), Co(II), Ni(II), Cu(II), Cd(II) and Pb(II) ions from environmental samples by flame atomic absorption spectrometry (FAAS)

Energy Technology Data Exchange (ETDEWEB)

Duran, Celal [Department of Chemistry, Faculty of Art and Science, Karadeniz Technical University, 61080 Trabzon (Turkey); Gundogdu, Ali [Department of Chemistry, Faculty of Art and Science, Karadeniz Technical University, 61080 Trabzon (Turkey); Bulut, Volkan Numan [Department of Chemistry, Giresun Faculty of Art and Science, Karadeniz Technical University, 28049 Giresun (Turkey); Soylak, Mustafa [Department of Chemistry, Faculty of Art and Science, Erciyes University, 38039 Kayseri (Turkey)]. E-mail: soylak@erciyes.edu.tr; Elci, Latif [Department of Chemistry, Faculty of Art and Science, Pamukkale University, 20020 Denizli (Turkey); Sentuerk, Hasan Basri [Department of Chemistry, Faculty of Art and Science, Karadeniz Technical University, 61080 Trabzon (Turkey); Tuefekci, Mehmet [Department of Chemistry, Faculty of Art and Science, Karadeniz Technical University, 61080 Trabzon (Turkey)

2007-07-19

A new method using a column packed with Amberlite XAD-2010 resin as a solid-phase extractant has been developed for the multi-element preconcentration of Mn(II), Co(II), Ni(II), Cu(II), Cd(II), and Pb(II) ions based on their complex formation with the sodium diethyldithiocarbamate (Na-DDTC) prior to flame atomic absorption spectrometric (FAAS) determinations. Metal complexes sorbed on the resin were eluted by 1 mol L{sup -1} HNO{sub 3} in acetone. Effects of the analytical conditions over the preconcentration yields of the metal ions, such as pH, quantity of Na-DDTC, eluent type, sample volume and flow rate, foreign ions etc. have been investigated. The limits of detection (LOD) of the analytes were found in the range 0.08-0.26 {mu}g L{sup -1}. The method was validated by analyzing three certified reference materials. The method has been applied for the determination of trace elements in some environmental samples.

Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77

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Wajdi Thebti

2016-01-01

Full Text Available A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1.

Average [O II] nebular emission associated with Mg II absorbers: dependence on Fe II absorption

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Joshi, Ravi; Srianand, Raghunathan; Petitjean, Patrick; Noterdaeme, Pasquier

2018-05-01

We investigate the effect of Fe II equivalent width (W2600) and fibre size on the average luminosity of [O II] λλ3727, 3729 nebular emission associated with Mg II absorbers (at 0.55 ≤ z ≤ 1.3) in the composite spectra of quasars obtained with 3 and 2 arcsec fibres in the Sloan Digital Sky Survey. We confirm the presence of strong correlations between [O II] luminosity (L_{[O II]}) and equivalent width (W2796) and redshift of Mg II absorbers. However, we show L_{[O II]} and average luminosity surface density suffer from fibre size effects. More importantly, for a given fibre size, the average L_{[O II]} strongly depends on the equivalent width of Fe II absorption lines and found to be higher for Mg II absorbers with R ≡W2600/W2796 ≥ 0.5. In fact, we show the observed strong correlations of L_{[O II]} with W2796 and z of Mg II absorbers are mainly driven by such systems. Direct [O II] detections also confirm the link between L_{[O II]} and R. Therefore, one has to pay attention to the fibre losses and dependence of redshift evolution of Mg II absorbers on W2600 before using them as a luminosity unbiased probe of global star formation rate density. We show that the [O II] nebular emission detected in the stacked spectrum is not dominated by few direct detections (i.e. detections ≥3σ significant level). On an average, the systems with R ≥ 0.5 and W2796 ≥ 2 Å are more reddened, showing colour excess E(B - V) ˜ 0.02, with respect to the systems with R < 0.5 and most likely trace the high H I column density systems.

Discrimination of thermophilic and mesophilic proteins

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Vaisman Iosif I

2010-05-01

Full Text Available Abstract Background There is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. Understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts. Results We have systematically tested a large number of sequence and structure derived quantities for their ability to discriminate thermostable proteins from their non-thermostable orthologs using sets of mesophile-thermophile ortholog pairs. Most of the quantities tested correspond to properties previously reported to be associated with thermostability. Many of the structure related properties were derived from the Delaunay tessellation of protein structures. Conclusions Carefully selected sequence based indices discriminate better than purely structure based indices. Combined sequence and structure based indices improve performance somewhat further. Based on our analysis, the strongest contributors to thermostability are an increase in ion pairs on the protein surface and a more strongly hydrophobic interior.

Electrical properties and Raman studies of phase transitions in ferroelectric [N(CH3)4]2CoCl2Br2

Science.gov (United States)

Ben Mohamed, C.; Karoui, K.; Bulou, A.; Ben Rhaiem, A.

2018-03-01

The present paper accounted for the synthesis, electric properties and vibrational spectroscopy of [N(CH3)4]2CoCl2Br2. The dielectric spectra were measured in the frequency range 10-1-105 Hz and temperature interval from 223 to 393 K. The dielectical properties confirm the ferroelectric-paraelectric phase transition at 290 K, which is reported by Abdallah Ben Rhaiem et al. (2013). The equivalent circuit based on the Z-View-software was proposed and the conduction mechanisms were determined. The obtained results have been discussed in terms of the correlated barrier hopping model (CBH) in phase I and non-overlapping small polaron tunneling model (NSPT) in phases II and III. Raman spectra as function temperature have been used to characterize the phase transitions and their nature, which indicates a change of the some peak near the transitions phase.

Electrical properties of thermally evaporated nickel-dimethylglyoxime thin films

Science.gov (United States)

Dakhel, A. A.; Ali-Mohamed Ahmed, Y.

2005-06-01

Thin Bis-(dimethylglyoximato)nickel(II) [Ni(DMG)2] films of amorphous and crystalline structures were prepared by vacuum deposition on Si (P) substrates. The films were characterised by X-ray fluorescence and X-ray diffraction. The constructed Al/Ni(DMG)2/Si(P) metal-insulator-semiconductor devices were characterised by the measurement of the gate-voltage dependence of their capacitance and ac conductance, from which the surface states density Dit of insulator/semiconductor interface and the density of the fixed charges in the oxide were determined. The ac electrical conduction and dielectric properties of the Ni(DMG)2-Silicon structure were studied at room temperature. The data of the ac measurements of the annealed films follow the correlated barrier-hopping CBH mode, from which the fundamental absorption bandgap, the minimum hopping distance, and other parameters of the model were determined.

Structural information on the coordination compounds formed by manganese(II), cobalt(II), nickel(II), zinc(II), cadmium(II) and mercury(II) thiocyanates with 4-cyanopyridine N-oxide from their magnetic moments, electronic and infrared spectra

Science.gov (United States)

Ahuja, I. S.; Yadava, C. L.; Singh, Raghuvir

1982-05-01

Coordination compounds formed by the interaction of 4-cyanopyridine. N-oxide (4-CPO), a potentially bidentate ligand, with manganese(II), cobalt(II), nickel(II), zinc(II), cadmium(II) and rnercury(II) thiocyanates have been prepared and characterized from their elemental analyses, magnetic susceptibilities, electronic and infrared spectral studies down to 200 cm -1 in the solid state. The compounds isolated are: Mn(4-CPO) 2(NCS) 2, Co(4-CPO) 2(NCS) 2,Ni(4-CPO) 2(NCS) 2,Zn(4-CPO) 2(NCS) 2, Cd(4-CPO)(NCS) 2 and Hg(4-CPO) 2(SCN) 2. It is shown that 4-CPO acts as a terminal N-oxide oxygen bonded monodentate ligand in all the metal(II) thiocyanate complexes studied. Tentative stereochemistries of the complexes in the solid state are discussed. The ligand field parameters 10 Dq, B, β and λ calculated for the manganese(II), cobalt(II) and nickel(II) complexes are consistent with their proposed stereochemistries.

Macrocyclic receptor showing extremely high Sr(II)/Ca(II) and Pb(II)/Ca(II) selectivities with potential application in chelation treatment of metal intoxication.

Science.gov (United States)

Ferreirós-Martínez, Raquel; Esteban-Gómez, David; Tóth, Éva; de Blas, Andrés; Platas-Iglesias, Carlos; Rodríguez-Blas, Teresa

2011-04-18

Herein we report a detailed investigation of the complexation properties of the macrocyclic decadentate receptor N,N'-Bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6 (H(2)bp18c6) toward different divalent metal ions [Zn(II), Cd(II), Pb(II), Sr(II), and Ca(II)] in aqueous solution. We have found that this ligand is especially suited for the complexation of large metal ions such as Sr(II) and Pb(II), which results in very high Pb(II)/Ca(II) and Pb(II)/Zn(II) selectivities (in fact, higher than those found for ligands widely used for the treatment of lead poisoning such as ethylenediaminetetraacetic acid (edta)), as well as in the highest Sr(II)/Ca(II) selectivity reported so far. These results have been rationalized on the basis of the structure of the complexes. X-ray crystal diffraction, (1)H and (13)C NMR spectroscopy, as well as theoretical calculations at the density functional theory (B3LYP) level have been performed. Our results indicate that for large metal ions such as Pb(II) and Sr(II) the most stable conformation is Δ(δλδ)(δλδ), while for Ca(II) our calculations predict the Δ(λδλ)(λδλ) form being the most stable one. The selectivity that bp18c6(2-) shows for Sr(II) over Ca(II) can be attributed to a better fit between the large Sr(II) ions and the relatively large crown fragment of the ligand. The X-ray crystal structure of the Pb(II) complex shows that the Δ(δλδ)(δλδ) conformation observed in solution is also maintained in the solid state. The Pb(II) ion is endocyclically coordinated, being directly bound to the 10 donor atoms of the ligand. The bond distances to the donor atoms of the pendant arms (2.55-2.60 Å) are substantially shorter than those between the metal ion and the donor atoms of the crown moiety (2.92-3.04 Å). This is a typical situation observed for the so-called hemidirected compounds, in which the Pb(II) lone pair is stereochemically active. The X-ray structures of the Zn(II) and Cd(II) complexes show that

A structured understanding of cellobiohydrolase I binding to poplar lignin fractions after dilute acid pretreatment.

Science.gov (United States)

Yao, Lan; Yoo, Chang Geun; Meng, Xianzhi; Li, Mi; Pu, Yunqiao; Ragauskas, Arthur J; Yang, Haitao

2018-01-01

Cellulase adsorption to lignin is considered a cost barrier for bioethanol production; however, its detailed association mechanism is still not fully understood. In this study, two natural poplar variants with high and low sugar release performance were selected as the low and high recalcitrant raw materials (named L and H , respectively). Three different lignin fractions were extracted using ethanol, followed by p -dioxane and then cellulase treatment from the dilute acid pretreated poplar solids (fraction 1, 2, and 3, respectively). Each lignin fraction had different physicochemical properties. Ethanol-extracted lignin had the lowest weight average molecular weight, while the molecular weights for the other two lignin fractions were similar. 31 P NMR analysis revealed that lignin fraction with higher molecular weight contained more aliphatic hydroxyl groups and less phenolic hydroxyl groups. Semi-quantitative analysis by 2D HSQC NMR indicated that the lignin fractions isolated from the natural variants had different contents of syringyl (S), guaiacyl (G) and interunit linkages. Lignin extracted by ethanol contained the largest amount of S units, the smallest amounts of G and p -hydroxybenzoate (PB) subunits, while the contents of these lignin subunits in the other two lignin fractions were similar. The lignin fraction obtained after cellulase treatment was primarily comprised of β- O -4 linkages with small amounts of β-5 and β-β linkages. The binding strength of these three lignin fractions obtained by Langmuir equations were in the order of L 1  >  L 3  >  L 2 for the low recalcitrance poplar and H 1  >  H 2  >  H 3 for the high recalcitrance poplar. Overall, adsorption ability of lignin was correlated with the sugar release of poplar. Structural features of lignin were associated with its binding to CBH. For natural poplar variants, lignin fractions with lower molecular weight and polydispersity index (PDI) exhibited more CBH adsorption

Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

Science.gov (United States)

Hoang Thi Thu, Duyen; Razavi, Bahar S.

2016-04-01

Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil

Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

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Nadia Andrea Andreani

2016-12-01

Full Text Available Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

Solid Phase Extraction of Trace Al(III), Fe(II), Co(II), Cu(II), Cd(II) and Pb(II) Ions in Beverages on Functionalized Polymer Microspheres Prior to Flame Atomic Absorption Spectrometric Determinations.

Science.gov (United States)

Berber, Hale; Alpdogan, Güzin

2017-01-01

In this study, poly(glycidyl methacrylate-methyl methacrylate-divinylbenzene) was synthesized in the form of microspheres, and then functionalized by 2-aminobenzothiazole ligand. The sorption properties of these functionalized microspheres were investigated for separation, preconcentration and determination of Al(III), Fe(II), Co(II), Cu(II), Cd(II) and Pb(II) ions using flame atomic absorption spectrometry. The optimum pH values for quantitative sorption were 2 - 4, 5 - 8, 6 - 8, 4 - 6, 2 - 6 and 2 - 3 for Al(III), Fe(II), Co(II), Cu(II), Cd(II) and Pb(II), respectively, and also the highest sorption capacity of the functionalized microspheres was found to be for Cu(II) with the value of 1.87 mmol g -1 . The detection limits (3σ; N = 6) obtained for the studied metals in the optimal conditions were observed in the range of 0.26 - 2.20 μg L -1 . The proposed method was successfully applied to different beverage samples for the determination of Al(III), Fe(II), Co(II), Cu(II), Cd(II) and Pb(II) ions, with the relative standard deviation of <3.7%.

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

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Steven M. Swift

2015-06-01

Full Text Available Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

Science.gov (United States)

Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

2010-11-01

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

Synthesis, characterization and thermal studies of nickel (II), copper (II), zinc (II) and cadmium (II) complexes with some mixed ligands

International Nuclear Information System (INIS)

Mitra, Samiran; Kundu, Parimal; Singh, Rajkumar Bhubon

1998-01-01

Dichloro-(DCA) and trichloroacetate(TCA) -cyclic ligand morpholine (Morph)/thiomorpholine (Tmorph)/methylmorpholine (Mmorph)/dimethyl-piperazine (DMP) complexes of nickel (II), copper (II), zinc (II) and cadmium (II) with the compositions [Ni(tmorph) 2 (DCA) 2 ], [Ni(tmorph) 2 (TCA) 2 ].2H 2 O, [Cu(DMP) 2 (TCA) 2 ],[ML 2 X 2 ].nH 2 O where M=Zn II or Cd II , L=Morph, DMP or tmorph and X=DCA or TCA and n=O except in case of [Cd (Morph) 2 (TCA) 2 ] where n=1 have been synthesised. Some intermediate complexes have been isolated by temperature arrest technique (pyrolysis) and characterised. Configurational and conformational changes have been studied by elemental analyses, IR and electronic spectra, magnetic moment data (in the case of Ni(II) and Cu(II) complexes) and thermal analysis. E a * , ΔH, and ΔS for the decomposition reaction of these complexes are evaluated and the stability of the complexes with respect to activation energy has also been compared. The linear correlation has been found between E a * and ΔS for the decomposition of the complexes. (author)

Competition from Cu(II), Zn(II) and Cd(II) in Pb(II) binding to Suwannee River Fulvic Acid

NARCIS (Netherlands)

Chakraborty, P.; Chakrabarti, C.L.

2008-01-01

This is a study of trace metal competition in the complexation of Pb(II) by well-characterized humic substances, namely Suwannee River Fulvic Acid (SRFA) in model solutions. It was found that Cu(II) seems to compete with Pb(II) for strong binding sites of SRFA when present at the same concentration

Synthesis, investigation and spectroscopic characterization of piroxicam ternary complexes of Fe(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) with glycine and DL-phenylalanine

Science.gov (United States)

Mohamed, Gehad G.; El-Gamel, Nadia E. A.

2004-11-01

The ternary piroxicam (Pir; 4-hydroxy-2-methyl- N-(2-pyridyl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide) complexes of Fe(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) with various amino acids (AA) such as glycine (Gly) or DL-phenylalanine (PhA) were prepared and characterized by elemental analyses, molar conductance, IR, UV-Vis, magnetic moment, diffuse reflectance and X-ray powder diffraction. The UV-Vis spectra of Pir and the effect of metal chelation on the different interligand transitions are discussed in detailed manner. IR and UV-Vis spectra confirm that Pir behaves as a neutral bidentate ligand coordinated to the metal ions via the pyridine- N and carbonyl group of the amide moiety. Gly molecule acted as a uninegatively monodentate ligand and coordinate to the metal ions through its carboxylic group, in addition PhA acted as a uninegatively bidentate ligand and coordinate to the metal ions through its carboxylic and amino groups. All the chelates have octahedral geometrical structures while Cu(II)- and Zn(II)-ternary chelates with PhA have square planar geometrical structures. The molar conductance data reveal that most of these chelates are non electrolytes, while Fe(III)-Pir-Gly, Co(II)-, Ni(II)-, Cu(II)- and Zn(II)-Pir-PhA cheletes were 1:1 electrolytes. X-ray powder diffraction is used as a new tool to estimate the crystallinity of chelates as well as to elucidate their geometrical structures.

Coordination compounds of cobalt(II), nickel(II), copper(II), and zinc(II) with pantothenic acid

Energy Technology Data Exchange (ETDEWEB)

Shabilalov, A.A.; Yunuskhodzhaev, A.N.; Khodzhaev, O.F.; Azizov, M.A.

1986-11-01

The compounds Ni(PANA - H)/sub 2/ x 4H/sub 2/O (PANA stands for pantothenic acid, and - H indicates a deprotonated ligand), Cu(PANA - H)/sub 2/ x 2H/sub 2/O, Zn(PANA - H)/sub 2/ x H/sub 2/O, Co(PANA - H)Cl x H/sub 2/O, and Ni(PANA - H)Cl x 3H/sub 2/O have been synthesized on the basis of pantothenic acid and Co(II), Ni(II), Cu(II), and Zn(II) salts in aqueous media. The compounds have been identified by elemental and x-ray diffraction analysis. Some physicochemical properties (solubility, melting point, molar conductivity) of the compounds obtained have been studied. The structure of the compounds isolated has been established on the basis of an analysis of their IR, ESR, and electronic spectra, as well as derivatograms.

Spectroscopic and thermal degradation behavior of Mg(II), Ca(II), Ba(II) and Sr(II) complexes with paracetamol drug

OpenAIRE

Moamen S. Refat; Gehad G. Mohamed; Mohamed Y. El-Sayed; Hamada M.A. Killa; Hammad Fetooh

2017-01-01

Complexes of Mg(II), Ca(II), Ba(II) and Sr(II) with paracetamol drug were synthesized and characterized by elemental analysis, conductivity, UV–Vis, IR, and 1H NMR spectroscopy and thermal analysis, as well as screened for antimicrobial activity. The IR spectral data suggested that the ligand behaves as paracetamol behaves as a neutral bidentate ligand coordinated to the metal ions via the lone pair of electrons of nitrogen and carbonyl-O atoms of the amide group. From the microanalytical dat...

Optimization of simultaneous electrochemical determination of Cd(II), Pb(II), Cu(II) and Hg(II) at carbon nanotube-modified graphite electrodes.

Science.gov (United States)

Pikna, L'ubomír; Heželová, Mária; Kováčová, Zuzana

2015-01-01

The health of the environment is worsening every day. Monitoring of potentially toxic elements and remediation of environmental pollution are necessary. Therefore, the research and development of simple, inexpensive, portable and effective sensors is important. Electrochemistry is a useful component of the field of environment monitoring. The present study focuses on evaluating and comparing three types of electrodes (PIGE, PIGE/MWCNT/HNO3 and PIGE/MWCNT/EDTA/HNO3) employed for the simultaneous electrochemical determination of four potentially toxic elements: Cd(II), Pb(II), Cu(II) and Hg(II). Cyclic voltammograms were measured in an acetate buffer. The LOD, LOQ, the standard and relative precisions of the method and a prediction intervals were calculated (according to the technical procedure DIN 32 645) for the three electrodes and for each measured element. The LOD for PIGE/CNT/HNO3 (the electrode with narrowest calculated prediction intervals) was 2.98 × 10(-7) mol L(-1) for Cd(II), 4.83 × 10(-7) mol L(-1) for Pb(II), 3.81 × 10(-7) mol L(-1) for Cu(II), 6.79 × 10(-7) mol L(-1) for Hg(II). One of the benefits of this study was the determination of the amount of Hg(II) in the mixture of other elements.

Synthesis, spectral characterization thermal stability, antimicrobial studies and biodegradation of starch–thiourea based biodegradable polymeric ligand and its coordination complexes with [Mn(II), Co(II), Ni(II), Cu(II), and Zn(II)] metals

OpenAIRE

Nahid Nishat; Ashraf Malik

2016-01-01

A biodegradable polymer was synthesized by the modification reaction of polymeric starch with thiourea which is further modified by transition metals, Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). All the polymeric compounds were characterized by (FT-IR) spectroscopy, 1H NMR spectroscopy, 13C NMR spectroscopy, UV–visible spectra, magnetic moment measurements, thermogravimetric analysis (TGA) and antibacterial activities. Polymer complexes of Mn(II), Co(II) and Ni(II) show octahedral geometry, wh...

Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.

Science.gov (United States)

He, Dong; Luo, Wen; Wang, Zhiyuan; Lv, Pengmei; Yuan, Zhenhong; Huang, Shaowei; Xv, Jingliang

2017-07-01

Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T 50 60 -value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Extraction studies of Cd(II), Cu(II), Mn(II), Ni(II) and Zn(II) using N, N', N, N' -Bis((2-hydroxy-3,5-di-tert-butylbenzyl) (2-pyridylmethyl)) -ethylenediamine as a novel ligand

International Nuclear Information System (INIS)

Laus, R.; Anjos, A.D.; Naves, A.

2008-01-01

In the present study, the use of N,N',N,N'-bis((2-hydroxy-3,5-di-tert-butylbenzyl) (2- pyridylmethyl))-ethylenediamine (H2L) as ligand was evaluated in the liquid-liquid (water- chloroform) extraction of Cd(II), Cu(II), Mn(II), Ni(II) and Zn(II). Experiments were carried out to determine the pH for maximum extraction for each metal ion by ligand, maximum extraction capacity, extraction kinetics and extraction selectivity. The results revealed that the extraction of metal ions is dependent on the pH: maximum extraction maximum was obtained in the pH range of 4.5 - 6.0 for Cu(II) and 8.0 - 9.0 for Zn(II). Cd(II) and Mn(II) were best extracted at pH 9.0 and Ni(II) at 10.0. The ligand H2L was effective for the extraction of Cd(II), Cu(II) and Zn(II) (extraction efficient, %E equal 100%), whereas %E of 76% and 23.5% were observed for Mn(II) and Ni(II), respectively. The ligand presented high selectivity for the extraction of Cu(II) at pH 4.0. (author)

Preparation and Spectral Properties of Mixed-Ligand Complexes of VO(IV, Ni(II, Zn(II, Pd(II, Cd(II and Pb(II with Dimethylglyoxime and N-acetylglycine

Directory of Open Access Journals (Sweden)

Shayma A. Shaker

2010-01-01

Full Text Available A number of mixed-ligand complexes of the general formula [M(D(G] where D=dimethylglyoximato monoanion, G=N-acetylglycinato and M=VO(IV, Ni(II, Zn(II, Pd(II, Cd(II and Pb(II were prepared. Each complex was characterized by elemental analysis, determination of metal, infrared spectra, electronic spectra, (1H and 13C NMR spectra, conductivity and magnetic moments. All these complexes were not soluble in some of the organic solvent but highly soluble in dimethylformamide. The conductivity data showed the non-electrolytic nature of the complexes. The electronic spectra exhibited absorption bands in the visible region caused by the d-d electronic transition such as VO(IV, Ni(II and Pd(II. The IR and (1H, 13C NMR spectra which have indicate that the dimethylglyoxime was coordinated with the metal ions through the N and O atoms of the oxime group and N-acetylglycine was coordinated with metal ions through the N atom and terminal carboxyl oxygen atom.

Synthesis and characterization of polychelates of Cu(II), Ni(II), Co(II), Mn(II), Zn(II), oxovanadium(IV) and dioxouranium(VI) with 2,4-dihydroxybenzaldehyde-urea-formaldehyde polymer

International Nuclear Information System (INIS)

Patel, G.C.; Pancholi, H.B.; Patel, M.M.

1991-01-01

Polychelates of Cu(II), Ni(II), Co(II), Mn(II), Zn(II), oxovandium(IV) and dioxouranium(VI) with 2,4-dihydroxybenzaldehyde (2,4-DB)-urea(U)-formaldehyde(F) polymer (2,4-DBUF) have been prepared. Elemental analyses of the polychelates indicate a metal:ligand ratio of 1:2. The structures of the polychelates have been assigned on the basis of their elemental analyses, IR, reflectance spectra, magnetic moment, thermal data and their electrical conductivity behaviour. (author). 1 tab., 18 refs

Ni (II) and Cu(II) complexes of

African Journals Online (AJOL)

ADOWIE PERE

ABSTRACT: The objective of this study is to investigate the antimicrobial activity of novel. Schiff base metal complexes. The resistance of micro-organisms to classical antimicrobial compounds poses a challenge to effective management and treatment of some diseases. In line with this, copper (II), nickel (II) and cobalt (II) ...

Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges.

Science.gov (United States)

Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

2015-01-01

Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, psalt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (psalt-bridges and smaller volume nonpolar residues (Gly, Ala and Val) and lesser occurrence of bulky polar residues in the thermophilic proteins. A more stoichiometric relationship amongst these factors minimized the hindrance due to side chain burial and increased compactness and secondary structural stability in thermophilic proteins.

Evaluating the effects of buffer conditions and extremolytes on thermostability of granulocyte colony-stimulating factor using high-throughput screening combined with design of experiments.

Science.gov (United States)

Ablinger, Elisabeth; Hellweger, Monika; Leitgeb, Stefan; Zimmer, Andreas

2012-10-15

In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis. pH was identified as most critical factor regarding thermal stability. The most stabilizing buffer, sodium glutamate, and sodium acetate were determined for further investigations. Second we tested the effect of 6 naturally occurring extremolytes (trehalose, sucrose, ectoine, hydroxyectoine, sorbitol, mannitol) on the thermal stability of G-CSF, using a central composite circumscribed design. At low pH (3.8) and low buffer concentration (5 mM) all extremolytes led to a significant increase in thermal stability except the addition of ectoine which resulted in a strong destabilization of G-CSF. Increasing pH and buffer concentration led to an increase in thermal stability with all investigated extremolytes. The described systematic approach allowed to create a ranking of stabilizing extremolytes at different buffer conditions. Copyright © 2012. Published by Elsevier B.V.

Synthesis and characterization of chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), cadmium(II) and dioxouranium(VI) complexes of 4(2-pyridyl)-1-(2,4-dihydroxybenzaldehyde)-3-thiosemicarbazone

International Nuclear Information System (INIS)

Abu El-Reash, G.M.; Ibrahim, M.M.; Kenawy; El-Ayaan, Usama; Khattab, M.A.

1994-01-01

A few complexes of Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and dioxouranium(VI) with 4(2-pyridyl)-1-(2,4-dihydroxybenzaldehyde)-3-thiosemicarbazone have been synthesised and characterized on the basis of elemental analysis, IR, electronic NMR, and magnetic moment data. An octahedral structure is proposed for the Cr(III), Fe(III), Co(II) and Ni(H 3 PBT) 2 Cl 2 .2H 2 O complexes; a tetrahedral structure for the Mn(II) and Ni 2 (PBT)OAc.H 2 0 complexes and a square planar structure for the Cu(II) complexes. The antimicrobial and antifungal activities of H 3 PBT and of its metal(II) complexes are investigated. The results reveal that H 3 PBT exhibits greater antimicrobial activities than its complexes. (author). 34 refs., 4 figs., 2 tabs

Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass

Science.gov (United States)

Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH)...

Copper (II)

African Journals Online (AJOL)

CLEMENT O BEWAJI

Valine (2 - amino - 3 – methylbutanoic acid), is a chemical compound containing .... Stability constant (Kf). Gibb's free energy. ) (. 1. −. ∆. Mol. JG. [CuL2(H2O)2] ... synthesis and characterization of Co(ii), Ni(ii), Cu (II), and Zn(ii) complexes with ...

Synthesis and Spectral Investigations of Manganese(II, Cobalt(II, Nickel(II, Copper(II and Zinc(II Complexes of New Polydentate Ligands Containing a 1,8-Naphthyridine Moiety

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Sunkari Jyothi

2006-12-01

Full Text Available 2-(o-Hydroxyphenyl-1,8-naphthyridine (HN, 2-(4-hydroxy-6-methylpyran-2-one-3-yl-1,8-naphthyridine (HMPN and 2-(benzimidazol-2-yl-1,8-naphthyridine(BN react with acetates of Mn(II, Co(II, Ni(II, Cu(II and Zn(II to yield metal ioncomplexes of definite composition. These compounds were characterized by elementalanalyses, molar conductivity, magnetic susceptibility measurements, thermal studies, IR,UV-visible, NMR and mass spectral investigations. The complexes are found to have theformulae [M(HN2(H2O2], [M(HMPN2(H2O2] and [M(BN2(OAc2], respectively.

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.

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

Science.gov (United States)

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

2008-07-01

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

Coordination behavior of tetraaza [N4] ligand towards Co(II), Ni(II), Cu(II), Cu(I) and Pd(II) complexes: Synthesis, spectroscopic characterization and anticancer activity

Science.gov (United States)

El-Boraey, Hanaa A.

2012-11-01

Novel eight Co(II), Ni(II), Cu(II), Cu(I) and Pd(II) complexes with [N4] ligand (L) i.e. 2-amino-N-{2-[(2-aminobenzoyl)amino]ethyl}benzamide have been synthesized and structurally characterized by elemental analysis, spectral, thermal (TG/DTG), magnetic, and molar conductivity measurements. On the basis of IR, mass, electronic and EPR spectral studies an octahedral geometry has been proposed for Co(II), Ni(II) complexes and Cu(II) chloride complex, square-pyramidal for Cu(I) bromide complex. For Cu(II) nitrate complex (6), Pd(II) complex (8) square planar geometry was proposed. The EPR data of Cu(II) complexes in powdered form indicate dx2-y2 ground state of Cu(II) ion. The antitumor activity of the synthesized ligand and some selected metal complexes has been studied. The palladium(II) complex (8) was found to display cytotoxicity (IC50 = 25.6 and 41 μM) against human breast cancer cell line MCF-7 and human hepatocarcinoma HEPG2 cell line.

Using chiral liquid chromatography quadrupole time-of-flight mass spectrometry for the analysis of pharmaceuticals and illicit drugs in surface and wastewater at the enantiomeric level.

Science.gov (United States)

Bagnall, J P; Evans, S E; Wort, M T; Lubben, A T; Kasprzyk-Hordern, B

2012-08-03

This paper presents and compares for the first time two chiral LC-QTOF-MS methodologies (utilising CBH and Chirobiotic V columns with cellobiohydrolase and vancomycin as chiral selectors) for the quantification of amphetamine, methamphetamine, MDA (methylenedioxyamphetamine), MDMA (methylenedioxymethamphetamine), propranolol, atenolol, metoprolol, fluoxetine and venlafaxine in river water and sewage effluent. The lowest MDLs (0.3-5.0 ng L(-1) and 1.3-15.1 ng L(-1) for river water and sewage effluent respectively) were observed using the chiral column Chirobiotic V. This is with the exception of methamphetamine and MDMA which had lower MDLs using the CBH column. However, the CBH column resulted in better resolution of enantiomers (R(s)=2.5 for amphetamine compared with R(s)=1.2 with Chirobiotic V). Method recovery rates were typically >80% for both methodologies. Pharmaceuticals and illicit drugs detected and quantified in environmental samples were successfully identified using MS/MS confirmation. In sewage effluent, the total beta-blocker concentrations of propranolol, atenolol and metoprolol were on average 77.0, 1091.0 and 3.6 ng L(-1) thus having EFs (Enantiomeric Fractions) of 0.43, 0.55 and 0.54 respectively. In river water, total propranolol and atenolol was quantified on average at <10.0 ng L(-1). Differences in EF between sewage and river water matrices were evident: venlafaxine was observed with respective EF of 0.43 ± 0.02 and 0.58 ± 0.02. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea.

Science.gov (United States)

Sayed, Ahmed; Ghazy, Mohamed A; Ferreira, Ari J S; Setubal, João C; Chambergo, Felipe S; Ouf, Amged; Adel, Mustafa; Dawe, Adam S; Archer, John A C; Bajic, Vladimir B; Siam, Rania; El-Dorry, Hamza

2014-01-17

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Study of electrical properties of glassy Se100–xTex alloys

Indian Academy of Sciences (India)

Unknown

can be successfully explained by correlated barrier hopping (CBH) model. ... The results show that bipolaron hopping dominates over single-polaron hopping in this glassy system. This .... where ∆E is the activation energy and σ0 is called the.

Removal of nickel(II and palladium(II from surface waters

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V. Sharifzade

2013-04-01

Full Text Available A new sorbent was prepared using alumina and 5-Br-PADAP, and its adsorption ability for the removal of Ni(II and Pd(II from different waters was investigated. The procedure is based on retention of the analytes on the alumina load with 5-Br-PADAP at pH ~ 6. The separation/preconcentration conditions for the quantitative recoveries were investigated. The limit of detections (LOD based on three times the standard deviations of the blank, were 0.187 and 0.253 ng mL-1 for Ni(II and Pd(II, respectively. Obtained sorption capacities for 1 g sorbent were 6.0 mg Ni(II and 11.0 mg Pd(II. The linearity was maintained in the concentration range of 0.625 to 6.0 ng mL-1 for Ni(II and 0.416 to 7.0 ng mL-1 for Pd(II in the original solution. Eight replicate determinations of a mixture containing 2.0 µg mL-1 each of the elements in the final solution gave relative standard deviation of ±0.82 and ±1.12% for Ni(II and Pd(II, respectively. The proposed method was successfully applied to the determination trace amounts of Ni(II and Pd(II in the surface water samples.DOI: http://dx.doi.org/10.4314/bcse.v27i1.2

Evaluation of the computerized procedures Manual II (COPMA II)

International Nuclear Information System (INIS)

Converse, S.A.

1995-11-01

The purpose of this study was to evaluate the effects of a computerized procedure system, the Computerized Procedure Manual II (COPMA-II), on the performance and mental workload of licensed reactor operators. To evaluate COPMA-II, eight teams of two operators were trained to operate a scaled pressurized water reactor facility (SPWRF) with traditional paper procedures and with COPMA-II. Following training, each team operated the SPWRF under normal operating conditions with both paper procedures and COPMA-II. The teams then performed one of two accident scenarios with paper procedures, but performed the remaining accident scenario with COPMA-II. Performance measures and subjective estimates of mental workload were recorded for each performance trial. The most important finding of the study was that the operators committed only half as many errors during the accident scenarios with COPMA-II as they committed with paper procedures. However, time to initiate a procedure was fastest for paper procedures for accident scenario trials. For performance under normal operating conditions, there was no difference in time to initiate or to complete a procedure, or in the number of errors committed with paper procedures and with COPMA-II. There were no consistent differences in the mental workload ratings operators recorded for trials with paper procedures and COPMA-II

Efficiency of Chitosan for the Removal of Pb (II, Fe (II and Cu (II Ions from Aqueous Solutions

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Soheil Sobhanardakani

2014-09-01

Full Text Available Background: Heavy metals have been recognized as harmful environmental pollutant known to produce highly toxic effects on different organs and systems of both humans and animals. The aim of this paper is to evaluate the adsorption potential of chitosan for the removal of Pb(II, Fe(II and Cu(II ions from aqueous solutions. Methods: This study was conducted in laboratory scale. In this paper chitosan has been used as an adsorbent for the removal of Pb(II, Fe(II and Cu(II from aqueous solution. In batch tests, the effects of parameters like pH solution (1.0-8.0, initial metal concentrations (100-1000 mgL-1, contact time (5.0-150 min and adsorbent dose (1.0-7.0 g on the adsorption process were studied. Results: The results showed that the adsorption of Pb(II, Fe(II and Cu(II ions on chitosan strongly depends on pH. The experimental isothermal data were analyzed using the Langmuir and Freundlich equations and it was found that the removal process followed the Langmuir isotherm and maximum adsorption capacity for the adsorption of Pb(II, Fe(II and Cu(II ions by the chitosan were 55.5mg g−1, 71.4 mg g−1 and 59 mg g−1, respectively, under equilibrium conditions at 25±1 ºC. The adsorption process was found to be well described by the pseudo-second-order rate model. Conclusion: The obtained results showed that chitosan is a readily, available, economic adsorbent and was found suitable for removing Pb(II, Fe(II and Cu(II ions from aqueous solution.

Spectrophotometric determination of cobalt(II), nickel(II) and copper (II) with 1-(2 pyridylazo)-2-naphthol in micellar medium

International Nuclear Information System (INIS)

Shar, G.A.; Soomro, G.A.

2004-01-01

Spectrophotometric determination of cobalt(II), nickel(II) and copper(II) is carried out with 1-(2 pyridylazo)-2-naphthol as a complexing reagent in aqueous phase using non-ionic surfactant Tween 80. Beer's law is obeyed for Co(II), Ni(II) and Cu(II) over the range 0.5 - 4.0, 0.5 - 4.0 and 0.5 - 3.0 ngmL/sup -1/ with detection limit (2 σ) of 6.7, 3.2 and 3.9 ngmL/sup -1/. The max molar absorption, molar absorptivity, Sandell's sensitivity of Co(II), Ni(II) and Cu(II) are 580 nm, 570 nm and 555 nm; max (104 mol/sup -1/ cm /sup -1/) is 0.87, 1.8 and 1.6 and 6.8, 3.3 and 3.9 ng cm-2 respectively. The pH at which complex is formed for Co(II), Ni(II) and Cu(II) is 5, 5.5 and 6.5 respectively. The critical micelle concentration (CMC) of Tween 80 is 5%. The present method is compared with that of atomic absorption spectroscopy and no significant difference is noted between the two methods at 95% confidence level. The method has been applied to the determination of Co(II), Ni(II) and Cu(II) in industrial waste water and pharmaceutical samples. (author)

Synthesis, spectral characterization thermal stability, antimicrobial studies and biodegradation of starch–thiourea based biodegradable polymeric ligand and its coordination complexes with [Mn(II, Co(II, Ni(II, Cu(II, and Zn(II] metals

Directory of Open Access Journals (Sweden)

Nahid Nishat

2016-09-01

Full Text Available A biodegradable polymer was synthesized by the modification reaction of polymeric starch with thiourea which is further modified by transition metals, Mn(II, Co(II, Ni(II, Cu(II and Zn(II. All the polymeric compounds were characterized by (FT-IR spectroscopy, 1H NMR spectroscopy, 13C NMR spectroscopy, UV–visible spectra, magnetic moment measurements, thermogravimetric analysis (TGA and antibacterial activities. Polymer complexes of Mn(II, Co(II and Ni(II show octahedral geometry, while polymer complexes of Cu(II and Zn(II show square planar and tetrahedral geometry, respectively. The TGA revealed that all the polymer metal complexes are more thermally stable than their parental ligand. In addition, biodegradable studies of all the polymeric compounds were also carried out through ASTM-D-5338-93 standards of biodegradable polymers by CO2 evolution method which says that coordination decreases biodegradability. The antibacterial activity was screened with the agar well diffusion method against some selected microorganisms. Among all the complexes, the antibacterial activity of the Cu(II polymer–metal complex showed the highest zone of inhibition because of its higher stability constant.

Removal of Ni (II), Co (II) and Pb (II) ions from aqueous media using ...

African Journals Online (AJOL)

Removal of Ni (II), Co (II) and Pb (II) ions from aqueous media using Starch ... The results showed that 0.025 % loaded SSMNPs gave the optimal sorption ... constants (Lagergren and Pseudo-2nd-order) for Ni2+ and Co2+ adsorption were ... Langmuir correlation coefficients showed a better fit for the adsorption isotherms.

Selective Hg(II) adsorption from aqueous solutions of Hg(II) and Pb(II) by hydrolyzed acrylamide-grafted PET films.

Science.gov (United States)

Rahman, Nazia; Sato, Nobuhiro; Sugiyama, Masaaki; Hidaka, Yoshiki; Okabe, Hirotaka; Hara, Kazuhiro

2014-01-01

Selective Hg(II) adsorption from aqueous solutions of Hg(II) and Pb(II) using hydrolyzed acrylamide (AAm)-grafted polyethylene terephthalate (PET) films was examined to explore the potential reuse of waste PET materials. Selective recovery of Hg(II) from a mixture of soft acids with similar structure, such as Hg(II) and Pb(II), is important to allow the reuse of recovered Hg(II). An adsorbent for selective Hg(II) adsorption was prepared by γ-ray-induced grafting of AAm onto PET films followed by partial hydrolysis through KOH treatment. The adsorption capacity of the AAm-grafted PET films for Hg(II) ions increased from 15 to 70 mg/g after partial hydrolysis because of the reduction of hydrogen bonding between -CONH2 groups and the corresponding improved access of metal ions to the amide groups. The prepared adsorbent was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The absorbent film showed high selectivity for the adsorption of Hg(II) over Pb(II) throughout the entire initial metal concentration range (100-500 mg/L) and pH range (2.2-5.6) studied. The high selectivity is attributed to the ability of Hg(II) ions to form covalent bonds with the amide groups. The calculated selectivity coefficient for the adsorbent binding Hg(II) over Pb(II) was 19.2 at pH 4.5 with an initial metal concentration of 100 mg/L. Selective Hg(II) adsorption equilibrium data followed the Langmuir model and kinetic data were well fitted by a pseudo-second-order equation. The adsorbed Hg(II) and Pb(II) ions were effectively desorbed from the adsorbent film by acid treatment, and the regenerated film showed no marked loss of adsorption capacity upon reuse for selective Hg(II) adsorption.

New Mn(II, Ni(II, Cd(II, Pb(II complexes with 2-methylbenzimidazole and other ligands. Synthesis, spectroscopic characterization, crystal structure, magnetic susceptibility and biological activity studies

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Shayma A. Shaker

2016-11-01

Full Text Available Synthesis and characterization of Mn(II, Ni(II, Cd(II and Pb(II mixed ligand complexes of 2-methylbenzimidazole with other ligands have been reported. The structure of the ligands and their complexes was investigated using elemental analysis, IR, UV–Vis, (1H, 13C NMR spectroscopy, molar conductivity and magnetic susceptibility measurements. In all the studies of complexes, the 2-methylbenzimidazole behaves as a neutral monodentate ligand which is coordinated with the metal ions through the N atom. While benzotriazole behaves as a neutral bidentate ligand which is coordinated with the Ni(II ion through the two N atoms. Moreover, the N-acetylglycine behaves as a bidentate ligand which is coordinated with the Mn(II, Ni(II and Pb(II ions through the N atom and the terminal carboxyl oxygen atom. The magnetic and spectral data indicate the tetrahedral geometry for Mn(II complex, irregular tetrahedral geometry for Pb(II complex and octahedral geometry for Ni(II complex. The X-ray single crystal diffraction method was used to confirm a centrosymmetric dinuclear Cd(II complex as each two metal ions are linked by a pair of thiocyanate N = S bridge. Two 2-methylbenzimidazole N-atom donors and one terminal thiocyanate N atom complete a highly distorted square pyramid geometry around the Cd atom. Besides, different cell types were used to determine the inhibitory effect of Mn(II, Ni(II, Cd(II and Pb(II complexes on cell growth using MTT assay. Cd(II complex showed cytotoxic effect on various types of cancer cell lines with different EC50 values.

Chelation of Cu(II, Zn(II, and Fe(II by Tannin Constituents of Selected Edible Nuts

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Magdalena Karamać

2009-12-01

Full Text Available The tannin fractions isolated from hazelnuts, walnuts and almonds were characterised by colorimetric assays and by an SE-HPLC technique. The complexation of Cu(II and Zn(II was determined by the reaction with tetramethylmurexide, whereas for Fe(II, ferrozine was employed. The walnut tannins exhibited a significantly weaker reaction with the vanillin/HCl reagent than hazelnut and almond tannins, but the protein precipitation capacity of the walnut fraction was high. The SE-HPLC chromatogram of the tannin fraction from hazelnuts revealed the presence of oligomers with higher molecular weights compared to that of almonds. Copper ions were most effectively chelated by the constituents of the tannin fractions of hazelnuts, walnuts and almonds. At a 0.2 mg/assay addition level, the walnut tannins complexed almost 100% Cu(II. The Fe(II complexation capacities of the tannin fractions of walnuts and hazelnuts were weaker in comparison to that of the almond tannin fraction, which at a 2.5 mg/assay addition level, bound Fe(II by ~90%. The capacity to chelate Zn(II was quite varied for the different nut tannin fractions: almond tannins bound as much as 84% Zn(II, whereas the value for walnut tannins was only 8.7%; and for hazelnut tannins, no Zn(II chelation took place at the levels tested.

Stability constants of glutarate complexes of copper(II), zinc(II), cobalt(II) and uranyl(II) by paper electrophoresis

International Nuclear Information System (INIS)

Singh, R.K.P.; Yadava, J.R.; Yadava, K.L.

1981-01-01

Stability constants of Copper(II), Zinc(II), Cobalt(II) and Uranyl(II) glutarates have been determined by paper electrophoresis. Glutaric acid (0.005 mol dmsup(-3)) was added to the background electrolyte : 0.1 mol dmsup(-3) HClO 4 . The proportions of (CH 2 ) 3 COOH COO - and (CH 2 ) 3 C 2 O 4 2- were varied by changing the pH of the electrolyte. These anions yielded the complexes Cu(CH 2 ) 3 C 2 O 4 , [Zn(CH 2 ) 3 COOH COO] + [Co(CH 2 ) 3 COOH COO] + and UO 2 (CH 2 ) 3 C 2 O 4 whose stability constants are found to be 10sup(3.9), 10sup(2.9), 10sup(2.7) and 10sup(13.5) respectively. (author)

Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments.

Science.gov (United States)

Hesp, J R; Poolman, T M; Budge, C; Batten, L; Alexander, F; McLuckie, G; O'Brien, S; Wells, P; Raven, N D H; Sutton, J M

2010-02-01

Adenylate kinase (tAK), a thermostable enzyme, was assessed as a possible means of providing a quantitative measure of cleaning efficacy suitable for validating the performance of an automated washer disinfector (AWD) during routine use. Two indicator formulations were developed using either a commercially available washer disinfector soil or a protein-based soil. Each indicator consisted of 100 microg (in test soil) of tAK dried on to a steel or plastic surface. These indicators were placed in each basket of a washer disinfector and processed alongside soiled surgical instruments during a standard day's operation. After processing, remaining tAK activity was detected using a rapid enzyme assay (2 min detection time) in a handheld hygiene monitor. The amount of tAK remaining on each indictor after a full AWD cycle was found to range from 0.1 to 0.4 ng, which represented a mean log(10) removal of 5.8+/-0.3. There was no statistical difference in the residual tAK activity between individual runs or the position of the indicator in the machine. The tAK indicator was also used to analyse the protein removal within each component of the wash cycle. These results demonstrated that all phases of the wash process contributed to the removal of the protein load, with the main wash alone being responsible for 3.6-4.0 log(10) reductions in protein activity. We propose that a quantitative cleaning index using such rapid readout indicator devices would provide a valuable addition to the methodologies for validating cleaning processes.

Pb(II), Cd(II) and Zn(II) adsorption on low grade manganese ore ...

African Journals Online (AJOL)

Low grade manganese ore (LMO) of Orissa containing 58.37% SiO2, 25.05% MnO2, 8.8% Al2O3, and 5.03% Fe2O3 as the main constituents was taken to study its adsorption behaviour for Pb(II), Cd(II) and Zn(II) from aqueous solutions. The XRD studies showed the crystalline phases to be quartz, ß-MnO2, d-MnO2 and ...

Crystal Structure of Rat Carnitine Palmitoyltransferase II (CPT-II)

Energy Technology Data Exchange (ETDEWEB)

Hsiao,Y.; Jogl, G.; Esser, V.; Tong, L.

2006-01-01

Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the {beta}-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9 Angstroms resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.

Multi-metals column adsorption of lead(II), cadmium(II) and manganese(II) onto natural bentonite clay.

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Alexander, Jock Asanja; Surajudeen, Abdulsalam; Aliyu, El-Nafaty Usman; Omeiza, Aroke Umar; Zaini, Muhammad Abbas Ahmad

2017-10-01

The present work was aimed at evaluating the multi-metals column adsorption of lead(II), cadmium(II) and manganese(II) ions onto natural bentonite. The bentonite clay adsorbent was characterized for physical and chemical properties using X-ray diffraction, X-ray fluorescence, Brunauer-Emmett-Teller surface area and cation exchange capacity. The column performance was evaluated using adsorbent bed height of 5.0 cm, with varying influent concentrations (10 mg/L and 50 mg/L) and flow rates (1.4 mL/min and 2.4 mL/min). The result shows that the breakthrough time for all metal ions ranged from 50 to 480 minutes. The maximum adsorption capacity was obtained at initial concentration of 10 mg/L and flow rate of 1.4 mL/min, with 2.22 mg/g of lead(II), 1.71 mg/g of cadmium(II) and 0.37 mg/g of manganese(II). The order of metal ions removal by natural bentonite is lead(II) > cadmium(II) > manganese(II). The sorption performance and the dynamic behaviour of the column were predicted using Adams-Bohart, Thomas, and Yoon-Nelson models. The linear regression analysis demonstrated that the Thomas and Yoon-Nelson models fitted well with the column adsorption data for all metal ions. The natural bentonite was effective for the treatment of wastewater laden with multi-metals, and the process parameters obtained from this work can be used at the industrial scale.

Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

Science.gov (United States)

Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2015-04-08

Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

Antibacterial Co(II, Ni(II, Cu(II and Zn(II complexes with biacetyl-derived Schiff bases

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MUHAMMAD IMRAN

2010-08-01

Full Text Available The condensation reactions of biacetyl with ortho-hydroxyaniline and 2-aminobenzoic acid to form bidendate NO donor Schiff bases were studied. The prepared Schiff base ligands were further utilized for the formation of metal chelates having the general formula [ML2(H2O2] where M = Co(II, Ni(II, Cu(II and Zn(II and L = HL1 and HL2. These new compounds were characterized by conductance measurements, magnetic susceptibility measurements, elemental analysis, and IR, 1H-NMR, 13C-NMR and electronic spectroscopy. Both Schiff base ligands were found to have a mono-anionic bidentate nature and octahedral geometry was assigned to all metal complexes. All the complexes contained coordinated water which was lost at 141–160 °C. These compounds were also screened for their in vitro antibacterial activity against four bacterial species, namely: Escherichia coli, Staphylococcus aureus, Salmonella typhi and Bacillus subtilis. The metal complexes were found to have greater antibacterial activity than the uncomplexed Schiff base ligands.

SEPARATION OF Fe (III, Cr(III, Cu(II, Ni(II, Co(II, AND Pb(II METAL IONS USING POLY(EUGENYL OXYACETIC ACID AS AN ION CARRIER BY A LIQUID MEMBRANE TRANSPORT METHOD

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La Harimu

2010-06-01

Full Text Available Fe (III, Cr(III, Cu(II, Ni(II, Co(II, and Pb(II  metal ions had been separated using poly(eugenyl oxyacetic acid as an ion carrier by bulk liquid membrane transport method. The effect of pH, polyeugenyl oxyacetic acid ion carrier concentration, nitric acid concentration in the stripping solution, transport time, and metal concentration were optimized. The result showed that the optimum condition for transport of metal ions was at pH 4 for ion Fe(III and at pH 5 for Cr(III, Cu(II, Ni(II, Co(II, and Pb(II ions. The carrier volumes were optimum with concentration of 1 x 10-3 M at 7.5 mL for Cr(III, Cu (II,  Ni(II, Co(II ions and at 8.5 mL for Fe(III and Pb(II ions. The concentration of HNO3 in stripping phase was optimum at 2 M for Fe(III and Cu(II ions, 1 M for Cr(III, Ni(II and Co(II ions, and 0.5 M for Pb(II ion. The optimum transport times were 36 h for Fe(III and Co(II ions, and 48 h for Cr(III, Cu (II, Ni(II, and Pb(II ions. The concentration of metal ions accurately transported were 2.5 x 10-4 M for Fe(III and Cr(III ions, and 1 M for Cu (II, Ni(II, Co(II, and Pb(II ions. Compared to other metal ions the transport of Fe(III was the highest with selectivity order of Fe(III > Cr(III > Pb(II > Cu(II > Ni(II > Co(II. At optimum condition, Fe(III ion was transported through the membrane at 46.46%.   Keywords: poly(eugenyl oxyacetic acid, transport, liquid membrane, Fe (III, Cr(III, Cu(II, Ni(II, Co(II, and Pb(II ions

Synthesis and Characterization of Multimetallic Fe(II) and Mn(II ...

African Journals Online (AJOL)

Iron(II) and Manganese(II) complexes of the resulting ligand were obtained from its reactions with Fe(II) and Mn(II) salts in absolute methanol for the metal to ligand ratio 2:3. These complexes were characterized by Solubility, Conductivity, IR and UV-VIS spectrometry, elemental analysis and mass spectrometry. Keywords: ...

(II) complexes

African Journals Online (AJOL)

activities of Schiff base tin (II) complexes. Neelofar1 ... Conclusion: All synthesized Schiff bases and their Tin (II) complexes showed high antimicrobial and ...... Singh HL. Synthesis and characterization of tin (II) complexes of fluorinated Schiff bases derived from amino acids. Spectrochim Acta Part A: Molec Biomolec.

Fe (III), Co(II), Ni(II), Cu(II) and Zn(II) complexes of schiff bases based-on glycine and phenylalanine: Synthesis, magnetic/thermal properties and antimicrobial activity

Science.gov (United States)

Sevgi, Fatih; Bagkesici, Ugur; Kursunlu, Ahmed Nuri; Guler, Ersin

2018-02-01

Zinc (II), copper (II), nickel (II), cobalt (II) and iron (III) complexes of Schiff bases (LG, LP) derived from 2-hydroxynaphthaldehyde with glycine and phenylalanine were reported and characterized by 1H NMR, 13C NMR, elemental analyses, melting point, FT-IR, magnetic susceptibility and thermal analyses (TGA). TGA data show that iron and cobalt include to the coordinated water and metal:ligand ratio is 1:2 while the complex stoichiometry for Ni (II), Cu (II) and Zn (II) complexes is 1:1. As expected, Ni (II) and Zn (II) complexes are diamagnetic; Cu (II), Co (II) and Fe (III) complexes are paramagnetic character due to a strong ligand of LG and LP. The LG, LP and their metal complexes were screened for their antimicrobial activities against five Gram-positive (Staphylococcus aureus, Methicillin resistant Staphylococcus aureus (MRSA), Bacillus cereus, Streptococcus mutans and Enterococcus faecalis) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and one fungi (Candida albicans) by using broth microdilution techniques. The activity data show that ligands and their metal complexes exhibited moderate to good activity against Gram-positive bacteria and fungi.

DNA damage by the cobalt (II) and zinc (II) complexes of ...

African Journals Online (AJOL)

Using the single cell gel electrophoresis method, the tetraazamacrocycle Zn(II) complex (Zn(II)-L) and the tetraazamacrocycle Co(II) complex (Co(II)-L) were investigated focusing on their DNA damage to Tetrahymena thermophila. When the cells were treated with the 0.05, 0.25 and 0.50 mg/ml Zn(II)-L, the tail length ...

Syntheses and spectroscopic properties of mercury(II) and nickel(II ...

African Journals Online (AJOL)

Mercury(II) complex, [Hg2(BPTU-2H)Cl2] and nickel(II) complex, [Ni(BPTU-H)2] were prepared by reacting Bis(N-phenylthiourea), BPTU, with mercury(II) chloride and nickel(II) acetate respectively. The complexes were characterized by IR, diffuse reflectance, 1H NMR spectra and elemental analysis. BPTU acts as ...

Solar Type II Radio Bursts and IP Type II Events

Science.gov (United States)

Cane, H. V.; Erickson, W. C.

2005-01-01

We have examined radio data from the WAVES experiment on the Wind spacecraft in conjunction with ground-based data in order to investigate the relationship between the shocks responsible for metric type II radio bursts and the shocks in front of coronal mass ejections (CMEs). The bow shocks of fast, large CMEs are strong interplanetary (IP) shocks, and the associated radio emissions often consist of single broad bands starting below approx. 4 MHz; such emissions were previously called IP type II events. In contrast, metric type II bursts are usually narrowbanded and display two harmonically related bands. In addition to displaying complete dynamic spectra for a number of events, we also analyze the 135 WAVES 1 - 14 MHz slow-drift time periods in 2001-2003. We find that most of the periods contain multiple phenomena, which we divide into three groups: metric type II extensions, IP type II events, and blobs and bands. About half of the WAVES listings include probable extensions of metric type II radio bursts, but in more than half of these events, there were also other slow-drift features. In the 3 yr study period, there were 31 IP type II events; these were associated with the very fastest CMEs. The most common form of activity in the WAVES events, blobs and bands in the frequency range between 1 and 8 MHz, fall below an envelope consistent with the early signatures of an IP type II event. However, most of this activity lasts only a few tens of minutes, whereas IP type II events last for many hours. In this study we find many examples in the radio data of two shock-like phenomena with different characteristics that occur simultaneously in the metric and decametric/hectometric bands, and no clear example of a metric type II burst that extends continuously down in frequency to become an IP type II event. The simplest interpretation is that metric type II bursts, unlike IP type II events, are not caused by shocks driven in front of CMEs.

Synthesis and characterization of iron(III), manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) complexes of salicylidene-N-anilinoacetohydrazone (H2L1) and 2-hydroxy-1-naphthylidene-N-anilinoacetohydrazone (H2L2).

Science.gov (United States)

AbouEl-Enein, S A; El-Saied, F A; Kasher, T I; El-Wardany, A H

2007-07-01

Salicylidene-N-anilinoacetohydrazone (H(2)L(1)) and 2-hydroxy-1-naphthylidene-N-anilinoacetohydrazone (H(2)L(2)) and their iron(III), manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) complexes have been synthesized and characterized by IR, electronic spectra, molar conductivities, magnetic susceptibilities and ESR. Mononuclear complexes are formed with molar ratios of 1:1, 1:2 and 1:3 (M:L). The IR studies reveal various modes of chelation. The electronic absorption spectra and magnetic susceptibility measurements show that the iron(III), nickel(II) and cobalt(II) complexes of H(2)L(1) have octahedral geometry. While the cobalt(II) complexes of H(2)L(2) were separated as tetrahedral structure. The copper(II) complexes have square planar stereochemistry. The ESR parameters of the copper(II) complexes at room temperature were calculated. The g values for copper(II) complexes proved that the Cu-O and Cu-N bonds are of high covalency.

Heterologous expression of two Aspergillus niger feruloyl esterases in Trichoderma reesei for the production of ferulic acid from wheat bran.

Science.gov (United States)

Long, Liangkun; Zhao, Haoyuan; Ding, Dafan; Xu, Meijuan; Ding, Shaojun

2018-05-01

Feruloyl esterase (FAE)-encoding genes AnfaeA and AnfaeB were isolated from Aspergillus niger 0913. For overexpression of the two genes in Trichoderma reesei, constitutive and inductive expression plasmids were constructed based on parental plasmid pAg1-H3. The constructed plasmids contained AnfaeA or AnfaeB gene under the control of glyceraldehyde-3-phosphate dehydrogenase A gene (gpdA) promoter (from A. nidulans) or cellobiohydrolases I (cbh I) gene promoter (from T. reesei), and cbh I terminator from T. reesei. The target plasmids were transferred into T. reesei D-86271 (Rut-C30) by Agrobacterium tumefaciens-mediated transformation (ATMT), respectively. A high level of feruloyl esterase was produced by the recombinant fungal strains under solid-state fermentation, and the cbh I promoter was more efficient than the gpdA promoter in the expression of AnfaeA. The optimum temperatures and pH values were 50 °C and 5.0 for AnFAEA, and 35 °C and 6.0 for AnFAEB. The maximum production levels were 20.69 U/gsd for AnFAEA and 15.08 U/gsd for AnFAEB. The recombinant fungal enzyme systems could release 62.9% (for AnFAEA) and 52.2% (for AnFAEB) of total ferulic acids from de-starched wheat bran, which was higher than the 46.3% releasing efficiency of A. niger 0913. The supplement of xylanase from T. longibrachiatum in the enzymatic hydrolysis led to a small increment of the ferulic acids release.

Study on thermostabilizers for trivalent oral poliomyelitis vaccine

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M. L. F. Leal

1990-09-01

Full Text Available Different formulations of trivalent oral poliomyelitis vaccine were tested, in order to obtain better thermostability, reduce corrosion of machinery and improve production costs. Magnesium chloride, sucrose, arginine and 199-Hank's medium were used in the formulations. The most appropriate formulation was a mixture of MgCl2 and arginine, which was highly thermostable, and had low production costs. The low corrosive formulation was rejected, due to low thermostability on storage.

Pecan nutshell as biosorbent to remove Cu(II), Mn(II) and Pb(II) from aqueous solutions.

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Vaghetti, Julio C P; Lima, Eder C; Royer, Betina; da Cunha, Bruna M; Cardoso, Natali F; Brasil, Jorge L; Dias, Silvio L P

2009-02-15

In the present study we reported for the first time the feasibility of pecan nutshell (PNS, Carya illinoensis) as an alternative biosorbent to remove Cu(II), Mn(II) and Pb(II) metallic ions from aqueous solutions. The ability of PNS to remove the metallic ions was investigated by using batch biosorption procedure. The effects such as, pH, biosorbent dosage on the adsorption capacities of PNS were studied. Four kinetic models were tested, being the adsorption kinetics better fitted to fractionary-order kinetic model. Besides that, the kinetic data were also fitted to intra-particle diffusion model, presenting three linear regions, indicating that the kinetics of adsorption should follow multiple sorption rates. The equilibrium data were fitted to Langmuir, Freundlich, Sips and Redlich-Peterson isotherm models. Taking into account a statistical error function, the data were best fitted to Sips isotherm model. The maximum biosorption capacities of PNS were 1.35, 1.78 and 0.946mmolg(-1) for Cu(II), Mn(II) and Pb(II), respectively.

Physico - chemical investigation on Co(II), Ni(II), Cu(II), Zn(II), Cd(II), UO2+2 and VO+2 ions-O-(-N-3,5-dichloro-α-pyridone imino)

International Nuclear Information System (INIS)

Mathur, Praveen; Trivedi, Pradeep; Mehta, R.K.

1983-01-01

Studies on the interaction of newly synthesised ligand, O-(N-3, 5-dichloro-α-pyridone imino) benzene sulphonic acid (H 2 PB) with Co(II), Ni(II), Cu(II), Zn(II), Cd(II), UO 2 +2 and VO +2 have been carried out potentiometrically. Many physico-chemical studies on thermodynamics, elemental analysis, molecular weight, magnetic moment, conductance, electronic and IR spectra have also been made on the solid chelates and their adducts. The dissociation constants of H 2 PB and stabilities of its bivalent chelates have been evaluated potentiometrically at 25deg, 35deg and 45degC in aqueous medium (0.01M, 0.05M and 0.1M NaClO 4 ) by Bjerrum's method. The stability sequence is in agreement with the Irving-William's rule. (author)

Unsaturated b-ketoesters and their Ni(II, Cu(II and Zn(II complexes

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MUHAMMED BASHEER UMMATHUR

2009-03-01

Full Text Available A new series of b-ketoesters in which the keto group is attached to the olefinic linkage were synthesized by the reaction of methyl acetoacetate and aromatic aldehydes under specified conditions. The existence of these compounds predominantly in the intramolecularly hydrogen bonded enol form was well demonstrated from their IR, 1H-NMR and mass spectral data. Details on the formation of their [ML2] complexes with Ni(II, Cu(II and Zn(II and the nature of the bonding are discussed on the basis of analytical and spectral data.

Synthesis, spectral, thermal and antimicrobial studies on cobalt(II), nickel(II), copper(II), zinc(II) and palladium(II) complexes containing thiosemicarbazone ligand

Science.gov (United States)

El-Sawaf, Ayman K.; El-Essawy, Farag; Nassar, Amal A.; El-Samanody, El-Sayed A.

2018-04-01

The coordination characteristic of new N4-morpholinyl isatin-3-thiosemicarbazone (HL) towards Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) has been studies. The structures of the complexes were described by elemental analyses, molar conductivity, magnetic, thermal and spectral (IR, UV-Vis, 1H and 13C NMR and ESR) studies. On the basis of analytical and spectral studies the ligand behaves as monobasic tridentate ONS donor forming two five membered rings towards cobalt, copper and palladium and afforded complexes of the kind [M(L)X], (Mdbnd Co, Cu or Pd; Xdbnd Cl, Br or OAc). Whereas the ligand bound to NiCl2 as neutral tridentate ONS donor and with ZnCl2 as neutral bidentate NS donor. The newly synthesized thiosemicarbazone ligand and some of its complexes were examined for antimicrobial activity against 2 gram negative bacterial strains (Escherichia coli Pseudomonas and aeruginosa), 2 gram positive bacterial strains (Streptococcus pneumoniae and Staphylococcus aureus)} and two Pathogenic fungi (Aspergillus fumigatus and Candida albicans). All metal complexes possess higher antimicrobial activity comparing with the free thiosemicarbazone ligand. The high potent activities of the complexes may arise from the coordination and chelation, which tends to make metal complexes act as more controlling and potent antimicrobial agents, thus hindering the growing of the microorganisms. The antimicrobial results also show that copper bromide complex is better antimicrobial agent as compared to the Schiff base and its metal complexes.

Equilibrium and kinetic studies of Pb(II, Cd(II and Zn(II sorption by Lagenaria vulgaris shell

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Mitić-Stojanović Dragana-Linda

2012-01-01

Full Text Available The sorption of lead, cadmium and zinc ions from aqueous solution by Lagenaria vulgaris shell biosorbent (LVB in batch system was investigated. The effect of relevant parameters such as contact time, biosorbent dosage and initial metal ions concentration was evaluated. The Pb(II, Cd(II and Zn(II sorption equilibrium (when 98% of initial metal ions were sorbed was attained within 15, 20 and 25 min, respectively. The pseudo first, pseudo-second order, Chrastil’s and intra-particle diffusion models were used to describe the kinetic data. The experimental data fitted the pseudo-second order kinetic model and intra-particle diffusion model. Removal efficiency of lead(II, cadmium(II and zinc(II ions rapidly increased with increasing biosorbent dose from 0.5 to 8.0 g dm-3. Optimal biosorbent dose was set to 4.0 g dm-3. An increase in the initial metal concentration increases the sorption capacity. The sorption data of investigated metal ions are fitted to Langmuir, Freundlich and Temkin isotherm models. Langmuir model best fitted the equilibrium data (r2 > 0.99. Maximal sorption capacities of LVB for Pb(II, Cd(II and Zn(II at 25.0±0.5°C were 0.130, 0.103 and 0.098 mM g-1, respectively. The desorption experiments showed that the LVB could be reused for six cycles with a minimum loss of the initial sorption capacity.

Recovery of Cd(II), Co(II) and Ni(II) from Chloride Medium by Solvent Extraction Using CYANEX 923 and CYANEX 272 I

International Nuclear Information System (INIS)

Ahmed, M.; El Dessouky, S.I.; El-Nadi, Y.A.; Daoud, J.A.; Saad, E.A.

2008-01-01

The paper aims to study the extraction and separation of Cd(II), Co(II) and Ni(II) from their mixtures in hydrochloric acid medium with CYANEX 923 in kerosene. Preliminary investigations showed that only Cd(II) is extracted with CYANEX 923 while Co(II) and Ni(II) are not extracted. Different parameters affecting the extraction of Cd(II) with CYANEX 923 such as hydrochloric acid, hydrogen ion, extractant and metal concentrations, temperature investigations were also investigated. The stoichiometry of the extracted metal species investigated was found to be HCdCl 3 . 2 CYANEX 923. The stripping of the extracted Cd(II) species is obtained with 0.1 M HCl solution. Co(II) was found to be extracted with CYANEX 272 at ph 5.8 leaving Ni(II) in the solution. A developed process for the sequential of Cd(II), Co(II) and Ni(II) from their mixture in hydrochloric acid medium is proposed

The type II collagen fragments Helix-II and CTX-II reveal different enzymatic pathways of human cartilage collagen degradation

DEFF Research Database (Denmark)

Charni-Ben Tabassi, N; Desmarais, S; Jensen, Anne-Christine Bay

2008-01-01

human recombinant cathepsins (Cats) and matrix-metalloproteases (MMPs). Next, we analyzed the spontaneous release of Helix-II and CTX-II from cartilage sections of patients with knee OA who were immediately deep frozen after joint replacement to preserve endogenous enzyme activity until assay. Cartilage....... Cat D was unable to digest intact cartilage. MMPs-1, -3, -7, -9, and -13 efficiently released CTX-II, but only small amount of Helix-II. Neither CTX-II nor Helix-II alone was able to reflect accurately the collagenolytic activity of Cats and MMPs as reflected by the release of hydroxyproline. In OA...

Application of a novel alkali-tolerant thermostable DyP-type peroxidase from Saccharomonospora viridis DSM 43017 in biobleaching of eucalyptus kraft pulp.

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Wangning Yu

Full Text Available Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50-80°C. It also showed broad pH adaptability (>35% activity at pH 4.0-9.0 and alkali-tolerance (>80% activity after incubation at pH 5-10 for 1 h at 37°C, and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0. SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.

A novel mercuric reductase from the unique deep brine environment of atlantis II in the red sea

KAUST Repository

Sayed, Ahmed Anazadeh

2013-11-26

Aunique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+ in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel mercuric reductase from the unique deep brine environment of atlantis II in the red sea

KAUST Repository

Sayed, Ahmed Anazadeh; Ghazy, Mohamed A.; Ferreira, Ari José Scattone; Setú bal, Joã o Carlos; Chambergo, Felipe Santiago; Ouf, Amged; Adel, Mustafa; Dawe, Adam Sean; Archer, John A.C.; Bajic, Vladimir B.; Siam, Rania; El-Dorry, Hamza A A

2013-01-01

Aunique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+ in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Thermal, spectral, magnetic and biological studies of thiosemicarbazones complexes with metal ions: Cu(II), Co(II), Ni(II), Fe(III), Zn(II), Mn(II) and UO2(VI)

International Nuclear Information System (INIS)

Mashaly, M.M.; Seleem, H.S.; El-Behairy, M.A.; Habib, H.A.

2004-01-01

Thiosemicarbazones ligands, isatin-3-thiosemicarbazone(HIT) and N-acetylisatin-3-thiosemicarbazone (HAIT), which have tridentate ONN coordinating sites were prepared. The complexes of both ligands with Cu(II), Co(II), Ni(II), Fe(III), Zn(II), Mn(II) and UO 2 (VI) ions were isolated. The ligands and their metal complexes were characterized by elemental analysis, IR, UV-Vis and mass spectra, also by conductance, magnetic moment and TG-DSC measurements. All the transition metal complexes have octahedral configurations, except Cu-complexes which have planar geometry and the UO 2 (VI) complexes which have coordination number 8 and may acquire the distorted dodecahedral geometry. Thermal studies explored the possibility of obtaining new complexes. Inversion from octahedral to square-planar configuration occurred upon heating the parent Ni-HIAT complex to form the corresponding pyrolytic product. The antifungal activity against the tested organisms showed that some metal complexes enhanced the activity with respect to the parent ligands. (author)

Competitive adsorption of Pb(II), Cu(II), and Zn(II) ions onto hydroxyapatite-biochar nanocomposite in aqueous solutions

Science.gov (United States)

Wang, Yu-Ying; Liu, Yu-Xue; Lu, Hao-Hao; Yang, Rui-Qin; Yang, Sheng-Mao

2018-05-01

A hydroxyapatite-biochar nanocomposite (HAP-BC) was successfully fabricated and its physicochemical properties characterized. The analyses showed that HAP nanoparticles were successfully loaded on the biochar surface. The adsorption of Pb(II), Cu(II), and Zn(II) by HAP-BC was systematically studied in single and ternary metal systems. The results demonstrated that pH affects the adsorption of heavy metals onto HAP-BC. Regarding the adsorption kinetics, the pseudo-second-order model showed the best fit for all three heavy metal ions on HAP-BC. In both single and ternary metal ion systems, the adsorption isotherm of Pb(II) by HAP-BC followed Langmuir model, while those of Cu(II) and Zn(II) fitted well with Freundlich model. The maximum adsorption capacity for each tested metal by HAP-BC was higher than that of pristine rice straw biochar (especially for Pb(II)) or those of other reported adsorbents. Therefore, HAP-BC could explore as a new material for future application in heavy metal removal.

Biosorption characteristics of copper (II), chromium (III), nickel (II), and lead (II) from aqueous solutions by Chara sp. and Cladophora sp.

Science.gov (United States)

Elmaci, Ayşe; Yonar, Taner; Ozengin, Nihan

2007-09-01

The aim of this research was to expose individual removals of copper, chromium, nickel, and lead from aqueous solutions via biosorption using nonliving algae species, Chara sp. and Cladophora sp. Optimum pH values for biosorption of copper (II), chromium (III), nickel (II), and lead (II) from aqueous solutions were determined to be 6, 7, 7, and 3 for Cladophora sp. and 5, 3, 5, and 4 for Chara sp. respectively. Maximum adsorption capacities of Chara sp. [10.54 for chromium (III) and 61.72 for lead (II)] and Cladophora sp. [6.59 for chromium (III) and 16.75 and 23.25 for lead (II)] for chromium (III) and lead (II) are similar. On the other hand, copper (II) and nickel (II) biosorption capacity of Cladophora sp. [14.28 for copper (II) and 16.75 for nickel (II)] is greater than Chara sp. [6.506 for copper (II) and 11.76 for nickel (II)]. Significantly high correlation coefficients indicated for the Langmuir adsorption isotherm models can be used to describe the equilibrium behavior of copper, chromium, nickel, and lead adsorption onto Cladophora sp. and Chara sp.

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation.

Science.gov (United States)

Liang, Min; Chen, Min; Liu, Xinying; Zhai, Yafei; Liu, Xian-wei; Zhang, Houcheng; Xiao, Min; Wang, Peng

2012-02-01

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.

DNA damage by the cobalt (II) and zinc (II) complexes of ...

African Journals Online (AJOL)

STORAGESEVER

2008-09-03

Sep 3, 2008 ... distributed in grade 3. The results indicated that Co(II)-L induced a relatively high level of DNA damage in comparison with the level of damage induced by Zn(II)-L. Key words: Tetraazamacrocycle Zn(II) complex, tetraazamacrocycle Co(II) complex, Tetrahymena thermophila, DNA damage, the comet assay.

On-site cellulase production and efficient saccharification of corn stover employing cbh2 overexpressing Trichoderma reesei with novel induction system.

Science.gov (United States)

Li, Yonghao; Zhang, Xiaoyue; Xiong, Liang; Mehmood, Muhammad Aamer; Zhao, Xinqing; Bai, Fengwu

2017-08-01

Although on-site cellulase production offers cost-effective saccharification of lignocellulosic biomass, low enzyme titer is still a barrier for achieving robustness. In the present study, a strain of T. reesei was developed for enhanced production of cellulase via overexpression of Cellobiohydrolase II. Furthermore, optimum enzyme production was achieved using a novel inducer mixture containing synthesized glucose-sophorose (MGD) and alkali pre-treated corn stover (APCS). Within 60h, a remarkably higher cellulase productivity and activity were achieved in the fed-batch fermentation using the optimized ratio of MGD and APCS in the inducer mixture, compared to those reported using cellulosic biomass as the sole inducer. After the enzyme production, APCS was added directly into the fermentation broth at 20% solid loading, which produced 122.5g/L glucose and 40.21g/L xylose, leading to the highest yield reported so far. The improved enzyme titers during on-site cellulase production would benefit cost-competitive saccharification of lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and characterization of nickel(II), cobalt(II), copper(II), manganese(II), zinc(II), zirconium(IV), dioxouranium(VI) and dioxomolybdenum(VI) complexes of a new Schiff base derived from salicylaldehyde and 5-methylpyrazole-3-carbohydrazide

International Nuclear Information System (INIS)

Syamal, A.; Maurya, M.R.

1986-01-01

Synthesis of a new Schiff base derived from salicylaldehyde and 5-methylpyrazole-3-carbohydrazide, and its coordination compounds with nickel(II), cobalt(II), copper(II), manganese(II), zinc(II), zirconium(IV), dioxouranium(VI) and dioxomolybdenum(VI) are described. The ligand and the complexes have been characterized on the basis of analytical, conductance, molecular weight, i.r., electronic and n.m.r. spectra and magnetic susceptibility measurements. The stoichiometries of the complexes are represented as NiL . 3H 2 O, CoL . 2H 2 O, CuL, MnL . 2H 2 O, ZnL . H 2 O, Zr(OH) 2 (LH) 2 , Zr(OH) 2 L . 2MeOH, UO 2 L . MeOH and MoO 2 L . MeOH (where LH 2 =Schiff base). The copper(II) complex shows a subnormal magnetic moment due to antiferromagnetic exchange interaction while the nickel(II), cobalt(II) and manganese(II) complexes show normal magnetic moments at room temperature. The i.r. and n.m.r. spectral studies show that the Schiff base behaves as a dibasic and tridentate ligand coordinating through the deprotonated phenolic oxygen, enolic oxygen and azomethine nitrogen. (orig.)

Levels of Cd (II, Mn (II, Pb (II, Cu (II, and Zn (II in Common Buzzard (Buteo buteo from Sicily (Italy by Derivative Stripping Potentiometry

Directory of Open Access Journals (Sweden)

P. Licata

2010-01-01

Full Text Available The purpose of this study was to determine the concentrations of heavy metals (Cd, Pb, Cu, Mn, and Zn in different organs (liver, kidney, muscle, lung, skin, and feathers of buzzards (Buteo buteo, utilized as a “biological indicator” for environmental contamination, from different areas of Sicily and to investigate the relationships between birds sex, age, and weight and metal levels in these samples. All samples of common buzzards were collected at the “Recovery Center of Wild Fauna” of Palermo, through the Zooprophilactic Institute. Potentiometric stripping analysis (PSA was used to determine the content of Cd(II, Cu(II, Mn(II, Pb(II, and Zn(II in bird tissues. For toxic metals, the highest levels of Pb were in liver and those of Cd in lung; Zn levels were higher than Cu and Mn in all tissues analyzed. The concentrations in liver, lung, kidney, and muscle could be considered as an indicative of chronic exposure to metals while the presence of metals in skin could be consequential to storing and elimination processes. The found concentrations of metals in the studied matrices required a highly sensitive method for their determination and a simple sample preparation procedure, and the proposed method was well suited for this purpose.

Complexes cobalt(II, zinc(II and copper(II with some newly synthesized benzimidazole derivatives and their antibacterial activity

Directory of Open Access Journals (Sweden)

S. O. PODUNAVAC-KUZMANOVIC

1999-05-01

Full Text Available The preparation and properties of some complexes of cobalt(II, zinc(II and copper(II with several newly synthesized benzimidazole derivatives (L are reported. The complexes, of the general formula [MCl2L2] (M=Co(II, Zn(II and [CuCl2L(H2O], have a tetrahedral structure. The complexes were characterized by elemental analysis, molar conductivity, magnetic susceptibility measurements, IR and absorption electronic spectra. The antibacterial activitiy of the benzimidazoles and their complexes was evaluated against Erwinia carotovora subsp. carotovora and Erwinia amylovora. The complexes were found to be more toxic than the ligands.

Micellar effect on metal-ligand complexes of Co(II, Ni(II, Cu(II and Zn(II with citric acid

Directory of Open Access Journals (Sweden)

Nageswara Rao Gollapalli

2009-12-01

Full Text Available Chemical speciation of citric acid complexes of Co(II, Ni(II, Cu(II and Zn(II was investigated pH-metrically in 0.0-2.5% anionic, cationic and neutral micellar media. The primary alkalimetric data were pruned with SCPHD program. The existence of different binary species was established from modeling studies using the computer program MINIQUAD75. Alkalimetric titrations were carried out in different relative concentrations (M:L:X = 1:2:5, 1:3:5, 1:5:3 of metal (M to citric acid. The selection of best chemical models was based on statistical parameters and residual analysis. The species detected were MLH, ML2, ML2H and ML2H2. The trend in variation of stability constants with change in mole fraction of the medium is explained on the basis of electrostatic and non-electrostatic forces. Distributions of the species with pH at different compositions of micellar media are also presented.

Production of Cellulases by Rhizopus stolonifer from Glucose-Containing Media Based on the Regulation of Transcriptional Regulator CRE.

Science.gov (United States)

Zhang, Yingyiing; Tang, Bin; Du, Guocheng

2017-03-28

Carbon catabolite repression is a crucial regulation mechanism in microorganisms, but its characteristic in Rhizopus is still unclear. We extracted a carbon regulation gene, cre , that encoded a carbon catabolite repressor protein (CRE) from Rhizopus stolonifer TP-02, and studied the regulation of CRE by real-time qPCR. CRE responded to glucose in a certain range, where it could significantly regulate part of the cellulase genes ( eg, bg, and cbh2 ) without cbh1 . In the comparison of the response of cre and four cellulase genes to carboxymethylcellulose sodium and a simple carbon source (lactose), the effect of CRE was only related to the concentration of reducing sugars. By regulating the reducing sugars to range from 0.4% to 0.6%, a glucose-containing medium with lactose as the inducer could effectively induce cellulases without the repression of CRE. This regulation method could potentially reduce the cost of enzymes produced in industries and provide a possible solution to achieve the large-scale synthesis of cellulases.

Complexes of Cu(II), Ni(II), Co(II), oxovanadium(IV) and dioxouranium(VI) with N,N'-ethylenebis (2-hydroxy-4-methylpropiophenoneimine)

International Nuclear Information System (INIS)

Patel, M.M.; Patel, M.R.; Patel, M.N.; Patel, R.P.

1982-01-01

Complexes of Cu(II), Ni(II), Co(II), oxovanadium(IV) and dioxouranium(VI) with the schiff base, N,N'-ethylenebis(2-hydroxy-4-methylpropiophenoneimine)(4-MeOHPEN), have been synthesised and characterised on the basis of elemental analyses, conductivity, magnetic moment, electronic and infrared spectral data. Square-planar structures are suggested for Cu(II), Ni(II) and Co(II) complexes while a distorted square-pyramidal structure is suggested for the oxovanadium(IV) complex. (author)

Elaboration of a Highly Porous RuII,II Analogue of HKUST-1.

Science.gov (United States)

Zhang, Wenhua; Freitag, Kerstin; Wannapaiboon, Suttipong; Schneider, Christian; Epp, Konstantin; Kieslich, Gregor; Fischer, Roland A

2016-12-19

When the dinuclear Ru II,II precursor [Ru 2 (OOCCH 3 ) 4 ] is employed under redox-inert conditions, a Ru II,II analogue of HKUST-1 was successfully prepared and characterized as a phase-pure microcrystalline powder. X-ray absorption near-edge spectroscopy confirms the oxidation state of the Ru centers of the paddle-wheel nodes in the framework. The porosity of 1371 m 2 /mmol of Ru II,II -HKUST-1 exceeds that of the parent compound HKUST1 (1049 m 2 / mmol).

Dynamic adsorption of mixtures of Rhodamine B, Pb (II), Cu (II) and Zn(II) ions on composites chitosan-silica-polyethylene glycol membrane

Science.gov (United States)

Mahatmanti, F. W.; Rengga, W. D. P.; Kusumastuti, E.; Nuryono

2018-04-01

The adsorption of a solution mixture of Rhodamine B, Pb (II), Cu (II) and Zn(II) was studied using dynamic methods employing chitosan-silica-polyethylene glycol (Ch/Si/P) composite membrane as an adsorptive membrane. The composite Ch/Si/P membrane was prepared by mixing a chitosan-based membrane with silica isolated from rice husk ash (ASP) and polyethylene glycol (PEG) as a plasticizer. The resultant composite membrane was a stronger and more flexible membrane than the original chitosan-based membrane as indicated by the maximum percentage of elongation (20.5 %) and minimum Young’s Modulus (80.5 MPa). The composite membrane also showed increased mechanical and hydrophilic properties compared to the chitosan membranes. The membrane was used as adsorption membrane for Pb (II), Cu (II), Cd (II) ions and Rhodamine B dyes in a dynamic system where the permeation and selectivity were determined. The permeation of the components was observed to be in the following order: Rhodamine B > Cd (II) > Pb (II) > Cu (II) whereas the selectivity was shown to decrease the order of Cu (II) > Pb (II) > Cd (II) > Rhodamine B.

Serum insulin-like growth factor II (IGF-II) in chronic heart failure

International Nuclear Information System (INIS)

Tong Lijun; Chen Donghai; Ji Naijun; Fan Bifu; Wang Chengyao; Mei Yibin; Li Fuyuan; Kao Yan

2004-01-01

Objective: To investigate the clinical significance of changes of serum insulin-like growth factor II (IGF-II) levels in patients with chronic heart failure. Methods: Serum IGF-II levels were measured with RIA in 132 cases of chronic heart failure and 45 controls. Results: Serum IGF-II levels were significantly higher in patients with chronic heart failure than those in the controls (t=0.033, P<0.001). IGF-II levels were highest in grade IV CHF patients (vs grade II t=3.963, P<0.01; vs grade III, t=3.578, P<0.01). In the twelve patients died in hospital, the serum IGF-II levels were significantly higher than those patients recovered (t=7.141, P<0.01). Conclusion: Serum IGF-II levels were increased in CHF patients and were highest in the most severe cases. (authors)

Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07.

Science.gov (United States)

Gaur, Rajeeva; Tiwari, Soni

2015-03-19

The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml(-1)) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca(2+), mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml(-1) and 769.230 μg ml(-1) min(-1), respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for

Ni(II, Pd(II and Pt(II complexes with ligand containing thiosemicarbazone and semicarbazone moiety: synthesis, characterization and biological investigation

Directory of Open Access Journals (Sweden)

SULEKH CHANDRA

2008-07-01

Full Text Available The synthesis of nickel(II, palladium(II and platinum(II complexes with thiosemicarbazone and semicarbazone of p-tolualdehyde are reported. All the new compounds were characterized by elemental analysis, molar conductance measurements, magnetic susceptibility measurements, mass, 1H-NMR, IR and electronic spectral studies. Based on the molar conductance measurements in DMSO, the complexes may be formulated as [Ni(L2Cl2] and [M(L2]Cl2 (where M = Pd(II and Pt(II due to their non-electrolytic and 1:2 electrolytic nature, respectively. The spectral data are consistent with an octahedral geometry around Ni(II and a square planar geometry for Pd(II and Pt(II, in which the ligands act as bidentate chelating agents, coordinated through the nitrogen and sulphur/oxygen atoms. The ligands and their metal complexes were screened in vitro against fungal species Alternaria alternata, Aspergillus niger and Fusarium odum, using the food poison technique.

Ergodic time-reversible chaos for Gibbs' canonical oscillator

International Nuclear Information System (INIS)

Hoover, William Graham; Sprott, Julien Clinton; Patra, Puneet Kumar

2015-01-01

Nosé's pioneering 1984 work inspired a variety of time-reversible deterministic thermostats. Though several groups have developed successful doubly-thermostated models, single-thermostat models have failed to generate Gibbs' canonical distribution for the one-dimensional harmonic oscillator. A 2001 doubly-thermostated model, claimed to be ergodic, has a singly-thermostated version. Though neither of these models is ergodic this work has suggested a successful route toward singly-thermostated ergodicity. We illustrate both ergodicity and its lack for these models using phase-space cross sections and Lyapunov instability as diagnostic tools. - Highlights: • We develop cross-section and Lyapunov methods for diagnosing ergodicity. • We apply these methods to several thermostatted-oscillator problems. • We demonstrate the nonergodicity of previous work. • We find a novel family of ergodic thermostatted-oscillator problems.

Anti-inflammatory drugs interacting with Zn(II), Cd(II) and Pt(II) metal ions.

Science.gov (United States)

Dendrinou-Samara, C; Tsotsou, G; Ekateriniadou, L V; Kortsaris, A H; Raptopoulou, C P; Terzis, A; Kyriakidis, D A; Kessissoglou, D P

1998-09-01

Complexes of Zn(II), Cd(II) and Pt(II) metal ions with the anti-inflammatory drugs, 1-methyl-5-(p-toluoyl)-1H-pyrrole-2-acetic acid (Tolmetin), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (Ibuprofen), 6-methoxy-alpha-methylnaphthalene-2-acetic acid (Naproxen) and 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid (indomethacin) have been synthesized and characterized. In the structurally characterized Cd(naproxen)2 complex the anti-inflammatory drugs acts as bidentate chelate ligand coordinatively bound to metal ions through the deprotonated carboxylate group. Crystal data for 1: [C32H26O8Cd], orthorhombic, space group P22(1)2(1), a = 5.693(2) (A), b = 8.760(3) (A), c = 30.74(1) (A), V = 1533(1) A3, Z = 2. Antibacterial and growth inhibitory activity is higher than that of the parent ligands or the platinum(II) diamine compounds.

Synthesis, spectroscopic and antimicrobial properties of Co(II), Ni (II ...

African Journals Online (AJOL)

The objective of this study is to investigate the antimicrobial activity of novel Schiff base metal complexes. The resistance of micro-organisms to classical antimicrobial compounds poses a challenge to effective management and treatment of some diseases. In line with this, copper (II), nickel (II) and cobalt (II) complexes of ...

RTNS-II [Rotating Target Neutron Source II] operational summary

International Nuclear Information System (INIS)

Heikkinen, D.W.

1988-09-01

The Rotating Target Neutron Source II facility (RTNS-II) operated for over nine years. Its purpose was to provide high intensities of 14 MeV neutrons for materials studies in the fusion energy program. For the period from 1982-1987, the facility was supported by both the US (Department of Energy) and Japan (Ministry of Education, Culture, and Science). RTNS-II contains two accelerator-based neutron sources which use the T(d,n) 4 He reaction. In this paper, we will summarize the operational history of RTNS-II. Typical operating parameters are given. In addition, a brief description of the experimental program is presented. The current status and future options for the facility are discussed. 7 refs., 5 tabs

Chakraborty, Prof. Ranajit

Indian Academy of Sciences (India)

Elected: 2006 Honorary. Chakraborty, Prof. Ranajit. Date of birth: 1946. Address: Director, Center for Computational Genomics, University of North Texas, HSC, 3500 Camp Bowie Blvd, CBH 249, Fort Worth, TX 76107, U.S.A.. Contact: Office: (+1-817) 735 2421. Fax: (+1-817) 735 5016. Email: ranajit.chakraborty@unthsc.

[Evaluation of serum PIVKA-II by Lumipulse PrestoII assay].

Science.gov (United States)

Hiramatsu, Kumiko; Tanaka, Yasuhito; Takagi, Kazumi; Kani, Satomi; Goto, Takaaki; Takasaka, Yoshimitsu; Matsuura, Kentaro; Sugauchi, Fuminaka; Moriyama, Kazushige; Murakami, Hiroshi; Kitajima, Sachiko; Mizokami, Masashi

2009-03-01

Measurements of serum concentrations of Des-gamma-carboxy Prothrombin (PIVKA-II) are widely used for diagnosing hepatocellular carcinoma (HCC). Recently, in Lumipulsef assay, it was reported that antibodies against alkaline phosphatase (ALP) derived from anti bleeding sheets led false high values of PIVKA-II in the patients with HCC resection. To improve the previous issue, newly developed Lumipulse PrestoII assay was examined. (1) The assay was reliable and positively correlated with the previous assays (Lumipulse f and Picolumi, R = 0.997 and 0.994 (n=115), respectively). (2) Eleven cases, which had false high values of PIVKA-II by the Lumipulsef assay, were examined by the PrestoII assay with excess of inactive ALP. The false high values of 10 cases were improved, but only one was still high. False reactivity of this case was stronger than other cases, more effective adsorption was required. (3) Comparing the absorbent activity of inactive ALP among 6 different kinds, we found inactive ALP with much higher adsorbent activity. When this inactive ALP was applied to assay, false high values of PIVKA-II were improved in all 11 cases. In conclusion, the PrestoII assay, which applies the inactive ALP with high activity, is reliable and useful for clinical screening.

(II) COMPLEX COMPOUND

African Journals Online (AJOL)

user

electrochemical sensors, as well as in various chromatographic ... were carried out using Jenway pH meter Model 3320 and a conductivity ... Figure 1: the proposed molecular structure of the copper (II) Schiff base complex. M = Cu (II) or Mn (II).

IGF-II receptors and IGF-II-stimulated glucose transport in human fat cells

International Nuclear Information System (INIS)

Sinha, M.K.; Buchanan, C.; Raineri-Maldonado, C.; Khazanie, P.; Atkinson, S.; DiMarchi, R.; Caro, J.F.

1990-01-01

Insulin-like growth factor II (IGF-II) receptors have been described in rat but not in human adipocytes. In both species, IGF-II has been reported to stimulate glucose transport by interacting with the insulin receptor. In this study, we have unequivocally demonstrated the presence of IGF-II receptors in human adipocytes. 125I-labeled IGF-II specifically binds to intact adipocytes, membranes, and lectin-purified detergent solubilized extracts. Through the use of 0.5 mM disuccinimidyl suberate, 125I-IGF-II is cross-linked to a 260-kDa protein that is identified as the IGF-II receptor by displacement experiments with unlabeled IGF-II, IGF-I, and insulin and either by immunoprecipitation or by Western blot analysis with mannose 6-phosphate receptor antibodies. The concentrations of IGF-II required for half-maximal and maximal stimulation of glucose transport in human adipocytes are 35 and 100 times more than that of insulin. The possibility of IGF-II stimulating glucose transport by interacting predominantly with the insulin receptor is suggested by the following: (1) the concentration of IGF-II that inhibits half of insulin binding is only 20 times more than that of insulin; (2) the lack of an additive effect of IGF-II and insulin for maximal stimulation of glucose transport; (3) the ability of monoclonal insulin receptor antibodies to decrease glucose transport stimulated by submaximal concentrations of both IGF-II and insulin; and (4) the ability of IGF-II to stimulate insulin receptor autophosphorylation albeit at a reduced potency when compared with insulin

Emodin (1,3,8-trihydroxy-6-methylanthraquinone): a spectrophotometric reagent for the determination of beryllium(II), magnesium(II) and calcium(II)

International Nuclear Information System (INIS)

Pal, T.; Jana, N.R.

1993-01-01

The individual determination of Be II , Mg II or Ca II by conventional spectrophotometry and simultaneous determination of Mg II and Ca II in mixtures by first-derivative spectrophotometry are possible at trace levels, using emodin (1,3,8-trihydroxy-6-methylanthraquinone) as spectrophotometric reagent. Interference from other metal species, application of these methods to rock samples and statistical analysis of the results are discussed. (author)

Synthesis and studies on Cu(II), Co(II), Ni(II) complexes of Knoevenagel β-diketone ligands

Science.gov (United States)

Sumathi, S.; Tharmaraj, P.; Sheela, C. D.; Anitha, C.

2012-11-01

Transition metal complexes of various acetylacetone based ligands of the type ML [where M = Cu(II), Ni(II), Co(II); L = 3-(aryl)-pentane-2,4-dione] have been synthesized. The structural features have been derived from their elemental analysis, magnetic susceptibility, molar conductance, IR, UV-Vis, 1H NMR, Mass and ESR spectral studies. Conductivity measurements reveal that all the complexes are non-electrolytic in nature. Spectroscopic and other analytical data of the complexes suggest octahedral geometry for other metal(II) complexes. The redox behavior of the copper(II) complexes have been studied by cyclic voltammetry. The free ligands and their metal complexes have been screened for their in vitro biological activities against the bacteria Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus as well as the fungus Candida albicans by well diffusion method. The zone of inhibition value indicates that the most of the metal(II) complexes are found to possess increased activities compared to those of the free ligands. All synthesized compounds may serve as potential photoactive materials as indicated from their characteristic fluorescence properties. The second harmonic generation (SHG) efficiency of the ligands (L1-L3) was found to be considerable effect than that of urea and KDP (potassium dihydrogen phosphate).

Characterization of a Highly Thermostable and Organic Solvent-Tolerant Copper-Containing Polyphenol Oxidase with Dye-Decolorizing Ability from Kurthia huakuii LAM0618T.

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Xiang Guo

Full Text Available Laccases are green biocatalysts that possess attractive advantages for the treatment of resistant environmental pollutants and dye effluents. A putative laccase-like gene, laclK, encoding a protein of 29.3 kDa and belonging to the Cu-oxidase_4 superfamily, was cloned and overexpressed in Escherichia coli. The purified recombinant protein LaclK (LaclK was able to oxidize typical laccase substrates such as 2,6-dimethoxyphenol and l-dopamine. The characteristic adsorption maximums of typical laccases at 330 nm and 610 nm were not detected for LaclK. Cu2+ was essential for substrate oxidation, but the ratio of copper atoms/molecule of LaclK was determined to only be 1:1. Notably, the optimal temperature of LaclK was 85°C with 2,6-dimethoxyphenol as substrates, and the half-life approximately 3 days at 80°C. Furthermore, 10% (v/v organic solvents (methanol, ethanol, isopropyl alcohol, butyl alcohol, Triton x-100 or dimethyl sulfoxide could promote enzymatic activity. LaclK exhibited wide-spectrum decolorization ability towards triphenylmethane dyes, azo dyes and aromatic dyes, decolorizing 92% and 94% of Victoria Blue B (25 μM and Ethyl Violet (25 μM, respectively, at a concentration of 60 U/L after 1 h of incubation at 60°C. Overall, we characterized a novel thermostable and organic solvent-tolerant copper-containing polyphenol oxidase possessing dye-decolorizing ability. These unusual properties make LaclK an alternative for industrial applications, particularly processes that require high-temperature conditions.

Bis(tripyrazol-1-ylmethanenickel(II tetracyanidonickelate(II dihydrate

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Sean Parkin

2009-12-01

Full Text Available The title complex, [Ni(C10H10N62][Ni(CN4]·2H2O, contains an octahedral nickel(II cation and a square-planar nickel(II anion. Both the cation and the anion reside on a crystallographic center of inversion. The NiII center in the cation is coordinated by six pyrazol-1-yl rings of two chelating tripyrazol-1-ylmethane [HC(pz3] ligands, with Ni—N distances that range between 2.0647 (19 and 2.0828 (19 Å. The NiII center in the anion is coordinated by four cyanide ligands, with Ni—C distances in the range 1.869 (2–1.869 (3 Å. The [Ni(CN4]2− anions are linked by inversion-related water molecules into extended chains that run parallel to the a axis.

Characterization of an organic solvent-tolerant thermostable glucoamylase from a halophilic isolate, Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production.

Science.gov (United States)

Yu, Hui-Ying; Li, Xin

2014-01-01

A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin-layer chromatography revealed that glucose was the sole end-product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0-100°C), pH (7.0-12.0), and NaCl concentration (0-20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers.

Synthesis and properties of complexes of copper(II), nickel(II), cobalt(II) and uranyl ions with 3-(p-tolylsulphonamido)rhodamine

International Nuclear Information System (INIS)

El-Bindary, A.A.; El-Sonbati, A.Z.

2000-01-01

Metal complexes of copper(II), nickel(II), cobalt(II) and uranyl ions with 3-(p-tolylsulphonamido)rhodamine (HL) have been prepared and characterized by chemical and thermal analyses, molar conductivity , magnetic susceptibility measurements, and infrared, electronic and EPR spectra. The visible and EPR spectra indicated that the Cu(II) complex has a tetragonal geometry. From EPR spectrum of the Cu(II) complex,various parameters were calculated. The crystal field parameters of Ni(II) complex were calculated and were found to agree fairly well with the values reported for known square pyramidal complexes. The infrared spectral studies showed a monobasic bidentate behaviour with the oxygen and nitrogen donor system. Thermal stabilities of the complexes are also reported. (author)

Adsorption study of cadmium (II) and lead (II) on radish peels

International Nuclear Information System (INIS)

Anwar, J.; Shafique, U.; Salman, M.; Zaman, W.; Memoona, M.

2009-01-01

The removal efficiency of heavy metals like Cd(II) and Pb(II) from aqueous solutions by adsorption on Raphanus sativus (Radish peels) has been studied. The effects of time, pH, concentration of adsorbent and agitation speed on adsorption have been evaluated. It is found that radish peels powder has high removal efficiency for both the metals. Batch adsorption study has shown that Cd(II) and Pb(II) has been removed up to 88% and 86% respectively. Adsorption equilibriums for both metals have been described by the Langmuir isotherm. The maximum amount of heavy metals (Q ) adsorbed at max equilibrium were 7.5 and 1.23 mg/g for Cd(II) and Pb(II) respectively as evaluated by Langmuir isotherm. It is concluded that waste materials like radish peels can be used for removal of heavy metals from aqueous streams. (author)

New complexes of Co(II, Ni(II, Cu(II with Schiff base N,N’-bis-(3-methoxy-saliciliden-3,3’-dimethylbenzidine

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Alan Ionela

2013-01-01

Full Text Available The new N,N’-bis-(3-methoxy-saliciliden-3,3’-dimetilbenzidine (H2L Schiff base and complexes with Co(II, Ni(II and Cu(II of type [M(HLCl(H2O] (M=Co(II, Cu(II [M2L(H2O4]X2 (M=Co(II, X=ClO4 and M=Cu(II, X=NO3 and [M2L(CH3COO2] (M=Co(II, Ni(II, Cu(II were synthesised. The ligand and complexes were characterized by elemental analysis, conductibility measurements, magnetic moments at room temperature, IR, NMR, UV-VIS-NIR, EPR spectra and thermogravimetric analysis. A molar ratio of 1:1 or 1:2 between ligand and metal was determined from the elemental analysis. Except for perchlorate complex that behave as electrolyte, the rest of complexes are non-electrolytes. The spectral data suggest a tetrahedral, pseudo-tetrahedral or square-planar stereochemistry respectively, data confirmed by magnetic behaviour of complexes. The antimicrobial tests indicate a fungicide effect both for ligand and complexes.

and copper(II)

Indian Academy of Sciences (India)

Unknown

(II) and copper(II)–zinc(II) complexes. SUBODH KUMAR1, R N PATEL1*, P V KHADIKAR1 and. K B PANDEYA2. 1 Department of Chemistry, APS University, Rewa 486 003, India. 2 CSJM University, Kanpur 208 016, India e-mail: (R N Patel) ...

Determination of Lead(II), Cadmium(II) and Copper(II) in Waste-Water and Soil Extracts on Mercury Film Screen-Printed Carbon Electrodes Sensor

International Nuclear Information System (INIS)

Mohd Fairulnizal Md Noh; Tothill, I.E.

2011-01-01

A sensor incorporating a three electrodes configuration have been fabricated using low cost screen-printing technology. These electrodes couples with Square Wave Stripping Voltammetry (SWSV) has provided a convenient screening tool for on-site detection of trace levels of Pb(II), Cd(II) and Cu(II). Modification of the graphite carbon surface based on in situ deposition of mercury film has been carried out. By appropriate choice of supporting medium and optimized parameters setting such as amount of mercury used the deposition potential, deposition time, frequency and scan rate, well resolved and reproducible response for Pb(II), Cd(II) and Cu(II) were obtained. The performance characteristics of the developed mercury film screen printed carbon electrode (MFSPCE) for 120 s deposition time showed that the linear range for Cd(II), Pb(II) and Cu(II) were 10 to 200 μg L -1 . The detection limit recorded for Cd(II), Pb(II) and Cu(II) were 2, 1 and 5 μg L -1 with relative standard deviation (RSD) of 6.5 %, 6.9 % and 7.5 %, respectively. Successful applications of the sensing device to waste-water and extracted soil samples were demonstrated. (author)

Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination.

Science.gov (United States)

Hong, Young-Ho; Lee, Dong-Woo; Pyun, Yu-Ryang; Lee, Sung Haeng

2011-12-28

Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.

Kinetic and spectroscopic investigation of CoII, NiII, and N-oxalylglycine inhibition of the FeII/α-ketoglutarate dioxygenase, TauD

International Nuclear Information System (INIS)

Kalliri, Efthalia; Grzyska, Piotr K.; Hausinger, Robert P.

2005-01-01

Co II , Ni II , and N-oxalylglycine (NOG) are well-known inhibitors of Fe II /α-ketoglutarate (αKG)-dependent hydroxylases, but few studies describe their kinetics and no spectroscopic investigations have been reported. Using taurine/αKG dioxygenase (TauD) as a paradigm for this enzyme family, time-dependent inhibition assays showed that Co II and Ni II follow slow-binding inhibition kinetics. Whereas Ni II -substituted TauD was non-chromophoric, spectroscopic studies of the Co II -substituted enzyme revealed a six-coordinate site (protein alone or with αKG) that became five-coordinate upon taurine addition. The Co II spectrum was not perturbed by a series of anions or oxidants, suggesting the Co II is inaccessible and could be used to stabilize the protein. NOG competed weakly (K i ∼ 290 μM) with αKG for binding to TauD, with the increased electron density of NOG yielding electronic transitions for NOG-Fe II -TauD and taurine-NOG-Fe II -TauD at 380 nm (ε 38 90-105 M -1 cm -1 ). The spectra of the NOG-bound TauD species did not change significantly upon oxygen exposure, arguing against the formation of an oxygen-bound state mimicking an early intermediate in catalysis

II-VI semiconductor compounds

CERN Document Server

1993-01-01

For condensed matter physicists and electronic engineers, this volume deals with aspects of II-VI semiconductor compounds. Areas covered include devices and applications of II-VI compounds; Co-based II-IV semi-magnetic semiconductors; and electronic structure of strained II-VI superlattices.

Estimated environmental loads of alpha-amylase from transgenic high-amylase maize

Energy Technology Data Exchange (ETDEWEB)

Wolt, Jeffrey D. [Department of Agronomy, Iowa State University, Ames, IA 50011 (United States); Biosafety Institute for Genetically Modified Agricultural Products, 164 Seed Science, Iowa State University, Ames, IA 50011 (United States); Karaman, Sule [Biosafety Institute for Genetically Modified Agricultural Products, 164 Seed Science, Iowa State University, Ames, IA 50011 (United States)

2007-11-15

Environmental exposure of plants bioengineered to improve efficiencies of biofuel production is an important consideration for their adoption. High-amylase maize genetically engineered to produce thermostable alpha-amylase in seed endosperm is currently in development, and its successful adoption will entail >1000 km{sup 2} of annual production in the USA. Environmental exposure of thermostable amylase will occur in production fields from preharvest and harvest dropped grain, with minor additional contributions from stover and root biomass. Mass loadings of thermostable alpha-amylase are projected to be 16 kg km{sup -2} and represent a potential source of increased alpha-amylase activity in receiving soils. An understanding of the degradation, persistence, accumulation, and activity of thermostable alpha-amylase introduced from transgenic high-amylase maize will be necessary in order to effectively manage transgenic crop systems intended or biofeedstock production. (author)

Syntheses, structures, and properties of imidazolate-bridged Cu(II)-Cu(II) and Cu(II)-Zn(II) dinuclear complexes of a single macrocyclic ligand with two hydroxyethyl pendants.

Science.gov (United States)

Li, Dongfeng; Li, Shuan; Yang, Dexi; Yu, Jiuhong; Huang, Jin; Li, Yizhi; Tang, Wenxia

2003-09-22

The imidazolate-bridged homodinuclear Cu(II)-Cu(II) complex, [(CuimCu)L]ClO(4).0.5H(2)O (1), and heterodinuclear Cu(II)-Zn(II) complex, [(CuimZnL(-)(2H))(CuimZnL(-)(H))](ClO(4))(3) (2), of a single macrocyclic ligand with two hydroxyethyl pendants, L (L = 3,6,9,16,19,22-hexaaza-6,19-bis(2-hydroxyethyl)tricyclo[22,2,2,2(11,14)]triaconta-1,11,13,24,27,29-hexaene), have been synthesized as possible models for copper-zinc superoxide dismutase (Cu(2),Zn(2)-SOD). Their crystal structures analyzed by X-ray diffraction methods have shown that the structures of the two complexes are markedly different. Complex 1 crystallizes in the orthorhombic system, containing an imidazolate-bridged dicopper(II) [Cu-im-Cu](3+) core, in which the two copper(II) ions are pentacoordinated by virtue of an N4O environment with a Cu.Cu distance of 5.999(2) A, adopting the geometry of distorted trigonal bipyramid and tetragonal pyramid, respectively. Complex 2 crystallizes in the triclinic system, containing two similar Cu-im-Zn cores in the asymmetric unit, in which both the Cu(II) and Zn(II) ions are pentacoordinated in a distorted trigonal bipyramid geometry, with the Cu.Zn distance of 5.950(1)/5.939(1) A, respectively. Interestingly, the macrocyclic ligand with two arms possesses a chairlike (anti) conformation in complex 1, but a boatlike (syn) conformation in complex 2. Magnetic measurements and ESR spectroscopy of complex 1 have revealed the presence of an antiferromagnetic exchange interaction between the two Cu(II) ions. The ESR spectrum of the Cu(II)-Zn(II) heterodinuclear complex 2 displayed a typical signal for mononuclear trigonal bipyramidal Cu(II) complexes. From pH-dependent ESR and electronic spectroscopic studies, the imidazolate bridges in the two complexes have been found to be stable over broad pH ranges. The cyclic voltammograms of the two complexes have been investigated. Both of the two complexes can catalyze the dismutation of superoxide and show rather high activity.

Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity.

Science.gov (United States)

Stewart, Alesha C; Bethel, Christopher R; VanPelt, Jamie; Bergstrom, Alex; Cheng, Zishuo; Miller, Callie G; Williams, Cameron; Poth, Robert; Morris, Matthew; Lahey, Olivia; Nix, Jay C; Tierney, David L; Page, Richard C; Crowder, Michael W; Bonomo, Robert A; Fast, Walter

2017-12-08

Use and misuse of antibiotics have driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here, we present evidence that New Delhi metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt(II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

Syntheses of polystyrene supported chelating resin containing the Schiff base derived from salicylaldehyde and triethylene tetramine and its copper(II), nickel(II), cobalt(II), iron(III), zinc(II), cadmium(II), molybdenum(VI), zirconium(IV) and uranium(VI) complexes

International Nuclear Information System (INIS)

Syamal, A.; Singh, M.M.

1998-01-01

A new polymer-anchored chelating ligand has been synthesized by the reaction of chloromethylated polystyrene (containing 0.94 mmol of Cl per gram of resin and 1% cross-linked with divinylbenzene) and the Schiff base derived from salicylaldehyde and triethylenetetramine. A new series of polystyrene supported, Cu(II), Ni(II), Co(II), Fe(III), Zn(II), Cd(II), Zr(IV), dioxomolybdenum (VI) and dioxouranium (VI) complexes of the formulae PS-LCu, PS-LNi, PS-LCo, PS-LFeCl.DMF, PS-LZn, PS-LCd, PS-LZr(OH) 2 . DMF, PS L MoO 2 and PS-LUO 2 (where PS-LH 2 = polymer-anchored Schiff base and DMF dimethyl-formamide) have been synthesized and characterised by elemental analysis, infrared, electronic spectra and magnetic susceptibility measurements. The complexes PS-LCu, PS-LNi and PS-LCo have square planar structure, PS-LFeCl.DMF, PS-LMoO 2 and PS-LUO 2 have octahedral structure, PS L Zn and PS-LCd are tetrahedral and PS-LZr(OH) 2 .DMF is pentagonal bipyramidal. The polymer-anchored Cu(II), Co(II) and Fe(III) complexes are paramagnetic while Ni(II), Zn(II), Cd(II), Zr(IV), dioxomolybdenum(VI) and dioxouranium(VI) complexes are diamagnetic. The negative shift of the v (C=N) (azomethine) and the positive shift of v (C--O)(phenolic) are indicative of ONNO donor behaviour of the polymer-anchored Schiff base. (author)

DNA binding and biological activity of mixed ligand complexes of Cu(II, Ni(II and Co(II with quinolones and N donor ligand

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S.M M Akram

2015-10-01

Full Text Available  AbstractMixed ligand complexes of  Cu(II, Ni(II and Co(II have been synthesized by using levofloxacin and bipyridyl and characterized using spectral and analytical techniques. The binding behavior of the Ni(II and Cu(II complexes with herring sperm DNA(Hs-DNA were determined using electronic absorption titration, viscometric measurements and cyclic voltammetry measurements. The binding constant calculated  for Cu(II and Ni(II complexes are 2.0 x 104 and 4.0 x 104 M-1 respectively. Detailed analysis reveals that these metal complexes interact with DNA through intercalative binding mode. The nuclease activity of  Cu(II and Ni(II complexes with ct-DNA was carried out using agarose gel electrophoresis technique. The antioxidant activities for the synthesized complexes have been tested and the antibacterial activity for Ni(II complex was also checked.Key words: Intercalation, hypochromism, red shift and  peak potential.

Mixed ligand complexes of alkaline earth metals: Part XII. Mg(II, Ca(II, Sr(II and Ba(II complexes with 5-chlorosalicylaldehyde and salicylaldehyde or hydroxyaromatic ketones

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MITHLESH AGRAWAL

2002-04-01

Full Text Available The reactions of alkaline earth metal chlorides with 5-chlorosalicylaldehyde and salicylaldehyde, 2-hydroxyacetophenone or 2-hydroxypropiophenone have been carried out in 1 : 1 : 1 mole ratio and the mixed ligand complexes of the type MLL’(H2O2 (where M = Mg(II, Ca(II, Sr(II and Ba(II, HL = 5-chlorosalicylaldehyde and HL’ = salicylaldehyde, 2-hydroxyacetophenone or 2-hydroxypropiophenone have been isolated. These complexes were characterized by TLC, conductance measurements, IR and 1H-NMR spectra.

Intrafibrillar Mineral May be Absent in Dentinogenesis Imperfecta Type II (DI-II)

Energy Technology Data Exchange (ETDEWEB)

Pople, John A

2001-03-29

High-resolution synchrotron radiation computed tomography (SRCT) and small angle x-ray scattering (SAXS) were performed on normal and dentinogenesis imperfecta type II (DI-II) teeth. Three normal and three DI-II human third molars were used in this study. The normal molars were unerupted and had intact enamel; donors were female and ranged in age from 18-21y. The DI-II specimens, which were also unerupted with intact enamel, came from a single female donor age 20y. SRCT showed that the mineral concentration was 33% lower on average in the DI-II dentin with respect to normal dentin. The SAXS spectra from normal dentin exhibited low-angle diffraction peaks at harmonics of 67.6 nm, consistent with nucleation and growth of the apatite phase within gaps in the collagen fibrils (intrafibrillar mineralization). In contrast, the low-angle peaks were almost nonexistent in the DI-II dentin. Crystallite thickness was independent of location in both DI-II and normal dentin, although the crystallites were significantly thicker in DI-II dentin (6.8 nm (s.d. = 0.5) vs 5.1 nm (s.d. = 0.6)). The shape factor of the crystallites, as determined by SAXS, showed a continuous progression in normal dentin from roughly one-dimensional (needle-like) near the pulp to two-dimensional (plate-like) near the dentin-enamel junction. The crystallites in DI-II dentin, on the other hand, remained needle-like throughout. The above observations are consistent with an absence of intrafibrillar mineral in DI-II dentin.

Adsorption of Cu(II), Hg(II), and Ni(II) ions by modified natural wool chelating fibers

International Nuclear Information System (INIS)

Monier, M.; Ayad, D.M.; Sarhan, A.A.

2010-01-01

The graft copolymerization of ethyl acrylate (EA) onto natural wool fibers initiated by potassium persulphate and Mohr's salt redox initiator system in limited aqueous medium was carried out in heterogeneous media. Ester groups of the grafted copolymers were partially converted into hydrazide function groups followed by hydrazone formation through reaction with isatin. Also the application of the modified fibers for metal ion uptake was studied using Cu(II), Hg(II) and Ni(II). The modified chelating fibers were characterized using FTIR spectroscopy, SEM and X-ray diffraction.

Cd(II and Pb(II complexes of the polyether ionophorous antibiotic salinomycin

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Tanabe Makoto

2011-09-01

Full Text Available Abstract Background The natural polyether ionophorous antibiotics are used for the treatment of coccidiosis in poultry and ruminants. They are effective agents against infections caused by Gram-positive microorganisms. On the other hand, it was found that some of these compounds selectively bind lead(II ions in in vivo experiments, despite so far no Pb(II-containing compounds of defined composition have been isolated and characterized. To assess the potential of polyether ionophores as possible antidotes in the agriculture, a detailed study on their in vitro complexation with toxic metal ions is required. In the present paper we report for the first time the preparation and the structure elucidation of salinomycin complexes with ions of cadmium(II and lead(II. Results New metal(II complexes of the polyether ionophorous antibiotic salinomycin with Cd(II and Pb(II ions were prepared and structurally characterized by IR, FAB-MS and NMR techniques. The spectroscopic information and elemental analysis data reveal that sodium salinomycin (SalNa undergoes a reaction with heavy metal(II ions to form [Cd(Sal2(H2O2] (1 and [Pb(Sal(NO3] (2, respectively. Abstraction of sodium ions from the cavity of the antibiotic is occurring during the complexation reaction. Salinomycin coordinates with cadmium(II ions as a bidentate monoanionic ligand through the deprotonated carboxylic moiety and one of the hydroxyl groups to yield 1. Two salinomycin anions occupy the equatorial plane of the Cd(II center, while two water molecules take the axial positions of the inner coordination sphere of the metal(II cation. Complex 2 consists of monoanionic salinomycin acting in polydentate coordination mode in a molar ratio of 1: 1 to the metal ion with one nitrate ion for charge compensation. Conclusion The formation of the salinomycin heavy metal(II complexes indicates a possible antidote activity of the ligand in case of chronic/acute intoxications likely to occur in the stock

Cd(II) and Pb(II) complexes of the polyether ionophorous antibiotic salinomycin

Science.gov (United States)

2011-01-01

Background The natural polyether ionophorous antibiotics are used for the treatment of coccidiosis in poultry and ruminants. They are effective agents against infections caused by Gram-positive microorganisms. On the other hand, it was found that some of these compounds selectively bind lead(II) ions in in vivo experiments, despite so far no Pb(II)-containing compounds of defined composition have been isolated and characterized. To assess the potential of polyether ionophores as possible antidotes in the agriculture, a detailed study on their in vitro complexation with toxic metal ions is required. In the present paper we report for the first time the preparation and the structure elucidation of salinomycin complexes with ions of cadmium(II) and lead(II). Results New metal(II) complexes of the polyether ionophorous antibiotic salinomycin with Cd(II) and Pb(II) ions were prepared and structurally characterized by IR, FAB-MS and NMR techniques. The spectroscopic information and elemental analysis data reveal that sodium salinomycin (SalNa) undergoes a reaction with heavy metal(II) ions to form [Cd(Sal)2(H2O)2] (1) and [Pb(Sal)(NO3)] (2), respectively. Abstraction of sodium ions from the cavity of the antibiotic is occurring during the complexation reaction. Salinomycin coordinates with cadmium(II) ions as a bidentate monoanionic ligand through the deprotonated carboxylic moiety and one of the hydroxyl groups to yield 1. Two salinomycin anions occupy the equatorial plane of the Cd(II) center, while two water molecules take the axial positions of the inner coordination sphere of the metal(II) cation. Complex 2 consists of monoanionic salinomycin acting in polydentate coordination mode in a molar ratio of 1: 1 to the metal ion with one nitrate ion for charge compensation. Conclusion The formation of the salinomycin heavy metal(II) complexes indicates a possible antidote activity of the ligand in case of chronic/acute intoxications likely to occur in the stock farming

Synthesis and characterization of zinc(II), cadmium(II) and mercury(II) complexes with bis(bidentate) Schiff bases

International Nuclear Information System (INIS)

Das, Mrinal Kanti; Ghosh, Shyamali

1998-01-01

A few Zn(II), Cd(II) and Hg(II) complexes of the bis(bidentate) Schiff bases derived from p-phenylenediamine and salicylaldehyde (H 2 Salpphen), and o-phenylenediamine and o-vanillin (H 2 Vanophen), of the type MCl 2 .H 2 L(H 2 L = H 2 Salpphen or H 2 Vanophen) have been synthesised. The complexes have been characterized by elemental analysis, infrared, 1 H and 13 C NMR and mass spectra. (author)

Synthesis, characterization and biological studies of 2-(4-nitro phenylaminocarbonyl)benzoic acid and its complexes with Cr(III), Co(II), Ni(II), Cu(II) and Zn(II)

International Nuclear Information System (INIS)

Aqeel Ashraf, M.; Jamil Maah, M.; Yusuf, I.

2012-01-01

Cr(III), Co(II), Ni(II), Cu(II) and Zn(II) salts of 2-(4-nitro phenylaminocarbonyl)benzoic acid were characterized by physical, analytical and spectroscopic studies and checked for their in-vitro antimicrobial activity against three bacterial strains, Mycobacterium smegmatis (Gram +ve), Escherichia coli (Gram -ve), Pseudomonas aeuroginosa (Gram -ve) and three fungal strains, Nigrospora oryzae, Aspergillus niger and Candida albicans. The antimicrobial activities of the metal complexes - were found to be greater than those of 2-(4-nitro phenylaminocarbonyl)benzoic acid alone.

Adsorption of Cu(II), Hg(II), and Ni(II) ions by modified natural wool chelating fibers

Energy Technology Data Exchange (ETDEWEB)

Monier, M., E-mail: monierchem@yahoo.com [Chemistry Department, Drexel University, Philadelphia, PA (United States); Chemistry Department, Faculty of Science, Mansoura University, Mansoura (Egypt); Ayad, D.M.; Sarhan, A.A. [Chemistry Department, Faculty of Science, Mansoura University, Mansoura (Egypt)

2010-04-15

The graft copolymerization of ethyl acrylate (EA) onto natural wool fibers initiated by potassium persulphate and Mohr's salt redox initiator system in limited aqueous medium was carried out in heterogeneous media. Ester groups of the grafted copolymers were partially converted into hydrazide function groups followed by hydrazone formation through reaction with isatin. Also the application of the modified fibers for metal ion uptake was studied using Cu(II), Hg(II) and Ni(II). The modified chelating fibers were characterized using FTIR spectroscopy, SEM and X-ray diffraction.

Adsorption of Cu(II), Hg(II), and Ni(II) ions by modified natural wool chelating fibers.

Science.gov (United States)

Monier, M; Ayad, D M; Sarhan, A A

2010-04-15

The graft copolymerization of ethyl acrylate (EA) onto natural wool fibers initiated by potassium persulphate and Mohr's salt redox initiator system in limited aqueous medium was carried out in heterogeneous media. Ester groups of the grafted copolymers were partially converted into hydrazide function groups followed by hydrazone formation through reaction with isatin. Also the application of the modified fibers for metal ion uptake was studied using Cu(II), Hg(II) and Ni(II). The modified chelating fibers were characterized using FTIR spectroscopy, SEM and X-ray diffraction. 2009 Elsevier B.V. All rights reserved.

Kinetic study on adsorption of Cr(VI), Ni(II), Cd(II) and Pb(II) ions from aqueous solutions using activated carbon prepared from Cucumis melo peel

Science.gov (United States)

Manjuladevi, M.; Anitha, R.; Manonmani, S.

2018-03-01

The adsorption of Cr(VI), Ni(II), Cd(II) and Pb(II), ions from aqueous solutions by Cucumis melo peel-activated carbon was investigated under laboratory conditions to assess its potential in removing metal ions. The adsorption behavior of metal ions onto CMAC was analyzed with Elovich, intra-particle diffusion rate equations and pseudo-first-order model. The rate constant of Elovich and intra-particle diffusion on CMAC increased in the sequence of Cr(VI) > Ni(II) > Cd(II) > Pb(II). According to the regression coefficients, it was observed that the kinetic adsorption data can fit better by the pseudo-first-order model compared to the second-order Lagergren's model with R 2 > 0.957. The maximum adsorption of metal ions onto the CMAC was found to be 97.95% for Chromium(VI), 98.78% for Ni(II), 98.55% for Pb(II) and 97.96% for Cd(II) at CMAC dose of 250 mg. The adsorption capacities followed the sequence Ni(II) ≈ Pb(II) > Cr(VI) ≈ Cd(II) and Ni(II) > Pb(II) > Cd(II) > Cr(VI). The optimum adsorption conditions selected were adsorbent dosage of 250 mg, pH of 3.0 for Cr(VI) and 6.0 for Ni(II), Cd(II) and Pb(II), adsorption concentration of 250 mg/L and contact time of 180.

Standardization of radioimmunoassay for dosage of angiotensin II (ang-II) and its methodological evaluation; Padronizacao do radioimunoensaio para dosagem de angiotensina II (ang-II) e sua validacao metodologica

Energy Technology Data Exchange (ETDEWEB)

Mantovani, Milene; Mecawi, Andre S.; Elias, Lucila L.K.; Antunes-Rodrigues, Jose, E-mail: llelias@fmrp.usp.b, E-mail: antunes@fmrp.usp.b [Universidade de Sao Paulo (FMRP/USP), Ribeirao Preto, SP (Brazil). Faculdade de Medicina

2011-10-26

This paper standardizes the radioimmunoassay (RIA) for dosage of ANG-II of rats, after experimental conditions of saline hypertonic (2%), treating with losartan (antagonist of ANG-II), hydric privation, and acute hemorrhage (25%). After that, the plasmatic ANG-II was extracted for dosage of RIA, whose sensitiveness was of 1.95 pg/m L, with detection of 1.95 to 1000 pg/m L. The treatment with saline reduced the concentration of ANG-II, while the administration pf losartan, the hydric administration and the hemorrhage increase the values, related to the control group. Those results indicate variations in the plasmatic concentration of ANG-II according to the experimental protocols, validating the method for evaluation of activity renin-angiotensin

Pius II. a utrakvismus

OpenAIRE

Šimek, Milan

2009-01-01

Milan Šimek Pius II. a utrakvismus Pius II. and utraquism Based on sources work - out, the thesis aims the description and analysis of the attitude alternation of Enea Sylvio Piccolomini - Pius II to the utraquism. The conclusions stress the postulate that Pius II. did not change that attitude, but just did not succed in quelling the utraquist movement. In the sense of political background that finally led to fatal dissention among both leaders, king Jiří of Poděbrady and pope Pius II.

Biosorption of Cd(II) and Zn(II) by nostoc commune: isotherm and kinetics studies

Energy Technology Data Exchange (ETDEWEB)

Morsy, Fatthy M. [Faculty of Science, Botany Department, Assiut University, Assiut (Egypt); Hassan, Sedky H.A. [Department of Biological Environment, Kangwon National University, Kangwon-do (Korea, Republic of); Koutb, Mostafa [Faculty of Science, Botany Department, Assiut University, Assiut (Egypt); Umm Al-Qura University, Faculty of Applied Science, Biology Department, Mecca (Saudi Arabia)

2011-07-15

In this study, Nostoc commune (cyanobacterium) was used as an inexpensive and efficient biosorbent for Cd(II) and Zn(II) removal from aqueous solutions. The effect of various physicochemical factors on Cd(II) and Zn(II) biosorption such as pH 2.0-7.0, initial metal concentration 0.0-300 mg/L and contact time 0-120 min were studied. Optimum pH for removal of Cd(II) and Zn(II) was 6.0, while the contact time was 30 min at room temperature. The nature of biosorbent and metal ion interaction was evaluated by infrared (IR) technique. IR analysis of bacterial biomass revealed the presence of amino, carboxyl, hydroxyl, and carbonyl groups, which are responsible for biosorption of Cd(II) and Zn (II). The maximum biosorption capacities for Cd(II) and Zn(II) biosorption by N. commune calculated from Langmuir biosorption isotherm were 126.32 and 115.41 mg/g, respectively. The biosorption isotherm for two biosorbents fitted well with Freundlich isotherm than Langmuir model with correlation coefficient (r{sup 2} < 0.99). The biosorption kinetic data were fitted well with the pseudo-second-order kinetic model. Thus, this study indicated that the N. commune is an efficient biosorbent for the removal of Cd(II) and Zn(II) from aqueous solutions. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Cadmium(II) and lead(II) adsorption onto hetero-atom functional mesoporous silica and activated carbon

Science.gov (United States)

Machida, Motoi; Fotoohi, Babak; Amamo, Yoshimasa; Mercier, Louis

2012-07-01

Adsorption of cadmium(II) and lead(II) on amino-, mercapto-functionalized mesoporous silica (HMS) and carboxylic-functionalized activated carbon (AC) were examined. The resultant isotherms fitted the Langmuir model and amino-functionalized HMS exhibited the highest adsorption capacity for both cadmium(II) and lead(II). Adsorption affinities for cadmium(II) were always greater than those for lead(II) in all three adsorbent types, while the difference between the two values was the largest for mercapto-functionalized HMS indicating a selective adsorption of cadmium(II). Influence of equilibrium solution pH on adsorption of cadmium(II), lead(II) and their binary mixtures was also studied. Carboxylic-functionalized AC adsorbed cadmium(II) and lead(II) in a wide pH range than conditions for the mercapto-functionalized HMS. It was concluded that each functional group had its own characteristics and advantages for adsorption of heavy metal ions; amino-groups showed high adsorption capacity, while mercapto-groups had good selectivity toward cadmium(II) adsorption and a wide solution pH in adsorption by carboxylic-groups were established in this study.

Cadmium(II) and lead(II) adsorption onto hetero-atom functional mesoporous silica and activated carbon

International Nuclear Information System (INIS)

Machida, Motoi; Fotoohi, Babak; Amamo, Yoshimasa; Mercier, Louis

2012-01-01

Adsorption of cadmium(II) and lead(II) on amino-, mercapto-functionalized mesoporous silica (HMS) and carboxylic-functionalized activated carbon (AC) were examined. The resultant isotherms fitted the Langmuir model and amino-functionalized HMS exhibited the highest adsorption capacity for both cadmium(II) and lead(II). Adsorption affinities for cadmium(II) were always greater than those for lead(II) in all three adsorbent types, while the difference between the two values was the largest for mercapto-functionalized HMS indicating a selective adsorption of cadmium(II). Influence of equilibrium solution pH on adsorption of cadmium(II), lead(II) and their binary mixtures was also studied. Carboxylic-functionalized AC adsorbed cadmium(II) and lead(II) in a wide pH range than conditions for the mercapto-functionalized HMS. It was concluded that each functional group had its own characteristics and advantages for adsorption of heavy metal ions; amino-groups showed high adsorption capacity, while mercapto-groups had good selectivity toward cadmium(II) adsorption and a wide solution pH in adsorption by carboxylic-groups were established in this study.

Synthesis, characterization and biological studies of metal complexes of Co (II), Ni (II), Cu (II), Zn (II) with sulphadimidine-benzylidene

International Nuclear Information System (INIS)

Tahira, F.; Imran, M.; Iqbal, J.

2009-01-01

Some novel complexes of Co (II), Ni (II), Cu (II), and Zn (II) have been synthesized with a Schiff base ligand derived from sulphadimidine and benzaldehyde. The structural features of the complexes have been determined by elemental analysis, magnetic susceptibility, conductance measurement, UV/ Vis. and infrared spectroscopy. IR studies revealed that the Schiff base ligand Sulphadimidine-benzylidene has monoanionic bidendate nature and coordinate with metal ions through nitrogen atom of azomethine (>C = N) and deprotonated -NH group. All the complexes were assigned octahedral geometry on the basis of magnetic moment and electronic spectroscopic data. Low value of conductance supports their non-electrolytic nature. The ligand, as well as its complexes were checked for their in vitro antimicrobial activities against two gram positive bacterial strains, Bacillus subtillus. Staphylococcus aureus and one gram negative Salmonella typhae and five fungal strains, Nigrospora oryzae, Curvularia lunata, Drechslera rostrata, Aspergillus niger and Candida olbicans by disc diffusion method and agar plate technique, respectively. Both the antibacterial and antitungal activities of the synthesized metal complexes were found to be more as compared to parent drug and uncomplexed ligand. All the complexes contain coordinated water, which is lost at 141-160 degree C. (author)

Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency

DEFF Research Database (Denmark)

Olpin, S E; Afifi, A; Clark, S

2003-01-01

Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients...

Elizabeth II uus kunstigalerii

Index Scriptorium Estoniae

1999-01-01

Tähistamaks oma troonile asumise 50. aastapäeva, avab Elizabeth II 6. II 2002 Buckinghami palees uue kunstigalerii, mis ehitatakse palee tiibhoonena. Arhitekt John Simpson. Elizabeth II kunstikogust

Electrochemical, spectroscopic, and photophysical properties of structurally diverse polyazine-bridged Ru(II),Pt(II) and Os(II),Ru(II),Pt(II) supramolecular motifs.

Science.gov (United States)

Knoll, Jessica D; Arachchige, Shamindri M; Wang, Guangbin; Rangan, Krishnan; Miao, Ran; Higgins, Samantha L H; Okyere, Benjamin; Zhao, Meihua; Croasdale, Paul; Magruder, Katherine; Sinclair, Brian; Wall, Candace; Brewer, Karen J

2011-09-19

Five new tetrametallic supramolecules of the motif [{(TL)(2)M(dpp)}(2)Ru(BL)PtCl(2)](6+) and three new trimetallic light absorbers [{(TL)(2)M(dpp)}(2)Ru(BL)](6+) (TL = bpy = 2,2'-bipyridine or phen = 1,10-phenanthroline; M = Ru(II) or Os(II); BL = dpp = 2,3-bis(2-pyridyl)pyrazine, dpq = 2,3-bis(2-pyridyl)quinoxaline, or bpm = 2,2'-bipyrimidine) were synthesized and their redox, spectroscopic, and photophysical properties investigated. The tetrametallic complexes couple a Pt(II)-based reactive metal center to Ru and/or Os light absorbers through two different polyazine BL to provide structural diversity and interesting resultant properties. The redox potential of the M(II/III) couple is modulated by M variation, with the terminal Ru(II/III) occurring at 1.58-1.61 V and terminal Os(II/III) couples at 1.07-1.18 V versus Ag/AgCl. [{(TL)(2)M(dpp)}(2)Ru(BL)](PF(6))(6) display terminal M(dπ)-based highest occupied molecular orbitals (HOMOs) with the dpp(π*)-based lowest unoccupied molecular orbital (LUMO) energy relatively unaffected by the nature of BL. The coupling of Pt to the BL results in orbital inversion with localization of the LUMO on the remote BL in the tetrametallic complexes, providing a lowest energy charge separated (CS) state with an oxidized terminal Ru or Os and spatially separated reduced BL. The complexes [{(TL)(2)M(dpp)}(2)Ru(BL)](6+) and [{(TL)(2)M(dpp)}(2)Ru(BL)PtCl(2)](6+) efficiently absorb light throughout the UV and visible regions with intense metal-to-ligand charge transfer (MLCT) transitions in the visible at about 540 nm (M = Ru) and 560 nm (M = Os) (ε ≈ 33,000-42,000 M(-1) cm(-1)) and direct excitation to the spin-forbidden (3)MLCT excited state in the Os complexes about 720 nm. All the trimetallic and tetrametallic Ru-based supramolecular systems emit from the terminal Ru(dπ)→dpp(π*) (3)MLCT state, λ(max)(em) ≈ 750 nm. The tetrametallic systems display complex excited state dynamics with quenching of the (3)MLCT emission at

Synthesis, characterization and polymerization of methacrylates of copper (II), cobalt (II) and molybdenum (II). Generation of new materials

International Nuclear Information System (INIS)

Rojas Bolanos, Omar

2006-01-01

Coordination compounds of the species copper (II), cobalt (II) and molybdenum (II) with methacrylic acid were synthesized and characterized. Besides, it realized reactions of bromine addition to the doubles links of the species obtained previously, also too like reactions with dry HCl. Finally, it got hybrids materials by polymerization of the first compounds in an acrylic matrix. Research concluded with the characterization of all the products. (author) [es

Synthesis of amino functionalized magnetic graphenes composite material and its application to remove Cr(VI), Pb(II), Hg(II), Cd(II) and Ni(II) from contaminated water

International Nuclear Information System (INIS)

Guo, Xiaoyao; Du, Bin; Wei, Qin; Yang, Jian; Hu, Lihua; Yan, Liangguo; Xu, Weiying

2014-01-01

Highlights: • Graphenes magnetic composite nanoparticles (Fe 3 O 4 -GS) were used to adsorb metal ions. • The adsorption of metal ions onto Fe 3 O 4 -GS could be well interpreted by the Freundlich equation. • The adsorption of metal ions onto Fe 3 O 4 -GS fit pseudo-second order kinetic model. • Thermodynamic studies illustrated that the adsorption process was endothermic and spontaneous in nature. - Abstract: In the present study, a kind of graphenes magnetic material (Fe 3 O 4 -GS) was prepared by compositing graphene sheet with ferroferric oxide, and shown to be effictive for removing Cr(VI), Pb(II), Hg(II), Cd(II) and Ni(II) ions from aqueous solution. The synthesized sorbent was characterized by SEM, TEM, FTIR, XRD, XPS and BET, respectively. The pH ZPC value of the sorbent was estimated to be 3.5 by alkaline-titration methods. Fe 3 O 4 -GS can be simply recovered from water with magnetic separation at low magnetic field within one minute. The sorption capacities of the metals were 17.29, 27.95, 23.03, 27.83 and 22.07 mg g −1 for Cr(VI), Pb(II), Hg(II), Cd(II) and Ni(II), respectively. Kinetic data showed good correlation with pseudo-second-order equation and the Freundlich model was found to fit for the isotherm data of all the heavy metal ions. It was found that the metals sorption was accomplished mainly via chelation or ion exchange. The results of thermodynamic studies illustrate that the adsorption process was endothermic and spontaneous in nature

Removal of Pb(II), Cu(II) and Cd(II) from aqueous solution by some fungi and natural adsorbents in single and multiple metal systems

International Nuclear Information System (INIS)

Shoaib, A.; Badar, T.; Aslam, N.

2011-01-01

Six fungal and 10 natural biosorbents were analyzed for their Cu(II), Cd(II) and Pb(II) uptake capacity from single, binary and ternary metal ion system. Preliminary screening biosorption of assays revealed 2 fungi (Aspergillus niger and Cunninghamella echinulata) and three natural [Cicer arietinum husk, Moringa oleifera flower and soil (clay)] adsorbents hold considerable high adsorption efficiency and capacity for 3 meta l ions amongst the adsorbents. Further biosorption trials with five elected adsorbents showed a considerable reduction in metal uptake capability of adsorbents in binary- and ternary systems as compared to singly metal system. Cd(II) manifested the highest inhibitory effect on the biosorption of other metal ions, followed by Pb(II) and Cu(II). On account of metal preference, the selectivity order for metal ion towards the studied biomass matrices was Pb(II) (40-90%) > Cd(II) (2-53%) > Cu(II) (2-30%). (author)

Syntheses and spectroscopic properties of mercury(II) and nickel(II ...

African Journals Online (AJOL)

Syntheses and spectroscopic properties of mercury(II) and nickel(II) ... The complexes were characterized by IR, diffuse reflectance, 1H NMR spectra and elemental ... coordinating through thiolato sulphur and hydrazinic nitrogen atoms.

Simultaneous production of raw starch degrading highly ...

African Journals Online (AJOL)

The widely used thermostable amylases were produced long time ago from Bacillus genus. Although, lactic acid bacteria (LAB) fermentation presents several advantages including the reduction of growth of pathogenic microorganisms, no study has yet reported thermostable amylases from lactic acid bacteria. An amylolytic ...

Low-temperature Synthesis of Tin(II) Oxide From Tin(II) ketoacidoximate Precursor

KAUST Repository

Alshankiti, Buthainah

2015-01-01

Sn (II) oxide finds numerous applications in different fields such as thin film transistors1, solar cells2 and sensors.3 In this study we present the fabrication of tin monoxide SnO by using Sn (II) ketoacid oximate complexes as precursors. Tin (II

Biosorption optimization of lead(II), cadmium(II) and copper(II) using response surface methodology and applicability in isotherms and thermodynamics modeling

International Nuclear Information System (INIS)

Singh, Rajesh; Chadetrik, Rout; Kumar, Rajender; Bishnoi, Kiran; Bhatia, Divya; Kumar, Anil; Bishnoi, Narsi R.; Singh, Namita

2010-01-01

The present study was carried out to optimize the various environmental conditions for biosorption of Pb(II), Cd(II) and Cu(II) by investigating as a function of the initial metal ion concentration, temperature, biosorbent loading and pH using Trichoderma viride as adsorbent. Biosorption of ions from aqueous solution was optimized in a batch system using response surface methodology. The values of R 2 0.9716, 0.9699 and 0.9982 for Pb(II), Cd(II) and Cu(II) ions, respectively, indicated the validity of the model. The thermodynamic properties ΔG o , ΔH o , ΔE o and ΔS o by the metal ions for biosorption were analyzed using the equilibrium constant value obtained from experimental data at different temperatures. The results showed that biosorption of Pb(II) ions by T. viride adsorbent is more endothermic and spontaneous. The study was attempted to offer a better understating of representative biosorption isotherms and thermodynamics with special focuses on binding mechanism for biosorption using the FTIR spectroscopy.

Biosorption optimization of lead(II), cadmium(II) and copper(II) using response surface methodology and applicability in isotherms and thermodynamics modeling

Energy Technology Data Exchange (ETDEWEB)

Singh, Rajesh; Chadetrik, Rout; Kumar, Rajender; Bishnoi, Kiran; Bhatia, Divya; Kumar, Anil [Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana (India); Bishnoi, Narsi R., E-mail: nrbishnoi@gmail.com [Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana (India); Singh, Namita [Department of Bio and Nanotechnology, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana (India)

2010-02-15

The present study was carried out to optimize the various environmental conditions for biosorption of Pb(II), Cd(II) and Cu(II) by investigating as a function of the initial metal ion concentration, temperature, biosorbent loading and pH using Trichoderma viride as adsorbent. Biosorption of ions from aqueous solution was optimized in a batch system using response surface methodology. The values of R{sup 2} 0.9716, 0.9699 and 0.9982 for Pb(II), Cd(II) and Cu(II) ions, respectively, indicated the validity of the model. The thermodynamic properties {Delta}G{sup o}, {Delta}H{sup o}, {Delta}E{sup o} and {Delta}S{sup o} by the metal ions for biosorption were analyzed using the equilibrium constant value obtained from experimental data at different temperatures. The results showed that biosorption of Pb(II) ions by T. viride adsorbent is more endothermic and spontaneous. The study was attempted to offer a better understating of representative biosorption isotherms and thermodynamics with special focuses on binding mechanism for biosorption using the FTIR spectroscopy.

Iodine capture by Hofmann-type clathrate Ni(II)(pz)[Ni(II)(CN)_4

International Nuclear Information System (INIS)

Massasso, Giovanni; Long, Jerome; Haines, Julien; Devautour-Vinot, Sabine; Maurin, Guillaume; Larionova, Joulia; Guerin, Christian; Guari, Yannick; Grandjean, Agnes; Onida, Barbara; Donnadieu, Bruno

2014-01-01

The thermally stable Hofmann-type clathrate framework Ni(II)(pz)[Ni(II)(CN)_4] (pz = pyrazine) was investigated for the efficient and reversible sorption of iodine (I_2) in the gaseous phase and in solution with a maximum adsorption capacity of 1 mol of I_2 per 1 mol of Ni(II)pz)[Ni(II)(CN)_4] in solution. (authors)

Adsorption characteristics of Pb(II) and Cu(II) ions by domestic clays

International Nuclear Information System (INIS)

Kwon, Ee Yol; Noh, Hea Ran

1990-01-01

This investigation was carried out to study the adsorption characteristics of Pb(II) and Cu(II) ions in aqueous solution by using clays of Gampo 35, Bentonite (chulwon) and Mangwoon 95 which were dug in the country. As the results, the adsorption of metal ions clays were reached equilibrium by shaking for about 40-60 minutes. In acidic solution, the adsorptivity of clays was increased as pH increased, however, Gampo 35 showed the high adsorptivity over 90% even at pH2-3. Pb(II) ion showed better removal efficiency than Cu(II) ion. The adsorptivities of adsorbents showed following order: Gampo 35>Bentonite> Mangwoon 95. The adsorption isotherms of Pb(II) ion on clays were well fitted in Freundlich's equation. Freundlich constantstion isotherms of Pb(II) ion on clays were well fitted in Freundlich's equation. Freundlich constants (1/n) of Gampo 35, Bentonite and Mangwoon 95 were 0.195, 0.271 and 0.314, respectively.(Author)

Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

Science.gov (United States)

Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

2015-12-01

Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly

Forest structure, diversity and regeneration potential along altitudinal gradient in Dhanaulti of Garhwal Himalaya

Energy Technology Data Exchange (ETDEWEB)

Saha, S.; Rajwar, G.S.; Kumar, M.

2016-07-01

Aim of the study: The aim of the present study was to understatnd the forest composition, structure, diversity and regeneration potential along altitudinal gradient. Area of study: The study was carried out in Dhanaulti forest which falls under temperate region of Garhwal Himalaya in Uttarakhand state, India. Material and Methods: Vegetation analysis was carried out using 10 quadrats at each altitude using a quadrate size of 10×10 m2. In each quadrate, categories of trees >30 cm cbh were considered as trees, 10-30cm cbh as saplings and <10 cm cbh as seedlings. The data were quantitatively analyzed. Main results: In upper and middle altitudes, Cedrus deodara was reported dominant tree whereas, in lower altitude Quercus leucotrichophora was reported dominant. Tree density was highest in lower altitude which reduced middle and upper altitudes whereas, total basal cover increased with increasing altitude. The increasing total basal cover with altitude could be because of the presence of Cedrus deodara trees having higher girth classes. In tree, sapling and seedling layers, diversity (H) and equitabiltiy (EC) decreased with increasing altitude. However, concentrations of dominace (CD) and beta diversity (BD have shown reverse trend with H and EC which increased with increasing altitudes, in each layer of tree, sapling and seedling. The distribution pattern of most species in all layers of trees, saplings and seedlings was contagious. The regeneration potential of the species has shown that some of the species in the absence of tree layer are still regenerating particularly, Rhododendron arboreum, Benthamidia capitata, Neolitsea pallens etc. It indicates that most of the species are shifting upward as they are getting suitable conditions. Research highlights: Altitude influence species composition, diversity and regeneration potential of species. (Author)